pretreatment of pulp mill wastewater ... abstract pretreatment of pulp mill wastewater treatment...
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PRETREATMENT OF PULP MILL WASTEWATER TREATMENT RESIDUES TO
IMPROVE THEIR ANAEROBIC DIGESTION
by
Nicholas Wood
A thesis submitted in conformity with the requirements
for the degree of Masters of Applied Science
Department of Chemical Engineering and Applied Chemistry
University of Toronto
© Copyright by Nicholas Wood 2008
ii
Abstract
Pretreatment of Pulp Mill Wastewater Treatment Residues to Improve Their Anaerobic
Digestion
by
Nicholas Wood
Masters of Applied Science
Department of Chemical Engineering and Applied Chemistry
University of Toronto, 2008
Anaerobic digestion of excess biological wastewater treatment sludge (WAS) from pulp
mills has the potential to reduce disposal costs and to generate energy through biogas production.
The organic matter in WAS is highly structured, which normally hinders biogas production. This
study investigated three methods of pretreating WAS from two different pulp mills before
anaerobic digestion to improve biogas yield and production rate. The three pretreatment
methods tested were: i) thermal pretreatment at 170oC, ii) caustic pretreatment at 140oC and pH
12, and iii) sonication at 20 kHz and 1 W/mL. Thermal pretreatment proved to be the most
effective, increasing biogas yield by 280% and 50% and increasing production rates 300-fold and
10-fold for the two samples, respectively. Caustic pretreatment showed similar results, but
resulted in the formation of soluble non-biodegradable compounds. Sonication was the least
effective pretreatment and did not substantially increase biogas yield, but increased biogas
production rate.
iii
Acknowledgements
I would like to thank my supervising professors Prof. Emma Master and Prof. Honghi
Tran, for their guidance and encouragement. I would like to thank NSERC, Tembec Inc., the
research consortium on "Alternative Fuels for Lime Kilns", and DMI for the financial support
and technical expertise they provided throughout the project. I would also like to thank the
following people that helped me with experimental techniques, solving problems, allowed me
access to their equipment, and for providing guidance and encouragement throughout my
project: Luke Pestl, Sonam Mahajan, and everyone else in Prof. Master’s lab group; Chris
Goode, Ivy Yang, and everyone else in Prof. Allen’s lab group; Melanie Duhamel, Cheryl
Washer, and everyone else in Prof. Edwards’ lab group; Ilya Perederiy, Sammy Peters, and
everyone else in Prof. Papangelakis’ lab group; Prof. Elizabeth Edwards; Prof. Grant Allen; Prof.
Vladimiros Papangelakis; Bert Wasmund; and Paul Jowlabar. Further, I would like to thank
Mohan Pandit, Angelina Tan, and Adrew Barquin for their hard work as summer research
students and their contribution to this project. Finally, I would like to thank my friends and
family for their support and encouragement over the course of the project.
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Table of Contents
Abstract ........................................................................................................................................... ii
Acknowledgements........................................................................................................................ iii
Table of Contents........................................................................................................................... iv
List of Tables ................................................................................................................................ vii
List of Figures ................................................................................................................................ ix
1.0 Introduction......................................................................................................................... 1
1.1 The Aerobic Wastewater Treatment Sludge (WAS) Problem........................................ 1
1.2 Potential for Anaerobic Digestion of WAS .................................................................... 1
1.3 Challenges and Opportunities ......................................................................................... 2
1.4 Objectives of the Project ................................................................................................. 3
2.0 Literature Survey ................................................................................................................ 4
2.1 Aerobic Biological Wastewater Treatment Systems ...................................................... 4
2.1.1 Wastewater Treatment Systems in the Pulp and Paper Industry ............................ 6
2.2 Waste Aerobic Wastewater Treatment Sludge (WAS)................................................... 8
2.2.1 Physical and Biological Structure........................................................................... 8
2.2.2 Composition.......................................................................................................... 10
2.2.3 Dewatering and Disposal ...................................................................................... 10
2.3 Anaerobic Digestion ..................................................................................................... 11
2.3.1 Microbiology......................................................................................................... 13
2.3.2 Biogas Properties .................................................................................................. 14
2.3.3 Reactor Configurations ......................................................................................... 15
2.3.4 Anaerobic Digestion in the Pulp and Paper Industry............................................ 17
2.4 Anaerobic Digestion of WAS ....................................................................................... 18
2.4.1 Previous Studies.................................................................................................... 18
2.4.2 Challenges to Digestion ........................................................................................ 19
2.4.3 Pretreatment Technologies.................................................................................... 21
2.4.3.1 Thermal Pretreatment........................................................................................ 21
2.4.3.2 Chemical Pretreatment...................................................................................... 23
2.4.3.3 Sonication ......................................................................................................... 25
2.4.3.4 Other Physical and Chemical Pretreatments..................................................... 27
2.4.3.5 Enzymatic and Biological Pretreatments .......................................................... 28
v
3.0 Methodology..................................................................................................................... 29
3.1 Biomass Sample Collection and Storage ...................................................................... 29
3.1.1 Aerobic Wastewater Treatment Sludge Samples.................................................. 29
3.1.2 Anaerobic Granule Samples ................................................................................. 29
3.2 Measurement of Physical and Chemical Properties...................................................... 30
3.2.1 Soluble Fraction .................................................................................................... 30
3.2.2 Suspended Solids .................................................................................................. 30
3.2.3 COD ...................................................................................................................... 31
3.2.4 Carbohydrates ....................................................................................................... 32
3.2.5 Protein ................................................................................................................... 33
3.2.6 Total Organic Carbon ........................................................................................... 35
3.2.7 Other Measurements ............................................................................................. 36
3.3 Pretreatments................................................................................................................. 36
3.3.1 Thermal ................................................................................................................. 36
3.3.2 Caustic................................................................................................................... 37
3.3.3 Sonication ............................................................................................................. 37
3.4 Anaerobic Digestion Experiments ................................................................................ 38
3.4.1 Biochemical Methane Potential Assay Setup ....................................................... 38
3.4.2 Nutrient Medium................................................................................................... 40
3.4.3 Sparging to Make Bottles Anaerobic.................................................................... 41
3.4.4 Anaerobic Glovebox ............................................................................................. 42
3.4.5 Biogas Production Measurement .......................................................................... 42
3.4.5.1 Syringe Method................................................................................................. 42
3.4.5.2 Liquid Displacement Method ........................................................................... 43
3.4.5.3 Pressure Transducer Method............................................................................. 44
3.4.5.4 CH4 Measurement............................................................................................. 44
3.5 Calculations................................................................................................................... 45
3.5.1 Statistical Significance.......................................................................................... 45
3.5.2 Error Calculations ................................................................................................. 45
3.5.3 Extent of Degradation ........................................................................................... 46
3.5.4 Reaction Rates ...................................................................................................... 46
4.0 Results and Discussion ..................................................................................................... 48
4.1 Biomass Properties........................................................................................................ 48
vi
4.1.1 Waste Aerobic Wastewater Treatment Sludge ..................................................... 48
4.1.2 Anaerobic Granules .............................................................................................. 50
4.2 BMP Assays Performed................................................................................................ 51
4.3 Untreated WAS Anaerobic Digestibility ...................................................................... 53
4.3.1 Kraft Mill and Sulphite Mill WAS ....................................................................... 53
4.3.2 WAS Digestion Compared to High-Rate Anaerobic Digester Feed..................... 57
4.3.3 WAS Toxicity ....................................................................................................... 57
4.3.4 WAS Digestion without Anaerobic Granules – Self Digestion............................ 58
4.4 Pretreatment Effects...................................................................................................... 59
4.4.1 NaOH Requirements to Bring WAS to pH 12...................................................... 59
4.4.2 Physical and Chemical Changes ........................................................................... 59
4.4.3 Anaerobic Digestion ............................................................................................. 64
4.4.3.1 Extent of Digestion ........................................................................................... 64
4.4.3.2 Total Biogas Production and CH4 Content ....................................................... 66
4.4.3.3 Biogas Production Rate..................................................................................... 71
4.5 Comparison of WAS Properties to Pretreatment Performance..................................... 74
4.6 Energy Balance ............................................................................................................. 79
4.7 Economic Analysis of Pretreatments ............................................................................ 80
4.7.1 WAS Disposal through Combustion..................................................................... 83
4.7.2 WAS Disposal by Landfill.................................................................................... 84
5.0 Conclusion ........................................................................................................................ 87
5.1 Summary....................................................................................................................... 87
5.2 Implications................................................................................................................... 88
5.3 Recommendations......................................................................................................... 89
6.0 Abbreviations.................................................................................................................... 91
7.0 References......................................................................................................................... 92
vii
List of Tables
Table 2.2.1 Summary of chemical composition of secondary sludge from different sources. All values in terms of
percent dry solids.........................................................................................................................................................10
Table 2.3.1 Typical compositions of biogas produced during anaerobic digestion (Deublein & Steinhauser, 2008). 15
Table 2.4.1 Summary of typical results of thermal pretreatment on the anaerobic digestion of WAS from municipal
sources. ........................................................................................................................................................................22
Table 2.4.2 Summary of typical results of thermochemical pretreatment on the anaerobic digestion of WAS from
various sources. ...........................................................................................................................................................24
Table 2.4.3 Summary of typical results of sonication pretreatment on the anaerobic digestion of WAS from
municipal sources. .......................................................................................................................................................26
Table 3.4.1 Concentrations of components in nutrient medium used in biochemical methane potential assays. Stock
concentration refers to the amount of a particular nutrient in its concentrated stock solution. Medium concentration
refers to the amount of a particular nutrient in the final medium mixture. ..................................................................41
Table 4.1.1 Summary of waste activated sludge properties.........................................................................................48
Table 4.1.2 Waste activated sludge biological macromolecule content. .....................................................................49
Table 4.1.3 Elemental analysis of waste activated sludge samples. ............................................................................50
Table 4.1.4 Physical and chemical properties of anaerobic granule samples used in anaerobic digestion experiments
in this study. ................................................................................................................................................................51
Table 4.2.1 Description of BMP assay experiments performed in this study..............................................................52
Table 4.3.1 Total biogas production from S WAS and K WAS. Each substrate was digested in triplicate during the
BMP assay. ..................................................................................................................................................................55
Table 4.3.2 Biogas yield in experiment 1 subtracting blank control values for S WAS, the soluble fraction of S WAS,
and feed sent to the reactor granules were sampled from. ...........................................................................................57
Table 4.3.3 Total biogas produced during the WAS toxicity BMP assay in experiment 2. ........................................58
Table 4.3.4 Total biogas production for samples containing no granules and the blank control which contained
anaerobic granules but no substrate in experiment 1. ..................................................................................................59
Table 4.4.1 NaOH requirements of WAS for caustic pretreatment. ............................................................................59
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Table 4.4.2 Total biogas produced, volume fraction of CH4 in the biogas, and substrate COD and VSS added in
experiment #4 where K WAS was digested. Each value represents an average of a triplicate. .................................70
Table 4.4.3 Total biogas produced, volume fraction of CH4 in the biogas, and substrate COD and VSS added in
experiment #5 where S WAS was digested. Each value represents and average of a triplicate. ................................70
Table 4.4.4 Summary of first-order reaction kinetics regressions. R2 represents the coefficient of determination and
goodness of fit of the regressions. Y0 represents the use of COD at time at infinity and k represents the reaction rate
constant........................................................................................................................................................................72
Table 4.4.5 Summary of regressions performed on different portions of the data from experiment 5 on S WAS.
Range of regression refers to the portion of the data to which the regression was applied. ........................................74
Table 4.5.1 Summary of the slope, intercept, and correlation coefficient of regressions of total biogas production
versus the contents of each assay bottle. “All Data” refers to the data set from both experiment 4 and experiment 5.
.....................................................................................................................................................................................78
Table 4.6.1 Energy requirements and increase in energy from biogas as a result of pretreatment of K WAS in
experiment 4. Sonication (actually applied to WAS) is based on the actual amount of energy passed to the WAS
through sonication. ......................................................................................................................................................80
Table 4.6.2 Energy requirements and increase in energy from biogas as a result of pretreatment of S WAS in
experiment 5. Sonication (actually applied to WAS) is based on the actual amount of energy passed to the WAS
through sonication. ......................................................................................................................................................80
Table 4.7.1 Assumed values used to calculate biogas production from soluble fraction of S WAS. These values
were based on the results obtained from experiment 5 in this study. ..........................................................................82
Table 4.7.2 Assumed values for variables used in the economic analysis. Values were based on estimates provided
by Mill A. All values in Canadian dollars. .................................................................................................................82
Table 4.7.3 Economic analysis of thermal and caustic pretreatment in the case sludge solids were disposed of in a
boiler. All values represent Canadian dollars per m3 of WAS produced requiring pretreatment. Heat recovery refers
to 75% of the energy used during pretreatment being recycled. Improved dewatering refers to the scenario where
pretreatment improves the solid content of dewatered sludge to the point where the solids do not require
supplemental fuel for burning......................................................................................................................................84
Table 4.7.4 Economic analysis of thermal and caustic pretreatment in the case sludge solids were disposed of by
land filling. All values represent Canadian dollars per m3 of WAS produced requiring pretreatment. Heat recovery
refers to 75% of the energy used during pretreatment being recycled.........................................................................85
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List of Figures
Figure 2.1.1 Basic flow diagrams of two commonly used activated sludge system configurations. .............................6
Figure 2.1.2 Flow diagram of a typical wastewater treatment system in a pulp and paper mill. ...................................7
Figure 2.2.1 Photo of a WAS sample used in this study taken with a light microscope................................................9
Figure 2.3.1 Interactions within the community of microorganisms that produce methane during anaerobic digestion.
Adapted from (Deublein & Steinhauser, 2008). ..........................................................................................................13
Figure 2.3.2 Diagram of the major components of an UASB anaerobic digester........................................................17
Figure 3.4.1 Liquid displacement biogas measurement apparatus. .............................................................................43
Figure 4.3.1 Cumulative biogas production subtracting blank (H2O as substrate) values for S WAS and the soluble
fraction of S WAS in experiment 1. ............................................................................................................................53
Figure 4.3.2 Cumulative biogas production subtracting blank (H2O as substrate) values for K WAS and S WAS in
experiments 2, 4, and 5................................................................................................................................................54
Figure 4.4.1 These graphs show the total and soluble COD before and after pretreatment. This data is from two
experiments (4 and 5) with a total of 5 replicates........................................................................................................60
Figure 4.4.2 These graphs show the change in total and volatile suspended solids before and after pretreatment. This
data is from two experiments (4 and 5) with a total of 5 replicates. ............................................................................61
Figure 4.4.3 These graphs show the total and soluble carbohydrates before and after pretreatment. This data is from
two experiments (4 and 5) with a total of 6 replicates. ................................................................................................63
Figure 4.4.4 These graphs show the amount of soluble protein before and after pretreatment. This data is from two
experiments (4 and 5) with a total of 8 or 16 replicates...............................................................................................63
Figure 4.4.5 Percent of total VSS removed after anaerobic digestion of untreated and pretreated WAS samples......65
Figure 4.4.6 Percent of substrate COD removed after anaerobic digestion. Values were calculated based on the total
amount COD required to produce measured methane production. Sample calculations can be found in Appendix C.
.....................................................................................................................................................................................66
Figure 4.4.7 Cumulative biogas production during experiment 4 (K WAS) subtracting blank control values. Data
points represent the mean of triplicate values and error bars represent the standard error in the mean.......................67
x
Figure 4.4.8 Cumulative biogas production during experiment 5 (S WAS) subtracting blank control values. Data
points represent the mean of triplicate values and error bars represent standard error in the mean. ...........................68
Figure 4.4.9 Cumulative biogas production during experiment 5 (S WAS) subtracting blank values. Each data point
represents the mean value of a triplicate and error bars represent standard error in the mean. ...................................69
Figure 4.4.10 COD consumption for caustic #2 pretreated S WAS in experiment 5. The lines represent two first-
order kinetics regressions performed on different parts of the data to account for the different kinetics. COD
consumption was calculated based on the theoretical amount of COD required to produced measured amounts of
methane. ......................................................................................................................................................................73
Figure 4.5.1 Cumulative methane production in experiment 4 (K WAS) and 5 (S WAS) compared with the potential
for methane production based on soluble COD content. .............................................................................................75
Figure 4.5.2 Net increase in methane produced compared with untreated samples during anaerobic digestion
showing the methane potential of soluble carbohydrate and protein content. Sonicated S WAS produced less
methane than predicted by methane potential of soluble.............................................................................................76
Figure 4.5.3 Regression of total biogas production versus soluble carbohydrate content of assay bottles in
experiment 4 and 5. .....................................................................................................................................................77
Figure 4.5.4 Regression of total biogas production versus soluble COD content of assay bottles in experiment 4 and
5...................................................................................................................................................................................77
Figure 4.7.1 Assumed process arrangement of pretreatment and dewatering used for economic analysis of operating
costs. ............................................................................................................................................................................81
1
1.0 Introduction
1.1 The Aerobic Wastewater Treatment Sludge (WAS) Problem
Biological wastewater treatment systems are widely used in municipal and industrial
applications, including the pulp and paper industry. The most commonly used configurations are
activated sludge systems and aerated stabilization basins. These systems use aerobic bacteria to
remove organic matter in wastewater by converting it to carbon dioxide, water, and biomass.
These bacteria exist as a suspension bound together in aggregates called flocs that readily settle
in clarifying tanks resulting in a clean effluent and a settled bacterial sludge. Any excess sludge
that is not recycled in the system requires disposal. This excess is known as secondary sludge or
waste activated sludge (WAS).
Disposal of WAS has been a major problem for aerobic biological treatment systems. In
the US alone, 4 million dry tonnes of sludge are produced each year by pulp and paper mills
(Scott & Smith, 1995). The most common methods for WAS disposal are land filling and
incineration. Because of the high water content of the sludge (92%-98%), it must be dewatered
before it can be disposed of (Gurjar, 2001). Dewatering usually requires the addition of
polymers or chemicals and mechanical pressing to remove water. This decreases the water
content of the sludge to about 70%. This relatively high water content leads to supplementary
fossil fuel requirements for incineration or that a large volume of water is transported in the case
of land filling. This results in sludge disposal costs accounting for as much as 50% of the total
wastewater treatment costs (Kyllönen, Lappi, Thun, & Mustranta, 1988).
The cost of WAS disposal is expected to increase over the coming years. Wastewater
treatment standards are going up in Europe and around the world requiring a higher degree of
organic matter removal from wastewater which leads to more sludge production. Landfill sites
are being filled to capacity and the construction of new sites faces public opposition and
stringent regulation. This will lead to increases in landfill tipping fees in the future as demand
outstrips supply (Scott & Smith, 1995). Incineration will also likely become expensive as the
price of supplementary fossil fuel increases.
1.2 Potential for Anaerobic Digestion of WAS
One alternative to traditional WAS disposal methods is another type of biological waste
treatment: anaerobic digestion. This system uses anaerobic microorganisms to convert the
2
organic matter in wastewater or sludge into biogas without the use of oxygen. Biogas is the main
byproduct of anaerobic digestion and contains about 60% methane by volume. The high
methane content makes biogas a useful fuel that can displace natural gas in pipelines and
industrial processes or be converted to electricity and heating (Deublein & Steinhauser, 2008).
Anaerobic digestion can reduce the amount of WAS in two ways. First, anaerobic
digestion is often used to treat wastewater before being sent to the aerobic treatment systems.
This produces biogas fuel and reduces the organic matter loading on the aerobic systems
resulting in less sludge biomass production (Gurjar, 2001). In a study of a pulp and paper mill in
Israel, a 75% reduction in WAS production was achieved by using a high-rate UASB type
anaerobic digester to treat all wastewater before an activated sludge system (Elliott & Mahmood,
2007). Second, WAS itself can be treated in an anaerobic digester to reduce solids and recover
energy in the form of biogas.
1.3 Challenges and Opportunities
Anaerobic digestion has been used for treating municipal sewage sludges for many years,
but has not been adopted for use in many industrial applications like the pulp and paper industry.
This is likely due to several disadvantages associated with anaerobic digestion. Anaerobic
digesters typically require long residence times as certain anaerobic microorganisms, such as
methanogens, have slow rates of growth. Also, WAS itself requires a long residence time of
around 30 days for digestion because of its complexity as a waste. Long residence times lead to
tanks and vessels that require large volumes. The high capital cost associated with building these
traditional anaerobic digesters would require long payback periods, discouraging their
application as the main disposal method for WAS.
Over the last few decades a newer type of anaerobic digester, known as the Upflow
Anaerobic Sludge Blanket (UASB) reactor, has been attracting industrial interest (Hobson &
Wheatley, 1993; Kleerebezem & Macarie, 2003). This type of reactor has a hydraulic retention
time of hours leading to a smaller reactor that retains a high efficiency. The anaerobic
microorganisms are in the form of “granules” that remain suspended within the reactor as
wastewater is pumped upward between them. There are thousands of installations of this type of
reactor being used to treat industrial wastewaters in combination with aerobic systems
(Kleerebezem & Macarie, 2003). In these cases, being able to send WAS to an existing UASB
reactor would greatly reduce the WAS disposal problem.
3
Sending WAS to a UASB reactor can be challenging. The majority of the
microorganisms that make up WAS are bound in microbial flocs where they are surrounded by a
matrix of biological polymers. These matrices, and the bacteria living within them, consist
mainly of lipids, carbohydrates, and proteins. Alone, these compounds are readily digested by
anaerobic bacteria. However, when they are arranged as flocs and bacterial cells, their
availability to bioconversion processes is greatly reduced. This leads to a waste with a high solid
content entering the reactor and long residence times to complete the bioconversion into biogas.
Various methods have been used in an attempt to improve the anaerobic digestibility of
municipal WAS in traditional anaerobic digesters. These methods include exposure to high
temperature, sonication, electric fields, high-pressure homogenization, microwave heating, and
the addition of acids and bases, or enzymes. Combinations of these methods have also been
evaluated. In all cases, the objective is to disrupt the bacterial floc structure and hydrolyse the
biological macromolecules in WAS to make them more soluble.
These tests have been mostly performed on WAS from municipal sources and have
produced varying results. Due to the variable nature of WAS it is difficult to compare results
between studies and few studies have compared various pretreatments on the same samples.
Anaerobic digestion of WAS with UASB granules has also not been thoroughly studied. This
study will be the first to test the improvement of anaerobic digestion of pulp and paper mill WAS
using several pretreatments in a side-by-side comparison. Potentially, industrial plants using
high-rate anaerobic digesters along with aerobic sludge systems could pretreat and anaerobically
digest WAS, producing biogas and reducing sludge disposal costs.
1.4 Objectives of the Project
The objective of this study was to investigate several physiochemical methods to pretreat
pulp mill WAS to make it more anaerobically digestible by UASB granules. The objective of
the pretreatments is to disrupt and hydrolyse the components of WAS, thereby increasing the
availability of the organic content in WAS and increasing the rate and extent of anaerobic
digestion. Specifically, this study investigated three pretreatments that have been found to be
effective at improving the anaerobic digestion of WAS from municipal sources in traditional
anaerobic digesters. The three pretreatments were: i) high temperature (170oC); ii) a high pH
(12) at a high temperature (140oC), and iii) sonication (at 20kHz and high intensity). The WAS
used was obtained from the aerobic wastewater treatment systems of two industrial sources: a
Kraft pulp mill and a sulphite pulp mill.
4
2.0 Literature Survey
2.1 Aerobic Biological Wastewater Treatment Systems
Biological wastewater treatment with aerobic microorganisms has been performed for
many years. The process was discovered in 1914 by Ardern and Lockett and came into heavy
use throughout the 1950s and 1960s as models and design principles for the system were
developed (Rittmann & McCarty, 2001). Wastewaters contain organic and other matter that can
cause serious environmental problems if sent directly to natural bodies of water. In an aerobic
biological treatment system, the wastewater is pumped into a tank or pond that contains a
suspension of aerobic bacteria. The aerobic bacteria oxidize the organic matter forming CO2,
H2O, and more bacteria. Collectively, all the organic matter is referred to as the biological
oxygen demand, or BOD, of the wastewater. BOD can be determined in the laboratory by
measuring total oxygen consumption of aerobic bacteria digesting a waste over a defined period
of time. The BOD of a waste determined over 5-days of incubation, for example, is referred to
as BOD5. Another measure of the organic matter is chemical oxygen demand (COD), which
correlates to BOD in most cases, but is measured using a chemical oxidation procedure. BOD
and COD refer to oxygen equivalents that are required in order to oxidize organic matter. This
oxidation can be tied to the transfer or electrons during biological metabolic processes (Rittmann
& McCarty, 2001).
The activated sludge and aerobic stabilization basin systems are the most commonly used
configurations for treating industrial and municipal wastewaters. The basic components of an
activated sludge system include an aeration tank, a settling tank, and a sludge recycle. The
wastewater is sent to an aerated tank along with nutrients required for microbial growth. In the
aeration tank, the well-mixed suspension of microorganisms that treat the wastewater exists in
the form insoluble aggregates known as flocs. Aeration is applied to satisfy the needs of the
microorganisms and is kept at a high enough concentration so not to be rate-limiting, which is
usually around 2 mg O2/L (Rittmann & McCarty, 2001). After the main tank, the effluent moves
into a settling tank where the bacterial flocs settle and are separated from the treated clean
effluent. The sludge is “activated” in the sense that the settled solids are recycled back to the
main aeration tank resulting in a high concentration of bacterial solids. This high concentration
of microorganisms in the reactor increases efficiency and allows a short hydraulic retention time
5
of several hours (Rittmann & McCarty, 2001). There is always an excess growth of sludge so
not all of it can be recycled. This excess sludge is known as waste activated sludge (WAS).
The most common configurations used today for activated sludge systems are: plug flow,
step aeration, completely mixed, and contact stabilization (Rittmann & McCarty, 2001). The
plug flow configuration is the conventional method used for activated sludge systems. The
aeration tank is long and narrow with the wastewater entering at one end and exiting at the other
as shown in Figure 2.1.1. Step aeration refers to a plug flow system where the influent
wastewater enters at several points along the length of the reactor. This is done to prevent high
concentrations of wastewater at any point in the reactor that would lead to possible inhibition as
is sometimes the case at the entrance of a plug flow system. In a completely mixed system, the
wastewater enters a single aeration tank that is completely mixed. In this configuration
organisms are never exposed to high levels of inhibitors, but removal efficiency is low. Contact
stabilization reactors involve a settling tank, a contact tank, and a return sludge flow as shown in
Figure 2.1.1. The return activated sludge (recycled bacterial flocs) is mixed with influent
wastewater and aerated for a short retention time in the contact tank, which is where the majority
of organic matter is broken down. The mixed liquor from the contact tank is then sent to a
settling tank where the clear effluent is removed and the sludge is collected at the bottom. This
sludge is sent to a stabilization tank where there is more aeration and adsorbed particles and
other organic matter are oxidized and then sent back to the contact tank as return activated sludge.
Typically activated sludge systems have solids retention times of less than 2 weeks and
remove about 90% to 95% of the total BOD5 in a wastewater (Rittmann & McCarty, 2001). A
typical loading for an activated sludge system would be between 0.6 (plug flow) to 1.0 (contact
stabilization) kg BOD5 m-3 day-1.
In contrast to activated sludge systems, aerated stabilization basins (ASB) are designed so
that biomass solids stay within the reactor so there is usually no clarifier after the basin to
remove excess suspended solids from the effluent that is produced. ASB systems usually consist
of aerated ponds or lakes with an area near the exit of the pond with no aeration where solids are
allowed to settle. Waste aerated stabilization basin sludge (WAS) is removed by periodic
dredging of the basins. Dredging is performed at different frequencies depending on the
operation of the basin and can be as frequent as several times per year or as infrequently as once
every two decades. The BOD reduction in aerated sludge basins is between 80% to 90%, which
is lower than in activated sludge systems. However, these systems often do not require the
addition of extra nutrients like activated sludge systems (Mahmood & Paice, 2006).
6
Figure 2.1.1 Basic flow diagrams of two commonly used activated sludge system configurations.
2.1.1 Wastewater Treatment Systems in the Pulp and Paper Industry
Aerobic biological treatment is widely used in the pulp and paper industry to treat
wastewater. Wastewater treatment in most pulp and paper mills first consists of a settling tank
where solid matter is removed. This is considered a primary treatment of the wastewater and the
solids produced are known as primary sludge. Primary sludge is mostly made up of cellulose,
hemicellulose, lignin, and other components of wood fibre (Rintala & Puhakka, 1994). After
primary settling, the rest of the wastewater is sent to a secondary treatment, which is usually
either an activated sludge system or aerobic stabilization basin. After aeration the mixed liquor
is sent to a second settling tank where clean effluent is separated from activated sludge that will
be recycled. The excess sludge produced is often referred to as secondary sludge. Figure 2.1.2
contains a schematic of a pulp and paper wastewater treatment system.
Aeration Tank Settling Tank Wastewater Effluent
Sludge Recycle WAS to disposal
Plug Flow Configuration
Settling Tank Wastewater Effluent
Return Sludge
WAS to disposal
Contact Stabilization Configuration
Contact Tank
Stabilization Tank Sludge Recycle
7
Figure 2.1.2 Flow diagram of a typical wastewater treatment system in a pulp and paper mill.
While primary sludge is easily dewatered and can be incinerated to generate energy,
secondary sludge has a high water content and its disposal is more difficult. Primary sludge is
often mixed with secondary sludge to improve the latter’s dewaterability. A recent survey of
Canadian pulp and paper mills found that on average mills were disposing of 50kg (dry)
wastewater treatment sludge per tonne of production with 30% of that being WAS and 70%
being primary sludge (Elliott & Mahmood, 2005). In the future, as pulping and papermaking
become more efficient, the amount of primary sludge produced is set to decrease, which will be
detrimental to the dewaterability of WAS.
Many Kraft mills in Canada utilize the aerated stabilization basin (ASB) for their
wastewater treatment (Mahmood & Paice, 2006). Although these systems are designed to retain
biological solids, these solids are still produced in excess and some systems require frequent
dredging or a secondary clarifier before effluent can be sent to any natural body of water
(Mahmood & Paice, 2006). In a survey of pulp and paper mills in Canada, it was found that
some mills with ASB systems produced less excess sludge than those with activated sludge
systems; in several cases ASB systems produced more (Elliott & Mahmood, 2005).
The wastewaters from pulp and paper mills have very different characteristics depending
on their origin and the processes used at the plant. There are large differences between pulping
factories and paper-making factories (Rintala & Puhakka, 1994). The sludges investigated in
this study were from a Kraft pulping mill and an ammonium sulphite pulping mill. In the Kraft
process wood chips are cooked under pressure and in a hot solution of caustic soda and sodium
Waste
Primary Sludge
Secondary
Sludge (WAS)
To dewatering
and disposal
Sludge Recycle
Secondary
Settling Aeration Tank Primary
Settling
Effluent
8
sulphide. In the ammonium bisulphite process cellulose is solubilised in acidic conditions. Both
processes lead to unique sets of wastewaters that would affect the operation of their wastewater
treatment systems and possibly affect the downstream anaerobic digestibility of the produced
secondary sludge.
Different pulping processes may produce different amounts of secondary sludge at a
given mill. On average, pulp and paper activated sludge systems produce excess sludge in the
ranges of 0.2 to 0.6 and 0.8 to 1.2 kg sludge solids per kg BOD7 removed in normal and high
loaded systems, respectively (Rintala & Puhakka, 1994). One review study comparing sludge
production from different types of pulping mills found total sludge production (primary and
secondary) for a sulphite pulp mill to be 58 kg sludge/tonne pulp, a Kraft pulp mill to be 102 kg
sludge/tonne pulp, and a deinking mill to be 234 kg sludge/tonne pulp (Scott & Smith, 1995).
2.2 Waste Aerobic Wastewater Treatment Sludge (WAS)
2.2.1 Physical and Biological Structure
The aerobic microorganisms in activated sludge and aerated basins exist in aggregates
called flocs that are held together by biological polymers and electrostatic forces (Rittmann &
McCarty, 2001). The main microorganisms degrading waste are heterotrophic and are a diverse
set of bacteria that breakdown a wide variety of organic matter. Most of the organisms are
secondary consumers, consuming the products of primary consumers that are actually breaking
down the organic matter of the wastewater. The species composition is continuously changing
as bacteria turn-over and compete for organic matter. Accordingly, there seems to be a large
redundancy in the species of microorganisms that breakdown organic substrates in wastewater.
Protozoa are present and have been found to be indicators of process performance, but are not
the main organic matter degraders (Rittmann & McCarty, 2001). Bacterial viruses and
multicellular microorganisms like nematodes are also present (Rittmann & McCarty, 2001).
The aggregates in activated sludge are composed of microbial cells, extracellular
polymeric substances (EPS), and inorganic cations and anions. Figure 2.2.1 gives an indication
of the nature of flocs and the intertwining of bacteria and biopolymers. EPS refers to the
collection of biopolymers that hold the bacterial sludges, slimes and films together. Extensive
research has been performed in determining the structure and composition of these biopolymers
(Frølund, Griebe, & Nielsen, 1995). Generally, EPS is composed of lectin-like proteins
covalently linked by polysaccharides. The EPS is also believed to be held together by the
9
electrostatic forces of inorganic ions such as Ca2+, Mg2+, and Fe2+/3+ (Park & Novak, 2007). The
amount of polysaccharides and proteins in EPS differs depending on extraction methods used
(cation exchange resin to remove Ca2+ and Mg2+, sulphide to remove Fe2+/3+, and pH 10.5
extraction for Al). The amount of EPS found in aerobic sludge has also been found to vary
depending on methods of analysis. In general, EPS represents 15% of the suspended solids of
activated sludge as determined by most extraction methods (Frølund, Palmgren, Keiding, &
Nielsen, 1996). However, a previous study using confocal laser spectroscopy found that just
over half of the organic matter in aerobic sludge was EPS (Liu & Fang, H. H. P., 2002). Proteins
are always present in higher amounts than polysaccharides; there can be two to four times as
many proteins than polysaccharides.
Figure 2.2.1 Photo of a WAS sample used in this study taken with a light microscope.
Water is also a major component of WAS and is found in several forms. Bacteria
themselves contain 70% to 80% water inside their cells (Rittmann & McCarty, 2001). The water
in sludge can be defined to exist in several forms: free water, interstitial water, surface water, and
bound water (Gurjar, 2001). Free water refers to the water not associated with the flocs.
Interstitial water refers to the water that is held within the flocs by capillary forces. The surface
water is water adsorbed to the surface of flocs and is held by surface forces. Bound water refers
to water that is chemically bonded to structures within the flocs. Some of this trapped water,
depending on its form, is difficult to remove and contributes largely to the difficulties associated
with dewatering WAS.
100μm
10
2.2.2 Composition
The chemical composition of WAS is similar to that of bacteria. It contains all the major
biomolecules including lipids, carbohydrates, and proteins, although relative amounts of these
components depends on the source of the WAS (Table 2.2.1). The properties of four pulp and
paper mill activated sludges were determined in the study by (Kyllönen et al., 1988). The
activated sludge from each mill showed a large variation in the percent of each component
(Table 2.2.1). A large percentage of the four sludges consisted of cellulose and lignin. This is
one way in which WAS from a pulp and paper mill will differ from municipal WAS and affect
the anaerobic digestibility of this feedstock.
Table 2.2.1 Summary of chemical composition of secondary sludge from different sources. All values in
terms of percent dry solids.
Source Municipal Municipal Municipal and
Industrial Pulp and
Paper Mills
Reference (Weemaes, M. P. J. & Verstraete, 1998)
(Frølund et al., 1996)
(Mikkelsen & Keiding, 2002)
(Kyllönen et al., 1988)
Carbohydrates 17% 10% 0 – 23%
Proteins 32 – 42% 46 – 52% 35% 22 – 52%
Lipids 5 – 12% 2 – 10%
Humic Acids 18 – 23% 6%
Cellulose 2 – 8%
Lignin 38 – 58%
pH 6.5 – 8 5.5 – 7
Heating Value [MJ/kg] 18.6 – 23.2 13 – 22
2.2.3 Dewatering and Disposal
Excess sludge disposal is a multi-step and often expensive procedure. Dewatering is
usually the first step in WAS disposal and is performed for several reasons. Dewatering sludge
decreases the volume requiring transportation, thereby reducing the cost of moving it to a landfill
site. Dewatering also reduces leachate production at the landfill site and is required before
incineration or combustion (Gurjar, 2001). The solids content of sludge must be increased to
35% – 45% w/v in order to reduce transportation costs and for it to have enough heating value to
be burned (Pere, Alen, Viikari, & Eriksson, 1993).
11
Dewatering can be achieved by several methods and usually involves the addition of
chemicals to improve coagulation and mechanical methods that involve filters or presses (Gurjar,
2001). Centrifuges are also sometimes used for solids separation in activated sludge plants, but
have several disadvantages including: high capital and electricity costs, and sensitivity to rapid
changes in loading and sludge properties (Rittmann & McCarty, 2001).
After dewatering the WAS is sent for its final disposal. The most popular methods for
disposal are land filling and incineration. In 1995, the most common methods of WAS disposal
by pulp and paper mills were: landfill, 69%, incineration, 27%, landspreading, 8%, and other
methods, 8% (Scott & Smith, 1995). A more recent survey of Canadian pulp and paper mills
found that 22% of wastewater treatment residues were sent to landfill, 40% was combusted, and
the remaining was disposed of by other methods such as spreading it on agricultural land (Elliott
& Mahmood, 2005).
There are several problems associated with each disposal method. Land filling is
considered the most ideal because it requires the least amount of sludge preparation (Gurjar,
2001). However, dewatered WAS can have high levels of pathogens, heavy metals, and toxic
organic compounds which can make land filling problematic and a possible liability in the future.
Incineration of sludges greatly reduces the volume required to be land filled, but with a solids
content of 30% or less, the addition of fuel is required (Gurjar, 2001). Incineration also has a
high environmental impact and is costly. Heavy metals are concentrated in the ash produced
during incineration and some heavy metals, such as mercury, can be transferred to the air (Gurjar,
2001). Ash disposal still requires the use of a landfill so high heavy metal content can be a
problem. Gaseous emissions must also be cleaned of NOx and SOx produced during incineration,
adding to the cost.
Various alternative methods have been investigated for disposing of WAS. These
include: composting, wet-oxidation, anaerobic digestion, and pyrolysis (Scott & Smith, 1995).
Anaerobic digestion gives the potential of converting WAS into an energy source rather than a
waste that requires energy to landfill or incinerate.
2.3 Anaerobic Digestion
Anaerobic digestion refers to the use of anaerobic microorganisms to convert organic
matter in municipal and industrial wastewaters into gases collectively known as biogas. In late
19th century Europe, anaerobic digestion was used to produce energy from municipal waste to
power street lighting in cities such as Paris (Deublein & Steinhauser, 2008). In 1923, biogas was
12
first put into the public gas works and sold to the public, a practice that became more and more
popular over the following decades (Deublein & Steinhauser, 2008). From then until the 1950s,
various improvements in understanding and reactor construction were developed and other types
of feedstocks, such as animal excrement and agricultural wastes, were introduced to improve
energy production (Deublein & Steinhauser, 2008).
Biogas production was greatly reduced after the 1950s due to the prevalence of cheap
heating oil resulting in most biogas plants being shut down. In the late 1970s, as now, rising fuel
prices led to an increase interest in anaerobic digestion as a combined water treatment and
energy production method. In Europe, many countries offer subsidies to offset the capital cost of
building anaerobic digestion plants which has lead to the plants becoming increasingly popular.
For example, the number of biogas plants in Germany has tripled since 1999 and contribute 3.2
TWh of energy generation (Deublein & Steinhauser, 2008).
Anaerobic digestion has also had a long history in China. For the past three decades, the
Chinese government has been encouraging the use of anaerobic digestion in rural communities
because it provides energy, improves hygiene by neutralizing pathogens in human and animal
waste, and also produces a fertilizer that can be used for farming. The government’s plan is to
have 50 million biogas plants in operation by 2010. In 2005, 10% of all farmers’ households
were using the biogas from their own biogas plants (Deublein & Steinhauser, 2008).
Anaerobic treatment has several advantages over other types of wastewater treatment.
The main advantage is the production of CH4 containing biogas that can be used as a fuel.
Anaerobic digestion has no requirements for aeration and low nutrient requirements. There is
also the possibility of high organic loadings of up to 10 times that of aerobic systems (Rittmann
& McCarty, 2001). Anaerobic digesters also produce one fifth to one tenth of the biological
solids produced by aerobic systems (Kleerebezem & Macarie, 2003).
There are several disadvantages associated with anaerobic digestion. These include the
low growth rate of microorganisms, possible odour production, buffering requirements
necessitating chemical addition, possible toxicity issues with industrial wastewaters, and poor
removal efficiency with dilute wastes (Hobson & Wheatley, 1993; Kleerebezem & Macarie,
2003). Anaerobic digesters also often require post-treatment such as an activated sludge system
because usually not all organic matter from the wastewater can be removed (Kleerebezem &
Macarie, 2003). Nevertheless, there are currently 1,600 full-scale anaerobic digesters in
operation worldwide and the full potential of anaerobic digestion has not yet been reached
(Kleerebezem & Macarie, 2003).
13
2.3.1 Microbiology
Anaerobic digestion of a substrate requires the concerted activity of several groups of
microorganisms (Figure 2.3.1). The first major group of organisms are the hydrolytic microbes
that break down complex organic matter like complex carbohydrates (cellulose, starch), fats, and
proteins into simple sugars (glucose), amino acids, and fatty acids (Rittmann & McCarty, 2001).
This is achieved through the use of extracellular enzymes secreted by these microorganisms
(Deublein & Steinhauser, 2008).
Figure 2.3.1 Interactions within the community of microorganisms that produce methane during anaerobic
digestion. Adapted from (Deublein & Steinhauser, 2008).
The second major group is the fermenting bacteria. These bacteria convert the simple
sugars, amino acids, and fatty acids into ethanol, organic acids such as acetic acid, and hydrogen
(Rittmann & McCarty, 2001). There are two types of fermenters: acidogens and acetogens.
Acidogenic bacteria degrade the compounds produced during hydrolysis into 1 to 5 carbon short-
chain organic acids, alcohols, hydrogen, and carbon dioxide. Acetogenic bacteria feed on the
organic acids produced by acidogens and produce acetate and H2. In order for this metabolic
pathway to be energetically favourable, H2 must be at a very low partial pressure in solution.
Hydrolysis
Fermentation
Acidogenesis
Acetogenesis
Methanogenesis
Nitrate Reduction
CH4, CO2, H2O H2S
NH3, NH4+
Complex organic matter:
proteins, carbohydrates, lipids
Volatile acids, acetate,
alcohols, CO2, H2
H2
CO2
Sulphate Reduction
Simple sugars, amino acids,
fatty acids, glycerol
14
This is facilitated by methanogens that use the H2 to produce methane. These organisms always
coexist and live in a very tight symbiotic relationship (Deublein & Steinhauser, 2008).
Methanogens are Archaea, and are the microorganisms responsible for producing CH4.
There are two groups of methanogens: acetate fermenters and hydrogen oxidizers (Rittmann &
McCarty, 2001). Acetate fermenters convert acetate to methane. Hydrogen oxidizers produce
methane from H2 and use CO2 as their carbon source. About 70% of the methane produced is
produced from acetic acid, the rest is from H2 (Deublein & Steinhauser, 2008). Methanogens
require strict anaerobic conditions and a slightly acidic pH to exist. Both groups of methanogens
are slow-growing and require long solids retention times in reactors. While activated sludge
bacteria have regeneration times around 2 hours, the regeneration time for methanogens is 5-16
days depending on the species (Rittmann & McCarty, 2001). Reactors need be designed with
this in mind so that methanogens are not washed out.
Other types of organisms in anaerobic digesters include nitrate reducers and sulphate
reducers. Nitrate reducing bacteria reduce nitrates to ammonia and ammonium. Sulphate
reduction occurs during anaerobic digestion whenever sulphur is present. Sulphur can come
from protein-containing wastes and sulphates in solution. Sulphate reduction leads to the
production of H2S, which is corrosive and has a strong odour. The H2S needs to be removed
from biogas before it can be burned to prevent SO2 formation during combustion. Notably,
sulphate reducers compete for acetate with methanogens.
2.3.2 Biogas Properties
The gases produced during anaerobic digestion are collectively known as “biogas”.
Biogas contains methane, carbon dioxide, and trace amounts of other compounds (Table 2.3.1)
(Deublein & Steinhauser, 2008). The ratio of methane to carbon dioxide is important as carbon
dioxide decreases the heating value of the biogas. The methane content in biogas typically needs
to be increased before it can be added to natural gas pipelines. This requires the removal of CO2
which may be performed by absorption into a liquid.
Usually before biogas is used in any process or added to a natural gas pipeline it must be
cleaned and refined. H2S must be removed from biogas as it is corrosive and results in SO2
production when the biogas is combusted. A biological method for removal involves stripping
the H2S from the biogas using water then using aerobic bacteria to convert the H2S to pure
sulphur. Chemical methods of removing H2S include precipitation with an iron salt or binding
15
with zinc. There are also several adsorption methods using chelates and activated carbon
(Deublein & Steinhauser, 2008).
Table 2.3.1 Typical compositions of biogas produced during anaerobic digestion (Deublein & Steinhauser,
2008).
Component vol. %
CH4 55 – 75%
CO2 20 – 35%
N2 0 – 5%
H2S 0 – 0.05%
Water vapour 1 – 5%
Siloxanes [mg/m3] 0 – 50
Heating Value [MJ/m3] 21.6 – 23.4
Water is also removed from the biogas to improve its heating value. The relative
humidity must be reduced to less than 60% to prevent the formation of condensation in piping
during transport. If biogas is to be supplied to a natural gas network, it needs to be dried to an
even higher degree. The water is usually removed by compression, cooling of the gas, or using
various absorption methods.
2.3.3 Reactor Configurations
There are several configurations of anaerobic digestion systems commonly used to treat
municipal and industrial wastewaters. The “completely mixed process” consists of a large
continuously-stirred tank reactor (CSTR) and is the type commonly used to treat domestic
wastewater (Rittmann & McCarty, 2001). These types of reactors run at 35oC and have retention
times of 15 to 25 days. The first of these types of reactors was built in Germany in 1927. They
require very concentrated wastewater streams and long retention times leading to very large
reactor volumes (Rittmann & McCarty, 2001). Long retention times are required mainly to
facilitate the growth of methanogens and other anaerobic microorganisms that grow very slowly
and would be continually washed out at shorter retention times.
Newer designs have attempted to separate biological solids retention time from the
wastewater retention time. This would satisfy the needs of methanogens and their slow growth
rate while permitting smaller reactor volumes (Rittmann & McCarty, 2001). These designs
include: i) anaerobic contact reactors that are similar to aerobic systems in that solids are
16
recycled after settling; ii) upflow and downflow packed beds where a medium such as gravel is
used for the biosolids to attach to and grow on; and, iii) fluidized and expanded beds which also
use a fine medium for biosolids to grow on.
The upflow anaerobic sludge blanket (UASB) reactor is a newer reactor design with
increasing importance for industrial wastewater treatment. The UASB reactor is considered a
“high-rate” anaerobic digester because of low hydraulic retention times and high efficiencies of
removal of organic contaminants in wastewaters. The design was developed in 1979 by Lettinga,
van Velsen, de Zeeuw, and Honma with the first full-scale operations started in the 1980s
(Rittmann & McCarty, 2001). The UASB is currently the dominant type of anaerobic digester,
with more than 800 industrial installations worldwide (Kleerebezem & Macarie, 2003). So far,
UASB reactors have mainly been used to treat wastes from the food industry, but installations
are emerging in other industries such as the pulp and paper and petrochemical industries
(Kleerebezem & Macarie, 2003).
In a UASB reactor, wastewater flows upward through a cylindrical reactor. The organic
matter in the wastewater is removed by anaerobic microorganisms in the reactor suspended by
the upward flow. Biosolids are retained within the reactor by inverted funnels, while liquid
effluent and biogas flow around them and are removed at the top of the reactor. Mixing occurs
within the reactor as the biogas is formed and bubbles to the top (Figure 2.3.2). Lettinga found
that the biosolids in the reactor form structures he called granules. Granules are about 0.5 mm to
3 mm in diameter and contain all the microorganisms required to perform methanogenesis from
many different substrates (Kleerebezem & Macarie, 2003). The granules have a high settling
velocity and stay within the reactor suspended in a similar fashion to a fluidized bed. The exact
reasons why granules form and their composition are still uncertain, but a variety of theories
exist that take into account selection pressure due to the flow regime in the reactor, microbial
constituents of the granules, and thermodynamic favourability of the granule structure (Hulshoff
Pol, de Castro Lopes, S. I., Lettinga, & Lens, P. N. L., 2004).
UASB reactors often remove about 60% to 70% of incoming BOD (La Motta, E. J., Silva,
Bustillos, Padrón, & Luque, 2007). In most cases this means that additional treatment is required
to polish the effluent leaving from this type of reactor. As a result, UASB reactors are often
found coupled with an aerobic treatment system. The combination of a UASB followed by an
activated sludge system for polishing has many advantages, including large decreases in excess
aerobic biological sludge production (Lettinga, 2005). In situations where wastewater treatment
is performed by an UASB reactor followed by an activated sludge system, it would be ideal to
17
convert excess sludge into biogas by sending it into the anaerobic digester. This concept has
been tested by several studies (La Motta, E. J. et al., 2007).
Figure 2.3.2 Diagram of the major components of an UASB anaerobic digester.
2.3.4 Anaerobic Digestion in the Pulp and Paper Industry
Anaerobic digestion has been investigated as a treatment method for wastewater streams
in the pulp and paper industry (Rintala & Puhakka, 1994). Several waste streams are amenable
to anaerobic digestion, but there are still few anaerobic digesters in operation in the industry.
The most common configurations include anaerobic digestion followed by an aerobic biological
treatment. This reduces BOD load on the aerobic system resulting in less sludge production and
produces energy in the form of biogas. In Canada there are four anaerobic bioreactors installed
to treat pulp and paper mill effluents, with only two in operation (Elliott & Mahmood, 2007).
Worldwide, there are around 100 anaerobic digesters at pulp and paper mills, with 75 being
UASBs (Kleerebezem & Macarie, 2003). In some cases UASBs have been used to replace
anaerobic lagoons leading to cost savings associated with the use of biogas in lime kilns and
other pulp and paper mill processes (Chinnaraj & Rao, 2006).
There are various configurations of UASB reactors that are commonly used. One
configuration is known as the internal circulation (IC) reactor commercialized by PaquesTM, a
company headquartered in the Netherlands. Installations of the PaquesTM IC reactor to digest
Anaerobic granule
sludge blanket
Biogas
Effluent
Wastewater Influent distribution
Gas-liquid-solid
separator
Degassing Unit
18
pulp and paper mill effluents before being sent to an activated sludge system have been
reasonably successful at the American Israeli Paper Mills (AIPM) recycled fibre paper mill in
Hadera, Israel and Tembec’s Temiscaming pulp mill in Quebec. Anaerobic digestion is not more
widely used likely due to the perception that many inhibitors to anaerobic digestion exist in pulp
and paper wastewaters (Rintala & Puhakka, 1994). Accordingly, there is a large untapped
potential for anaerobic digestion in the pulp and paper industry (Kleerebezem & Macarie, 2003).
2.4 Anaerobic Digestion of WAS
The digestibility of pulp and paper WAS at a laboratory and pilot-scale has been
investigated (Puhakka, 1992a). However, there are no full-scale anaerobic digesters in the pulp
and paper sector digesting solid residues such as WAS (Elliott & Mahmood, 2007). An
economic analysis of anaerobic digestion of sludge at a pulp and paper mill performed by (Elliott
& Mahmood, 2007) showed a payback period of 9 years. There is, however, the potential to
improve the economics of WAS digestion by pretreating it to reduce retention times in anaerobic
digesters and possibly by using it as a substrate in UASB reactors.
2.4.1 Previous Studies
Anaerobic digestion is widely used in the treatment of municipal wastewater sludges
before and after aerobic biological treatment. In a typical municipal wastewater treatment
facility, two thirds of the incoming BOD is removed by the anaerobic digestion system, with the
remainder being removed by an activated sludge system with the WAS produced by the system
also being pumped into the anaerobic digester (Speece, 1988). Low-rate systems used for
digesting WAS usually require residence times of 30 to 60 days, although newer high-rate
anaerobic digesters can shorten that time to 15 days (Gurjar, 2001). A typical municipal
anaerobic digester digesting WAS produces 0.146 to 0.217 mL CH4 / mg volatile solids added
(Bougrier, Delgenès, & Carrère, 2007) and leads to a 40-60% reduction in volatile solids (VS)
content of the WAS (Gurjar, 2001). Anaerobic digestion is also used to “stabilize” WAS.
Stabilization refers to reducing the VS and pathogen concentration of the WAS before final
disposal (Gurjar, 2001).
A UASB reactor followed by aerobic treatment has been found to be an effective method
to treat municipal sewage sludge. In a pilot-scale study, the addition of aerobic sludge solids to
the reactor did not produce any problems during operation (La Motta, E. J. et al., 2007). In this
19
case, the pilot-scale reactor produced 0.17 mL biogas / mg volatile suspended solids (VSS)
added with 60% methane in the biogas. The solids reduction was found to be poor in the UASB
with only 33% removal of VSS. There was very little accumulation of solids in the UASB
reactor, which is an important factor for good reactor performance. However, the suspended
solids content of the waste digested in the study was very low compared with the solids content
of most aerobic sludge wastes.
There have been only a few studies on anaerobic digestion of pulp and paper mill
biological sludges such as WAS. The study by (Puhakka, 1992a) looked at semi-continuous
pilot-scale anaerobic digestion of a Kraft mill WAS in Finland. In that study, high loading rates
of over 5kg VS m-3 day-1 were possible. Following a 25 day retention time, they achieved a
median VS removal of 40% and 0.220 mL of biogas produced per mg VS added. However, they
did not achieve steady state operation because of the large variability in sludge properties. In
another study (Puhakka, Viitasaari, Latola, & Määtä, 1988) chemithermomechanical pulp
(CMTP) mill WAS was anaerobically digested at a lab-scale. A VSS removal of 41% was
achieved with a biogas production of 0.09 mL per mg VSS added. The values found were
similar to the range of values found in municipal digesters, suggesting there is a good potential to
convert these residues into biogas.
2.4.2 Challenges to Digestion
High-rate anaerobic digesters are popular for industrial wastewater treatment because of
their smaller size and lower capital costs compared with traditional anaerobic digesters, but there
are a few difficulties in sending WAS directly to this type of reactor. There are also some
specific issues with pulp and paper mill WAS that may make its anaerobic digestion difficult.
One main challenge is the long residence times required for digestion. Hydrolysis of biological
macromolecules contained as microbial cells and EPS has been found to be a rate-limiting step in
anaerobic digestion (Milton & Arnold, 2003; Navia, Soto, Vidal, Bornhardt, & Diez, 2002). EPS
and the cell walls of microorganisms act as a physical barriers to enzymes produced by anaerobic
hydrolytic bacteria (Navia et al., 2002). WAS might also contain compounds that are not
degradable by anaerobic microorganisms. For instance, pulp and paper mill WAS can contain
lignin and other wood components that pass through aerobic treatment and adsorb to sludge flocs
(Ganczarczyk & Obiaga, 1974). It was found that mainly high molecular weight fractions of
lignin adsorbed to simulated sludge flocs and that Kraft lignin adsorbed more readily compared
with sulphite mill lignin and lignosulphonates (Ganczarczyk & Obiaga, 1974). When four
20
different pulp and paper mill WAS samples were tested directly, it was found that 38% to 58% of
WAS solids was lignin (Kyllönen et al., 1988). Lignin degradation under anaerobic conditions is
negligible and can hinder digestion of associated polysaccharides in lignocellulosic material
(Hobson & Wheatley, 1993; Rittmann & McCarty, 2001). Some anaerobic bacteria, however,
have been found to modify lignin. Desulfovibrio desulfuricans was found to partially
depolymerise Kraft lignin and reduce the sulphate portions of lignosulphonates (Ziomek &
Williams, 1989).
Complex wastes that contain large amounts of suspended solids can cause problems in
high-rate anaerobic digesters such as UASB reactors. UASB reactors are meant for digesting
soluble non-complex wastes or complex wastes that are highly soluble (Lettinga & Hulshoff Pol,
1991). It has been found that suspended solids can accumulate in the reactor and can lead to
variety of process upsets including the loss of anaerobic granules (Zeeman, Sanders, W. T. M.,
Wang, & Lettinga, 1997). One way to overcome this is to use a different configuration of
anaerobic digester to pretreat a complex waste like WAS before sending it to a UASB digester
(Zeeman et al., 1997).
Another challenge specific to the digestion of pulp and paper mill WAS is that it may
contain compounds that are toxic to the anaerobic microorganisms. As a result of the pulping
process, wood extractives are commonly found in the effluents from pulp and paper mills. Wood
extractives include: sterols, resin acids, terpenes, and other phenolic compounds (Kostamo,
Holmbom, & Kukkonen, J. V. K., 2004; Sierra-Alvarez & Lettinga, 1990). Resin acids, such as
dehydroabietic acid (DHA), can adsorb to activated sludge during the aerobic treatment
(Kostamo et al., 2004; Makris & Banerjee, 2002). Resin acids and other phenolic compounds
from wood can be toxic to methanogenic microorganisms (Sierra-Alvarez & Lettinga, 1990).
Resin acids identified in a chemithermomechanical pulp mill wastewater before activated sludge
treatment were also found to be toxic to anaerobic microorganisms. However, there seems to be
evidence that communities of anaerobic microorganisms in digesters can adapt to and breakdown
these compounds given sufficient time (McCarthy, Kennedy, & Droste, 1990). Still, these kinds
of chemicals and other aromatic-ring containing compounds can be inhibitory to anaerobic
digestion and prohibit the digestion of pulp and paper mill WAS if they are found in large
quantities (Hobson & Wheatley, 1993).
Heavy metals such as nickel, cadmium, lead, copper, and zinc can also be toxic to
anaerobic bacteria and methanogens (Hobson & Wheatley, 1993). These metals are present in
21
pulp mill WAS and are part of the reason land spreading of dewatered pulp mill sludges on
agricultural land is losing favour as a disposal method (Mahmood & Elliott, 2006).
Since, anaerobic digestion of WAS cannot completely destroy all aerobic sludge solids,
dewatering and disposal of a bacterial sludge will still be required. Moreover, anaerobic
digestion can decrease the dewaterability of WAS (Novak, Sadler, & Murthy, 2003). In a pilot-
scale study of anaerobic digestion of WAS from a Kraft mill, the dewaterability was decreased
(Puhakka, 1992b). This challenge could be addressed by a process design that involves
dewatering prior to anaerobic digestion of WAS. The soluble digestible organics could be
separated from suspended solids by a mechanical dewatering process and sent for anaerobic
digestion, while the remaining solids would be sent for sludge disposal.
2.4.3 Pretreatment Technologies
Pretreatment of WAS before being sent to anaerobic digestion has been suggested as a
possible method to overcome the challenges associated with its bioconversion to biogas. Several
different methods have been studied for the pretreatment of municipal WAS. These methods
rely on physical or chemical reactions to hydrolyse and solubilise the waste before anaerobic
digestion and include the application of thermal energy, addition of chemicals, sonication,
electrical fields, various biological processes, and mechanical forces. These pretreatments
decrease the amount of material requiring hydrolysis, the rate-limiting step in anaerobic
digestion, since the hydrolysis is performed beforehand rather than biologically within the
reactor. Pretreatments could make it economically feasible to anaerobically digest pulp and
paper sludges. A recent review of pretreatment technology in the context of pulp and paper mill
WAS digestion concluded that more experimentation using this type of waste is required (Elliott
& Mahmood, 2007).
2.4.3.1 Thermal Pretreatment
Thermal treatment of biological sludges was first suggested as a method to improve the
dewaterability of sludge (Stuckey & McCarty, 1984). Thermal treatment hydrolyses and disrupts
the components of bacterial flocs and releases bound water resulting in a sterilized waste that is
more easily dewatered. The optimum temperature range for this process has been found to be
around 200oC up to as high as 260oC. The use of thermal treatment to improve the dewatering of
pulp and paper mill sludges has also been found to be successful (Kyllönen et al., 1988).
22
However, this method has not been widely adopted due to the possibility of strong odour
production and the production of a coloured effluent. This type of dewatering facility also incurs
high capital costs and so is limited to only very large plants that produce more than 720 m3 h-1 of
sludge (Gurjar, 2001). Notably, the solubilisation of cell components was considered a
disadvantage in using this system to improve dewatering (Weemaes, M. P. J. & Verstraete, 1998).
Studies over the past several decades have investigated thermal treatment of various
municipal sludges to be used as a carbon source for wastewater denitrification and to improve
their digestibility in anaerobic digesters (Stuckey & McCarty, 1984; Weemaes, M. P. J. &
Verstraete, 1998). The studies found that thermal treatment promoted hydrolysis and split
complex nitrogen polymers reducing the amount of total solids. This led to an improvement in
biogas yields and COD removal in anaerobic digesters (Table 2.4.1).
Table 2.4.1 Summary of typical results of thermal pretreatment on the anaerobic digestion of WAS from
municipal sources.
Reference
(Stuckey & McCarty,
1984)
(Tanaka, Kobayashi, Kamiyama,
& Signey Bildan, L. N.,
Ma., 1997)
(Dohányos, Zábranska,
Kutil, & Jeníček,
2004)
(Valo, Carrère, &
Delgenès, 2004)
(Bougrier et al., 2007)
Sludge Type Municipal
WAS
Municipal WAS and a
mixed municipal/ industrial
WAS
Municipal WAS
Municipal WAS
Municipal WAS
Temperatures Investigated [oC] 150 – 275 115 - 180 120 – 170 130 – 170 135 – 190
Optimum Temperature [oC] 175 180 170 170 190oC
Contact Time [min] 60 15 1 15 – 60 35 – 50
Fraction Soluble COD After Treatment
55% 40% 60% 46%
Volatile Solids Reduction 30% 50%
Anaerobic Digestion Type (retention time, days)
Batch (34) Batch (20) Batch Continuous,
(20)
Semi-continuous,
(20)
Improvement in Methane Yield 27% 35 – 49% 45% 25%
Several studies have investigated the optimal conditions for the thermal hydrolysis of
municipal WAS in terms of contact time and temperature. As the thermal treatment temperature
is increased, the destruction of solids, solubilisation of COD, and biogas production are all
23
improved linearly. At temperatures of about 180oC and higher there is a sharp decrease in biogas
yield and degradability. It has been found that temperatures above 180oC results in decreased
anaerobic digestibility of all major cell components including protein and nucleic acids. It is
believed that nitrogen-containing compounds are converted into compounds that are not
biodegradable by or even toxic to anaerobic microorganisms (Stuckey & McCarty, 1984). The
creation of refractory compounds can be attributed to the Maillard reaction where sugars and
amino acids react in the presence of water (Bougrier et al., 2007; Penaud, Delgenès, & Moletta,
1999). Time has been found to be less of a factor in thermal pretreatment. Contact times of 60
seconds up to 200 minutes have been investigated and it appears that only a few minutes is
required to maximize improvements in biomass solubilisation and biogas production (Bougrier et
al., 2007; Kepp, Machenbach, Weisz, & Solheim, 2000; Valo et al., 2004). The optimal
conditions for thermal pretreatment seem to be around 170oC for at least 15 minutes.
Thermal pretreatment has also been commercialized at a full-scale. One example of a
commercially available thermal treatment process is the Cambi process sold by CambiTM, a
Norwegian company. A three year study of the process was run at a municipal wastewater
treatment plant using temperatures of 180oC or 165oC (Kepp et al., 2000). The thermal treatment
followed by anaerobic digestion was a net energy producer. The process greatly improved
dewaterability of sludge and decreased sludge solids requiring disposal by 25% and decreased
dewatering and disposal energy requirements by 50%. The study also reported that the Cambi
process has also been was successfully applied to pulp and paper mill WAS in laboratory
experiments. An independent study reviewed in (Elliott & Mahmood, 2007) found that this
process was effective at increasing biogas yield and decreased costs of sludge disposal from
$3,000,000 to $400,000. The cost of installing this process was around $3,000,000.
2.4.3.2 Chemical Pretreatment
The addition of chemicals to enhance pretreatment of WAS has also been studied. The
addition of alkaline agents such as lime, using pH of 12 or higher, has also been used to facilitate
dewatering, waste sterilization, and odour reduction (Gurjar, 2001). This method has also been
previously investigated for improving dewatering of pulp mill WAS (Kyllönen et al., 1988).
Previous studies have shown that the addition of an alkaline agent can reduce the temperature
required for thermal pretreatment while retaining the same effectiveness. The combination of
thermal and alkaline pretreatment was also found to be superior to alkaline pretreatment alone
24
(Kim et al., 2003; Penaud et al., 1999; Valo et al., 2004). Table 2.4.2 summarizes representative
results of thermochemical pretreatment from various studies.
Table 2.4.2 Summary of typical results of thermochemical pretreatment on the anaerobic digestion of WAS
from various sources.
Reference (Tanaka et al.,
1997) (Penaud et al.,
1999) (Kim et al.,
2003) (Valo et al.,
2004) (Navia et al.,
2002)
Sludge Type
Municipal WAS and a mixed
municipal/ industrial WAS
Industrial WAS Municipal WAS Municipal WAS Kraft pulp mill
WAS
Temperatures Investigated [oC]
115 - 180 140 121 130 – 170 Ambient
Chemicals Added NaOH NaOH, KOH,
Mg(OH)2, Ca(OH)2
NaOH, KOH, Mg(OH)2,
Ca(OH)2 KOH NaOH, KOH
Optimum Conditions
130oC, 1.85g/L NaOH
140oC, 5g/L NaOH (pH 12)
121oC, 9g/L NaOH
130oC. 168g/L KOH (pH 10)
2.4g/L NaOH
Contact Time [min] 5 30 30 15 – 60 0 – 30
Fraction Soluble COD After Treatment
75 – 80% 87% 60 – 80% 32%
Volatile Solids Reduction
40 – 50% (domestic) 70 – 80%
(domestic/industrial mix)
40 – 60% 21%
Anaerobic Digestion Type (retention time, days)
Batch (20) Batch Batch (7) Continuous, (20)
Improvement in Methane Yield
35% (domestic) 200%
(industrial/ domestic mix)
164% 38% 54%
Several studies have investigated the type of alkaline agent, the temperature, and the
contact time to determine the optimal conditions for the thermochemical pretreatment of
municipal WAS. Several studies have investigated NaOH, KOH, Ca(OH)2, and Mg(OH)2 as
alkaline agents (Kim et al., 2003; Penaud et al., 1999). Different cations have been found to
have different levels of toxicity to anaerobic bacteria, with sodium concentration being
especially important (Hobson & Wheatley, 1993). NaOH and KOH have been found to be the
most effective at solubilising WAS. A maximum of 5 g/L NaOH can be added before inhibition
of anaerobic bacteria occurs (Penaud et al., 1999). A range of temperatures has been studied in
25
combination with alkaline addition. Optimal temperatures have been found to be around 130oC
to 140oC with higher temperatures often improving COD solubilisation, but not biogas
production (Penaud et al., 1999; Valo et al., 2004). Time was also found not to be an important
factor as most of the solubilisation of organic matter occurred within the first 30 minutes of
pretreatment (Navia et al., 2002).
Few studies have looked at the pretreatment of WAS from pulp and paper mills.
Addition of NaOH and KOH to a Kraft mill WAS was investigated as a means to solubilise COD
and protein as well as reduce VSS content (Navia et al., 2002). NaOH or KOH was added in
varying amounts and allowed to hydrolyse the waste over varying lengths of time. The fraction
of COD that was soluble increased from 7.2% to a maximum of 32% at a pH of 13 and an alkali
dose of at least 60 meq/L (2.4 g/L NaOH) and a contact time of at least 30 minutes. There was
also no difference found between KOH and NaOH addition. The study did not investigate the
anaerobic digestion of the resulting sludge.
2.4.3.3 Sonication
Sonication refers to the use of ultrasound at high intensities to disrupt cellular matter. It
has been used for decades in research laboratories studying cellular components as a way to
disrupt cellular matter. Over the past decade it has been investigated as a possible method to
solubilise municipal sludge to improve anaerobic digestion (Khanal, Grewell, Sung, & Van
Leeuwen, 2007). Ultrasound refers to the application of vibrations at a frequency of 20 kHz or
higher, which is just above the limit of human hearing. The vibrations are applied by a
transducer in the form of a vibrating plate or probe. When the waves propagate through a liquid
they create alternating areas of low and high pressure which leads to the formation of
microbubbles that quickly collapse. As the microbubbles collapse they produce large shear
forces around them which disrupt any cellular matter in their vicinity. As they collapse they also
create localized areas of extremely high pressures and temperatures which result in the formation
of highly reactive hydroxyl radicals that can also interact with cellular matter (Khanal et al.,
2007). In the case of pretreating sludges for anaerobic digestion, radicals likely do not play a
large role in the disintegration of bacterial flocs because of the low frequencies that are often
used (Tiehm, Nickel, Zellhorn, & Neis, 2001).
Previous studies of sonication as a pretreatment for sludge have been performed over the
past 10 years at lab-scale, pilot-scale, and some full-scale operations (Grönroos et al., 2005). It
has been shown to be effective at solubilising organic matter as well as improving biogas
26
production (Table 2.4.3). The comparison of results between studies is difficult because
sonication has been found to depend highly on the solids concentration of waste, reactor
configuration, frequency of ultrasound, intensity, and other parameters. Additionally, the
measurement of solubilisation of sludge is usually represented in comparison to a reference
solubilisation performed by the addition of NaOH. The reference value is not consistent between
studies since different amounts of NaOH were added for different lengths of time and
solubilisation is affected by solids concentration and other factors (Khanal et al., 2007).
Table 2.4.3 Summary of typical results of sonication pretreatment on the anaerobic digestion of WAS from
municipal sources.
Reference
(Zhang, Zhang, &
Wang, 2007) (Grönroos et
al., 2005)
(Q. Wang, Kuninobu, Kakimoto, Ogawa, &
Kato, 1999) (Mao &
Show, 2006)
(Bougrier, Albasi,
Delgenès, & Carrère,
2006)
Sludge Type Municipal
WAS Municipal
WAS Municipal
WAS Municipal
WAS Municipal
WAS
Sonicator Type Probe Round steal
reactor Steal reactor Probe Probe
Frequency [kHz] 25 22 – 27, 40 9 20 20
Intensity [W/mL sludge] 0.5 0.1 – 0.3 2 0.18 – 0.52 0.45
Contact Time [min] 10 – 30 2.5 – 10 10 – 40
Fraction Soluble COD After Treatment
30.2% 7% 15%
Volatile Solids Reduction 26% 12%
Anaerobic Digestion Type (retention time, days)
Batch (19) Batch (11) Continuous
(2 – 8) Batch (24)
Improvement in Methane Yield 10% – 20% 64% 200% 47%
Several studies of sonication have examined the optimal conditions for its use as a
pretreatment. In general, most studies have found low frequencies at high intensities for long
periods of time result in the most solubilisation. In studies examining the relationship of
frequency to biogas production and organic matter solubilisation, it has been found that 20 kHz,
the very lowest frequency in the ultrasonic range, is the most effective frequency (Khanal et al.,
2007; Tiehm et al., 2001). Ultrasonic intensity refers to the amount of power applied to produce
the vibrational waves in the sludge. Studies have found that higher intensities lead to better
pretreatment results (Chu, Chang, Liao, Jean, & Lee, 2001; Khanal et al., 2007; Mao & Show,
2006; Zhang et al., 2007). The length of time of exposure to sonication also is an important
27
factor. One study found that sonication for more than 30 minutes did not lead to an increase in
methane production (Zhang et al., 2007). The length of time is also limited by temperature
increase. Without cooling, the temperature can increase drastically over the time of sonication.
For example, at a power density of 0.1W/mL, 120 minutes of sonication would result in a sludge
temperature of over 180oC (Khanal et al., 2007).
There have been a few commercial installations of sonication equipment to improve
anaerobic digestion at full-scale digesters. Four commonly used systems for sonication are the
SonicTM, MaXonicxTM, SonolyzerTM, and HielscherTM. They have been shown to improve
biogas production at some anaerobic digesters at municipal wastewater treatment facilities
(Elliott & Mahmood, 2007).
One possible negative aspect of sonication is that it has been found to significantly
deteriorate the dewaterability of sludge in most studies (Bougrier et al., 2006; Bougrier et al.,
2007; Chu et al., 2001; Na, Kim, & Khim, 2007). Dewaterability of pulp and paper mill sludges
deteriorated after they were sonicated in a 50 kHz ultrasound bath (Kyllönen et al., 1988). One
study of a full-scale SonixTM pretreatment system, however, found a small improvement in the
dewaterability of the resulting biomass solids (Hogan, Mormede, Clark, & Crane, 2004).
2.4.3.4 Other Physical and Chemical Pretreatments
There are many other physical and chemical pretreatment methods that have been tested
with varying degrees of success. Thermochemical pretreatment has been attempted with various
other chemical agents. Acids such as sulphuric acid, nitric acid, and hydrochloric acid have been
used (Chen, Jiang, Yuan, Zhou, & Gu, 2007; Perkins, Klasson, Counce, & Bienkowski, 2003).
The addition of these chemicals has been found to be effective in laboratory trials. Oxidizing
agents such as ozone, Fenton’s reagent, and hydrogen peroxide have also been tested (Bougrier
et al., 2006; Valo et al., 2004). These methods were found not to be very successful and do not
produce as good results as thermochemical pretreatment. Ozonation was also found to
deteriorate dewatering properties of the biological sludge (Böhler & Siegrist, 2004). Alternative
methods of heating such as the use of microwaves have also been investigated, however, they do
not appear to add any significant advantage to traditional thermal pretreatment (Eskicioglu,
Kennedy, & Droste, 2006; Eskicioglu, Terzianb, Kennedya, Drostea, & Hamodac, 2007).
Another method of pretreatment that has had some success at full-scale and has been
commercialized by several companies is the use of high-pressure homogenization. This
pretreatment involves pressurizing the WAS then rapidly depressurizing it. The rapid change in
28
pressure breaks apart the sludge and improves digestibility. The MicroSludgeTM system sold by
Paradigm Environmental Technologies Inc. is a high pressure homogenization system that is
currently on the market. A full-scale installation of the process is in operation at the Chilliwack
wastewater treatment plant in British Columbia and has been found to be very effective at
solubilising WAS (Elliott & Mahmood, 2007).
A proof of concept study has also been performed on the use of pulsed electric fields
similar to those used in food conservation to improve anaerobic digestion of municipal WAS
(Kopplow, Barjenbruch, & Heinz, 2004). It was found to be as effective at improving biogas
yield as thermal or high-pressure homogenization pretreatments, but required a very high energy
input.
2.4.3.5 Enzymatic and Biological Pretreatments
Several biological pretreatment methods have also been suggested. These methods
would involve a separate bioreactor containing hydrolytic microorganisms. Biological
pretreatment of cellulosic wastes by cellulytic or lignolytic fungi to make the cellulose more
available to anaerobic microorganisms has been attempted. This process was not found to be
very effective as it only increased the digestibility by a few percent (Hobson & Wheatley, 1993).
The separate addition of hydrolytic enzymes to hydrolyse various components of sludge
has also been attempted with some success. Large amounts of cellulase were required to achieve
only a small and variable increase in methane production (Hobson & Wheatley, 1993). However,
in a separate study where the addition of carbohydrase was tested, the addition of enzyme was
found to improve biogas production by about half as much as thermal (121oC for 60 minutes) or
high-pressure homogenization (600bar) pretreatment (Barjenbruch & Kopplow, 2003). One
interesting study used a centrifuge to lyse the cellular matter in WAS to liberate hydrolytic
enzymes contained within bacterial flocs (Dohányos, Zábranská, & Jenícek, 1997). Depending
on the sludge quality a large range of improvement in methane yield was produced. In some
cases the methane production increased by almost 90%. A study of enzymatic effects on several
pulp and paper WAS samples found no change in WAS characteristics after the addition of a
wide variety of enzymes including: Trichoderma (cellulose and hemi-cellulose hydrolysis),
Pektinex (pectine hydrolysis), Fungamyl (starch hydrolysis), Alcalase (protein hydrolysis), yeast
lipase (fat hydrolysis), albumin lysozyme (cell wall hydrolysis), and promozyme (hydrolysis of
polysaccharide slimes) (Kyllönen et al., 1988).
29
3.0 Methodology
3.1 Biomass Sample Collection and Storage
3.1.1 Aerobic Wastewater Treatment Sludge Samples
Aerobic biological wastewater treatment sludges were obtained from two pulp and paper
mills: Mill A and Mill B. Mill A produces 400 to 500 tonnes per day of ammonium bisulphite
pulp, 1 300 tonnes per day of bleached chemi thermo mechanical (BCTMP) pulp, and 800 tonnes
per day of paperboard. This mill uses a plug flow activated sludge wastewater treatment system
with a 24 hour retention time treating 90 000 m3 of wastewater per day at a loading of 300 tonnes
COD per day. The system achieves a BOD removal of 98% and produces about 40 tonnes of
WAS per day. The WAS from this mill will be abbreviated S WAS throughout this document.
Mill B is a Kraft mill that produces 650 tonnes per day of bleached hardwood pulp. This
mill uses a 626 000 m3 aerated stabilization basin to treat all of its wastewaters. The system has
an average loading of 60 tonnes COD per day with a COD removal of approximately 75%.
Biosolids are currently sent to a 525 000 m3 separate holding lagoon and are being considered for
several types of disposal including spreading on agricultural land. The excess sludge produced
by this mill will be abbreviated K WAS throughout this document.
Both samples of sludge were collected from the mills, sent in plastic pails by courier to
the University of Toronto, and placed in cold storage at 4oC until they were used in any
experiments.
3.1.2 Anaerobic Granule Samples
All samples of anaerobic granules were collected from the PaquesTM internal circulation
(IC) reactors at located at Mill A. The reactor treats acid condensate, post extraction washer
waste, and BCTMP production effluent at a rate of 15 000 m3 per day and a loading 150 tonnes
COD per day. The reactor has a hydraulic retention time of 8 hours and removes 50% to 60% of
the incoming COD producing biogas at a rate of 36 000 sm3 per day.
There are several collection ports in the IC reactor marking various heights from the
wastewater inlet at the bottom. Samples were taken from the ports located 6.2 m from the
bottom of the reactor. Granule samples were taken from the reactor and placed in 4 L plastic
30
bottles and shipped in coolers with ice packs to the university. Samples were taken at two times
during the year: October 2006 and September 2007.
At the university, the samples were divided into ~100 mL portions by pouring the
granules into 160 mL glass serum bottles. The bottles were then sealed with butyl rubber
stoppers and crimped and made anaerobic as described in section 3.4.3 with a sparging time of
30 to 45 minutes. Once anaerobic, the serum bottles were stored at 4oC until they were used for
experimentation. Storage of anaerobic granules at 4oC at industrial scales for up to 8 months has
been found not to negatively affect their biological activity or morphology (Shin, Bae, & Oh,
1993).
3.2 Measurement of Physical and Chemical Properties
3.2.1 Soluble Fraction
In this document the “soluble fraction” of any sample refers to the fraction of the sample
produced after centrifugation and filtration. Centrifugation was performed on 50 mL volumes of
sample placed in 50 mL polystyrene conical-bottom centrifuge tubes at 10,000 r.c.f. using a
Beckman Coulter Allegra 25R with a Beckman Coulter TA 10-250 rotor. The supernatant of the
centrifuged samples was then filtered by first drawing it up into a disposable 10 mL syringe and
then pushing the liquid through a sterile filter into a clean and dry glass vial. The syringe filters
used were Pall Acrodisc 25 mm sterile syringe filters with a Supor membrane and 0.45 µm pore
diameter.
3.2.2 Suspended Solids
Total suspended solids (TSS) refers to the weight of the residue left after passing a
sample through a filter of a specific type after drying in a 103oC oven. Volatile suspended solids
(VSS) refers to the weight loss after igniting that dried residue at 550oC and is a rough measure
of total solid organic matter in a sample. The method used in this study is based on the standard
method in the American Public Health Association (APHA) Standards Methods for the
Examination of Water and Wastewater (APHA, 1998). The filter used was the Whatman 934-
AH glass fibre filters with a pore diameter of 1.5 µm.
Filters were prepared by placing them into a metal weighing dish and then into a 550oC
oven for 30 minutes to remove any residue or water. Once cooled, the filters were weighed then
31
a known volume of sample was passed through the filter while under a vacuum. The filter and
residue was then dried at 103oC for at least 2 hours, weighed, ignited in an oven at 550oC for at
least 30 minutes, and then weighed again. TSS and VSS were calculated using the following
equations:
V
mmTSS
EmptyCAfter −=
103
V
mmVSS
CAfterCAfter 550103 −=
Where mAfter103C is the mass of the filter and residue after drying at 103oC, mEmpty is the
mass of the empty filter, mAfter550C was the mass of the filter and residue after ignition at 550oC,
and V refers to the volume of sample added to the filter.
3.2.3 COD
Chemical oxygen demand (COD) is a measure of the amount of oxygen required to
completely oxidize the contents of a sample. This gives a rough indication of the organic matter
available to microorganisms for digestion. The method used to measure COD in this study was
based on standard method 5220D in (APHA, 1998). In this method an excess of dichromate ion
(Cr2O72-) is added to a solution to oxidize samples in sulphuric acid. The chromium in the
dichromate ion is reduced to Cr3+ which absorbs strongly in the 600 nm region of the light
spectrum. Mercuric sulphate (HgSO4) is added to the mixture to complex with the chloride ion
to prevent its reduction to an elemental halide form, which would lead to overestimation of COD.
Large stocks of the digestion solution and sulphuric acid solution were prepared before
running the assay. The digestion solution was made by adding 10.216 g K2CrO7, previously
dried at 150oC for two hours, to 500 mL of MilliQ H2O in a 1 L volumetric flask. A 167 mL
aliquot of concentrated H2SO4 and 33.3 g HgSO4 were then added, and the mixture was allowed
to dissolve and cool to room temperature. The solution in the flask was then diluted to the mark
with MilliQ H2O. The sulphuric acid solution consisted of 5.5 g Ag2SO4 per kg concentrated
H2SO4. This solution was made in a glass bottle and required at least a day for the Ag2SO4 to
completely dissolve.
32
The assay was conducted in HACH COD 16 mm test tubes that have Teflon lined screw-
caps. A 2.5 mL aliquot of a standard or sample to be tested was added to each tube. The test
sample was diluted to contain an amount of COD that was within the standard curve
concentrations. A 1.5 mL aliquot of digestion solution and 3.5 mL sulphuric acid solution was
then added. The tubes were over turned several times and then placed in a heating block set at
150oC for 2 hours. After heating, the tubes were cooled to room temperature and the absorbance
of the resulting solution in the test tubes was measured in a spectrophotometer at 600 nm. A
standard curve corresponding to COD concentrations of 0.1 mg COD/mL to 1.0 mg COD/mL
was made using potassium hydrogen phthalate (KHP) that had been dried for over 2 hours at
110oC. KHP has a theoretical COD of 1.176 mg COD/mg. The standard curve created by the
KHP was linear and Beer’s law was used to calculate the COD of samples.
A low-range COD concentration method was also used in some measurements. This was
used for COD concentrations below 0.1 mg/mL. This method measured the absorbance of
remaining dichromate ion in solution at a wavelength of 420 nm. The digestion solution was
changed for this measurement: 1.022 g of K2Cr2O7 was added instead of 10.216 g.
COD tubes were cleaned after digestion using hot water and soap followed by rinsing
with 25% H2SO4 to eliminate any soap residues.
3.2.4 Carbohydrates
The method used to determine the amount of carbohydrates in samples was the anthrone
method. The anthrone method is a colorimetric method that has been found to be suitable for the
determination of total carbohydrates in activated sludge (Raunkjaer, Hvitved-Jacobsen, &
Nielsen, 1994). In this method, sulphuric acid was added to the sample to hydrolyse
polysaccharides to monosaccharides which react with the anthrone reagent to produce a complex
that absorbs strongly in the 625 nm region of the light spectrum. The original method is found in
(Morris, 1948) and the method used in this study is based on the method in (Raunkjaer et al.,
1994). The standard used in this study was glucose with concentrations from 0.01 mg/mL to
0.1 mg/mL. Anthrone reagent was prepared just before the experiment by placing 100 mg of
anthrone and 2.5 mL of anhydrous ethanol in a 50mL volumetric flask and diluting to the mark
with cold 75% H2SO4. Once the anthrone was dissolved, the reagent was placed in a refrigerator
at 4oC until it was used. The reagent was kept in ice during the entire assay.
33
The assay was performed in HACH 16 mm COD tubes with Teflon-lined screw-caps.
0.6 mL of each standard or sample was placed into each tube using dilutions to ensure the
concentration of carbohydrates was within the range of standard curve range. 3.0 mL of
anthrone reagent was added to each tube then the tube was capped. The tube was vortexed
several times to make a uniform mixture then placed in a heating block at 100oC for exactly 14
minutes. The assay is time sensitive so care was taken so that each tube would be in the heating
block for exactly the same amount of time. The tubes were then put on ice for 5 minutes then
allowed to come to room temperature. The absorbance of the solution in the tubes was then
measured in a spectrophotometer at 625 nm. The standard curve created was linear and Beer’s
law was used to calculate the carbohydrate content of samples.
3.2.5 Protein
Total protein content of the anaerobic granules and the waste activated sludge samples
was measured using the Kjeldahl method. The measurements were performed by SGS Lakefield
Research Limited located in Lakefield, ON. The Kjeldahl method measures total nitrogen
content (Total Kjeldahl Nitrogen, or TKN) of a sample by first completely digesting it in
sulphuric acid with a catalyst such as mercury and then measuring the resulting ammonia content.
To estimate the protein content of a sample, the following formula can be used (Kyllönen et al.,
1988):
Total Protein = 6.25 * (TKN – NH4-N)
Where TKN is the total Kjeldahl nitrogen in terms of mass of nitrogen and NH4-N refers
to the amount ammonia and ammonium in the sample before Kjeldahl digestion in terms mass of
nitrogen.
Soluble protein was measured by taking the soluble fraction of samples and subjecting
them to a modified Lowry protein assay. Only the soluble fraction of the samples was measured
due to interferences in the total fraction that could not be overcome, although a variety of
methods were attempted. More information on the protein assay modifications that were
performed is in Appendix B.
The Lowry assay used in these experiments is based on the microtiter method found in
(Peterson, 1977) including a modification presented in (Frølund et al., 1995). The method is
34
based on the reduction of the Folin-Ciocalteu phenol reagent by the tryptophan, tyrosine, peptide
bonds, and other constituents of peptides to produce a complex that absorbs visible light
(Peterson, 1979). Copper is added to facilitate electron transfer resulting in more colour
production (Peterson, 1979). To reduce interferences, several modifications were made to the
original Lowry method. The first modification, described in (Peterson, 1977), was to add the
detergent sodium dodecyl sulphate (SDS) to one of the reagents. This reduces interferences
caused by lipids and some sugars. The second modification, described in (Frølund et al., 1995),
involved running samples with reagents that contained copper or did not contain copper. Since
copper only enhances the colour produced by protein, by subtracting the absorbance of the
samples without copper added, the amount of colour produced just by proteins could be
determined.
The following stock solutions were prepared in MilliQ H2O ahead of time and could be
stored for two weeks at room temperature in the dark:
Stock 1a: 0.1% CuSO4 • 5 H2O
Stock 1b: 0.2% K-Tartrate; 10% Na2CO3
Stock 2: 10% sodium dodecyl sulphate (SDS)
Stock 3: 0.8 N NaOH
The day of the experiment the stock solutions were combined to make the following
reagents:
Reagent A-1: Stock 1a, Stock 1b, Stock 2, Stock 3, and MilliQ H2O in volume ratio
1:1:2:2:2.
Reagent A-2: Stock 1b, Stock 2, Stock 3, and MilliQ H2O in volume ratio 1:2:2:3.
Reagent B: 2N Folin-Ciocalteu reagent with MilliQ H2O in the volume ratio 1:5.
Calibration curves were made with bovine serum albumin (BSA) as the standard in
concentrations from 0.05 mg/mL to 0.1 mg/mL. The assay was performed in a polypropylene
96-well megatiter plate with 0.6 mL wells. 0.2 mL of samples diluted to contain protein within
the concentration range of the calibration curve was transferred by automated micropipette to
each well. Eight replicates of each sample and standard solution dilution were added to the
megatiter wells. To four of the replicates 0.2 mL of reagent A-1 was added and to the other four
35
0.2 mL of reagent A-2 was added. The plate was then shaken for 2 minutes and allowed to stand
at room temperature for 8 minutes. 0.1 mL of reagent B was then added to all wells and the plate
was shaken for 2 minutes and then allowed to stand at room temperature for 30 minutes.
0.28 mL of each well was then transferred to a 96-well polystyrene PCR plate. The plate was
read in a TECANTM Infinite M200 microplate spectrophotometer at 750 nm.
The calibration curve was made on a log/log plot of absorbance and concentration. A
linear regression of this line was used as the standard curve. The absorbances used were those
from the wells where reagent A-1, the reagent with copper, was added to the BSA.
The protein content of samples was found by plugging in a corrected absorbance into the
standard curve. The corrected absorbance was calculated using the following equation:
Acorr = [1 / (1 – X)] * (Atotal – Ablind)
Where Acorr refers to the corrected absorbance used with the calibration curve, Atotal was
the measured absorbance of the replicates of a sample with reagent A-1 (with copper) added,
Ablind was the measured absorbance of the replicates of a sample with reagent A-2 (without
copper added), and X refers to average ratio of absorbances of the BSA solutions with reagent A-
2 added over the absorbances of the BSA solutions with reagent A-1 added. The ratio the
variable X refers to was usually around 0.2. This means that the standard solutions of BSA
protein reacted with reagent A-2 absorb 20% of the light that those with reagent A-1 did. A full
derivation of this equation and sample calculations can be found in Appendix C.
3.2.6 Total Organic Carbon
Total organic carbon (TOC) was determined using a TOC-VCSH analyser manufactured
by Shimadzu Scientific Instruments, USA. The system uses the combustion catalytic
oxidation/nondispersiveinfrared (NDIR) method which works by oxidizing carbon in aqueous
samples and analysing the carbon dioxide content of the exhaust gases. The unit was previously
calibrated for total and inorganic carbon by laboratory personnel. The oven temperature was set
at 680oC, with a carrier gas (99.9% O2) flow rate of 150 mL/min and pressure of 200 kPa. 2 mL
aliquots of diluted sample were injected into the unit with total carbon concentrations between 0
and 500 ppm.
36
3.2.7 Other Measurements
Several biomass characterisation measurements were conducted by outside laboratories.
The ANALEST lab at the University of Toronto conducted the carbon, hydrogen, and nitrogen
(CHN) elemental analysis at their facilities. Biomass samples were dried in an oven overnight at
103oC in a crucible. The dried sample was placed in a vial and measured by ANALEST
technicians using the 2400 Series II CHNS Analyzer. The test used the Pregl and Dumas organic
analysis method.
Several other tests were performed by SGS Lakefield Research Limited located in
Lakefield, ON. These tests included solids content, inorganic and organic nitrogen content,
sulphate, sulphide, reactive phosphorus, and an elemental analysis of metal content. Biomass
samples were placed in containers provided by SGS that contained preservatives and were
shipped on ice to their facilities.
3.3 Pretreatments
Pretreatment conditions were based upon the optimal conditions found in previous
studies of the anaerobic digestion of municipal WAS. Pretreatments of biomass samples were
performed the same day the samples were going to be used in anaerobic digestion experiments.
Immediately after each pretreatment, the suspended solids content and soluble and total COD
were measured. After these measurements, 1.5 mL portions of the sample and the soluble
fraction of samples were placed in small screw-cap plastic vials and frozen with liquid N2. The
remainder of the samples were stored in 500 mL glass bottles with screw caps at 4oC until use in
anaerobic digestion tests. Frozen samples were stored in a -80oC freezer and carbohydrates and
soluble protein of the stored samples were measured at a later time.
3.3.1 Thermal
To heat the WAS samples for pretreatment, a Parr 2 litre titanium autoclave was used
with a Parr 4843 P80 controller. The samples were held within a glass liner inside the titanium
vessel. 400 mL of biomass sample was added to the glass liner of the autoclave. The headspace
of the reactor was flushed with N2 to remove O2 and prevent oxidation. The temperature in
autoclave was allowed to increase to 170oC and maintained at that temperature for 1 hour. The
sample was then allowed to cool to room temperature in the autoclave before removal. The pH
37
was measured after the pretreatment. In one experiment the pH had to be neutralized with drops
of 6 N HCl.
3.3.2 Caustic
During caustic pretreatment, NaOH was added to the WAS before being exposed to a
high temperature. 400 mL of biomass samples were added to a glass beaker along with a Teflon-
coated magnetic stir bar. NaOH pellets were added one at a time to the sample allowing each
pellet to completely dissolve with the help of the stir bar. In between addition of pellets, the pH
of the biomass was measured with a pH meter. When the sample reached pH 12 it was
transferred to the autoclave glass liner. This pH was chosen because previous studies have
reported that a pH higher than 12 does not result in any further improvement in WAS
solubilisation (Zhang et al., 2007). Pretreatments involving caustic were performed in the same
autoclave as thermal pretreatments. The temperature of the autoclave was allowed to increase to
140oC and maintained at that temperature for 1 hour. After autoclaving the sample was brought
to room temperature in the autoclave. The sample was brought to a pH 7 after pretreatment with
drops of 6 N HCl. The physical and chemical properties of the sample were measured after the
pH was neutralized.
3.3.3 Sonication
Sonication pretreatment was performed in a custom-built tubular reactor with a
sonicating plate transducer at the bottom. The reactor was manufactured by Advanced Sonics
Processing Systems and provided a frequency 20 kHz at a maximum input power of 450 W. The
reactor is acrylic with a 10.8 cm diameter and a 25 cm height. The actual power delivered to the
samples was tested previously (Yong, Farnood, Cairns, & Mao, 2008) and found to be
0.60 W/cm2, meaning 54.5 W was delivered directly to the WAS as ultrasonic energy when the
energy supplied to the sonicating reactor was 400 W.
400 mL of biomass samples was added to the reactor and the sonication was applied at
400 W for 30 minutes. This is an overall power density of 1 W/mL with an actual power density
applied to the WAS of 0.14 W/mL. The sonication was kept to 30 minutes to prevent excess
heat generation that would have damaged the reactor. The maximum operating temperature of
the reactor was 60oC and applying sonication for only 30 minutes ensured the temperature would
not exceed 50-55oC. This length of sonication should still be sufficient as previous studies found
38
that at an applied power of 0.44 W/mL, sludge flocs were completely disintegrated in that
amount of time (Chu et al., 2001). The bulk temperature was also not controlled as it has been
found to be an essential part of the effectiveness of sonication (Chu et al., 2001).
3.4 Anaerobic Digestion Experiments
3.4.1 Biochemical Methane Potential Assay Setup
The rate and extent of the anaerobic digestion of WAS was tested using the biochemical
methane potential assay (BMP) first described in (Owen, Stuckey, Healy, J. B., Jr., Young, &
McCarty, 1979). The BMP assay is a batch digestion where a substrate being tested for
anaerobic degradability is incubated in a sealed bottle with a sample of anaerobic
microorganisms in a defined nutrient medium. The volume of gas produced during the
incubation is measured and is an indication of the rate of substrate digestion. The method used
in this study is a slightly modified version of the method found in (Shelton & Tiedje, 1984). The
assays performed were 30 – 45 days long and run in triplicate.
The BMP assay was performed in 160 mL glass serum bottles that were cleaned with
soap and water and then soaked in a 30% nitric acid bath for 24 hours prior to use to remove any
organic residues in an attempt to improve the reproducibility of the assay. All bottles were
rinsed with MilliQ water several times before use. The total volume of liquid added to each assay
bottle was 100 mL, leaving approximately 60 mL of headspace. The 100 mL of liquid consisted
of diluted substrate (15 mL or 10 mL) and anaerobic granules diluted in nutrient medium (85 mL
or 90 mL).
Substrates were added to each bottle so that each assay bottle had the theoretical potential
to produce 20 mL of biogas assuming it would contain 70% methane. In theory, the production
of 1 mole of methane requires 64 g of COD. This would result in all assay bottles containing the
same amount of COD. Sample calculations can be found in Appendix C. In experiments 4 and 5,
some samples were added in terms of equal amounts of VSS rather than equal amounts of COD.
The COD of each substrate was measured the same day of the start of the experiments.
Substrates were then diluted so the same volume of diluted substrate could be added to each
assay bottle. Each substrate was diluted in a 100 mL volumetric flask with MilliQ H2O and
placed in a 160 mL serum bottle and sealed with butyl rubber stopper and crimped. Each of
39
these substrate bottles was made anaerobic as described in section 3.4.3. The substrates were
prepared immediately before the preparation of the BMP assays.
The BMP assays were prepared in the anaerobic atmosphere of the glovebox described in
section 3.4.4. Before starting the BMP assay the serum bottle containing the anaerobic granule
sample to be used was weighed to determine the volume of its contents. The contents of the
bottle was then sparged as described in section 3.4.3 to remove any residual methane and
hydrogen sulphide from the headspace.
The mixture of anaerobic granules and nutrient medium was prepared by diluting
anaerobic granule samples with nutrient medium that was previously prepared and transferred to
the glovebox. The contents of the serum bottle containing the granule sample was first
transferred a 500 mL glass bottle referred to in this document as “bottle #1”. To ensure all the
granules were transferred, the serum bottle was rinsed with nutrient medium and poured into
bottle #1. The total volume of the solution in bottle #1 was brought up to 300 mL or 200 mL
with nutrient medium. A magnetic stir bar was added to bottle #1 and was thoroughly mixed on
a stirring plate. The mixture of medium and granules that was to be added to each serum bottle
in the assay was then prepared in a second 500mL glass bottle referred to in this document as
bottle #2. An aliquot of the contents of bottle #1 was transferred with a 5 mL micropipette to
bottle #2. The micropipette tip was cut at 3 mm from the end to create a wider opening to
accommodate the granules. After the transfer to bottle #2, 300 mL of medium was added using a
graduated cylinder.
The granule-medium mixture in bottle #2 was continually stirred by a magnetic stir bar
and was transferred to all of the serum bottles in each assay using a 25 mL serological sterile
wide-mouth pipette. The previously prepared diluted substrates were then transferred to each
assay bottle using a 5 mL micropipette. The assay bottles were then sealed with a butyl rubber
stopper and crimped and moved to a shaking incubator. The assay was incubated at 35oC and
shaken at 200 rpm to ensure good mixing within the bottle. The time when the bottles were put
in the incubator was considered the start of the assay. Unused portions of bottle #1 and bottle #2
were preserved for later analysis in the same manner as pretreated samples and were stored in
a -80oC freezer.
The amount of granules in each assay bottle was chosen so that the ratio of VSS of the
granules to VSS of substrate was at least 2:1. This ratio is based on previous studies that found a
ratio of 2:1 or greater was ideal for BMP assays in terms of reproducibility and largest
40
conversion of substrate to biogas (Chynoweth, Turick, Owens, Jerger, & Peck, 1993; Raposo,
Banks, Siegert, Heaven, & Borja, 2006).
Every assay had a blank control and a positive control. In the blank control MilliQ water
was added instead of any substrate. This would give a correction for the background changes in
volume in the assay bottles not related to substrate digestion. The positive control contained a
mixture of glucose, sodium acetate, and sodium propionate as the substrate to determine the
maximum activity of the granules. This substrate was chosen to mimic the volatile fatty acid
content of the waste sent to the IC reactor that the granules were sampled from: 50% of the COD
was from glucose, 45% sodium acetate, and 5% propionate. Sample calculations for the
preparation of the positive control substrate can be found in Appendix C.
In experiment 1, each substrate was also added to assay bottles containing nutrient
medium without any granules. This added control was performed to see if there was any
methane production by the substrate samples on their own.
3.4.2 Nutrient Medium
The nutrient medium used for the BMP assays in this study was based on a previously
developed medium for methanogenic bacteria (Edwards & Grbić-Galić, 1994). A detailed
procedure for preparing the nutrient medium can be found in Appendix B. The medium was
prepared by mixing several previously prepared concentrated stock solutions. 10 mL of stock
MM1 (phosphate buffer), 10 mL of stock MM2 (salt solution), 2 mL of MM3 (trace mineral
solution), 2 mL of stock MM4 (magnesium chloride solution), and 1 mL of stock MM5
(resazurin indicator solution) were added to 500 mL of MilliQ water in a clean and dry glass
bottle. The mixture was then diluted with MilliQ water to about 970 mL. The bottle top was
covered in foil and autoclaved at 121oC for 20 minutes along with a screw cap and a glass
sparging rod. After autoclaving the medium was made anaerobic using the sparging rod as
described in section 3.4.3 for 30 to 45 minutes. After removing the sparging rod, the bottle was
capped tightly and transferred to an anaerobic glove box. In the glovebox 10 mL of stocks MM7
(vitamins), MM8 (amorphous ferrous sulphide), and MM6 (saturated bicarbonate) were added
using 10mL disposable sterile syringes. The medium volume was then brought up to 1L with
MilliQ water that was previously autoclaved and made anaerobic in a similar manner to the
medium. The final concentration of nutrients in the medium is shown in Table 3.4.1.
41
The medium is buffered by the equilibrium of CO2 in the anaerobic glovebox atmosphere
and sodium bicarbonate in the solution. The medium is buffered to pH 7. Ferrous sulphide is
used to reduce the medium to ensure it is anaerobic. Resazurin is added as an indicator of the
reducing potential of the medium; when the medium is clear the medium is reduced. The ferrous
sulphide appears as a fine black powder and was allowed to settle before the medium was used in
the BMP assay to ensure none of the powder was transferred to the assay bottles.
Table 3.4.1 Concentrations of components in nutrient medium used in biochemical methane potential assays.
Stock concentration refers to the amount of a particular nutrient in its concentrated stock solution. Medium
concentration refers to the amount of a particular nutrient in the final medium mixture.
Stock Compound
Stock Conc. [g/L]
Medium Conc.
[mg/L]
Stock Compound
Stock Conc. [g/L]
Medium Conc.
[mg/L]
MM1 KH2PO4 27.2 272 MM6 NaHCO3 6.9 69
K2HPO4 34.8 348 MM7 Biotin 0.02 0.2
MM2 NH4Cl 53.5 535 Folic acid 0.02 0.2
CaCl2•6H2O 7 70 Pyridoxine HCl 0.1 1.0
FeCl2•4H2O 2 20 Riboflavin 0.05 0.5
MM3 H3BO3 0.3 0.6 Thiamine 0.05 0.5
ZnCl2 0.1 0.2 Nicotinic acid 0.05 0.5
Na2MoO4•2H2O 0.1 0.2 Pantothenic acid 0.05 0.5
NiCl2•6H2O 0.75 1.5 PABA 0.05 0.5
MnCl2•4H2O 1 2.0
Cyanocobalamin (vitamin B12)
0.05 0.5
CuCl2•2H2O 0.1 0.2 Thioctic (lipoic) acid 0.05 0.5
CoCl2•6H2O 1.5 3.0 Coenzyme M 1 10
Na2SeO3 0.02 0.04 MM8 (NH4)2Fe(SO4)2•6H2O 39.2 392
Al2(SO4)3•18H2O 0.1 0.2 Na2S•9H2O 24 240
MM4 MgCl2•6H2O 50.8 101.6 Resulting FeS 2 20
MM5 Resazurin 1 1.00
3.4.3 Sparging to Make Bottles Anaerobic
Bottled liquids were made anaerobic by sparging with a gas mixture containing an 80/20
volume ratio of N2 and CO2. The gas was first passed through a heated glass tube containing
copper wool to remove any traces of O2 in the gas. In the case of sealed serum bottles, gas was
42
transferred to the bottle using 22.5 gauge sterile needles. Two needles were placed at right
angles from each other diagonally through the rubber stopper. The gas was passed through a
tube, through a Pall Acrodisc 25 mm 0.2µm pore-diameter sterile syringe filter, then into one of
the needles. The bottle was then placed on its side so the gas would bubble through the liquid in
the bottle.
3.4.4 Anaerobic Glovebox
BMP assays were set up in the anaerobic environment of a glovebox manufactured by
Coy Lab. Products Inc., Glasslake, MI, USA. Inside the glovebox was an atmosphere containing
80% N2, 10% CO2, and 10% H2 by volume. The H2 was present to react with a catalyst in the
glovebox to remove any trace amounts of oxygen. All syringes, pipettes, glass bottles, and
medium were placed in the glovebox several days before the actual start of the experiment to
ensure all equipment was oxygen-free.
3.4.5 Biogas Production Measurement
Biogas was measured at frequent intervals during each BMP assay. Several methods
were used to measure biogas production. They are presented here in the order of increasing
accuracy. Later experiments used the pressure transducer method. During the measurement of
biogas, the bottles were kept in a water bath set at their incubation temperature. Temperature
variation was found to have a large impact on the gas volume in the headspace of the assay
bottles.
3.4.5.1 Syringe Method
The method for measuring biogas suggested in (Owen et al., 1979) involved a lubricated
glass syringe. In this case, each BMP assay bottle is tilted onto its side and the needle of the
syringe was passed through the butyl rubber stopper. The piston of the glass syringe is pushed
out by the pressure inside the assay bottle until equilibrium with atmospheric pressure is reached.
This method was not used extensively in this study except for some preliminary measurements.
43
3.4.5.2 Liquid Displacement Method
The method used to measure biogas production in experiment 1 and 2 was based on the
volume displacement of a liquid using the apparatus in Figure 3.4.1. The apparatus consisted of a
1 litre Erlenmeyer flask containing 800 mL of an acidic salt solution containing 200 g of Na2SO4,
30 mL of H2SO4, and an indicator in MilliQ H2O as described in the APHA standard methods
(APHA, 1998). A Teflon stopper with a glass tube and a graduated pipette passing through it
was pushed onto the top of the flask. The glass tube was connected to a rubber tube with an
interchangeable sterile needle at the end. The other end of the glass tube was bent so that it went
under the level of the salt solution and up into the graduated pipette. The graduated pipette is
connected at the top to another rubber tube that is clamped tightly. Before each measurement,
the clamp is released and the salt solution is drawn up into the graduated cylinder through the
tube by a large syringe and then clamped shut.
Figure 3.4.1 Liquid displacement biogas measurement apparatus.
Biogas volume is measured by placing a needle connected to the apparatus through the
rubber stopper of the assay bottle. This releases the built up pressure in the headspace of the
bottle caused by biogas production, resulting in the gas moving through the needle and down the
rubber tube into the apparatus. The gas then bubbles through the displacement solution and
displaces the solution at the top of the graduated pipette tube. The volume displaced is equal to
Biogas from
assay bottle
Closed tube attached to syringe to
bring liquid level up to top graduation
Teflon stopper
Erlenmeyer flask
Glass tube
Graduated pipette
Displacement solution
44
the amount of biogas produced. An acidic (pH 2) strong salt solution is used so that CO2 and
other gases are not removed from the biogas being measured.
3.4.5.3 Pressure Transducer Method
In experiments 3, 4, and 5, the biogas production was measured using a pressure
transducer. The pressure transducer used was an Omega PX725 Industrial Pressure Transmitter
hooked up to an Omega DP24-E Process Meter. The pressure transducer worked by measuring
the change in resistance across a deforming silicon wafer. The range of the transducer was
0 inches H2O to 100 inches H2O (0 kPA to 24.9 kPa gauge pressure).
During the measurements, bottles were kept in a water bath at their incubation
temperature. A 22.5 gauge needle attached to a rubber tube leading to the transducer was pushed
through the rubber stopper in the assay bottle. The reading on the process meter was written
down and compared to a calibration curve to determine the amount of biogas produced.
A calibration curve was made for the pressure transducer by taking a sealed serum bottle
filled with 100mL of H2O at room temperature and adding different volumes of air to the bottle
with a gas-tight GC syringe. Volumes ranging from 0.1mL to 10mL were added to the
headspace of the bottle and measured with the transducer. The calibration curves can be found
in the Appendix B.
3.4.5.4 CH4 Measurement
After biogas production was measured, the methane concentration of the biogas was
measured using gas chromatography. If positive pressure was found inside the assay bottles,
they were brought to atmospheric pressure before they were sampled for measurement. This was
done by placing a needle into the stopper of the bottle and allowing the gases to escape. CH4
concentration was then measured by taking 0.3 mL samples of the headspace using a 0.5 mL gas-
tight glass syringe and injecting them into a GC.
The GC used was a Hewlett-Packard 5890 Series II Gas Chromatograph (GC) with a
GSQ 30 m x 0.53 mm I.D. PLOT column from J&W Scientific. The GC was equipped with a
flame ionization detector. The oven temperature was set to 190oC and kept constant throughout
the measurement. The carrier gas was helium and applied to the column at a constant pressure of
20 psi.
45
This measurement gave the concentration of methane in the headspace of the BMP assay
bottle. Based on a mass balance taking into account the amount of biogas being produced, the
total moles of CH4 produced between two biogas measurements (nCH4, produced) is calculated by:
nCH4, produced = CCH4, 1 * (Vbiogas, 1 + Vheadspace) – CCH4, 0 * Vheadspace
Where CCH4, t refers to the concentration of methane in the headspace measured at time
point t, Vbiogas, t refers to the volume of biogas produced at time point t, and Vheadspace is the
volume of the headspace in the assay bottle. Sample calculations can be found in Appendix C.
A calibration curve for the GC was made by adding known amounts of methane to a
serum bottle and performing measurements as described above. First, a serum bottle was
completely filled with 99% CH4 reference gas at atmospheric pressure. This was done by having
the serum bottle filled with water upside down in a tub of water. The methane was let out of the
gas cylinder and made to displace the water in the bottle. The bottle was then sealed with a
stopper and crimp. Second, various volumes of the gas in the first bottle were removed with a
gas-tight syringe and added to sealed serum bottles that contained 100mL of H2O. For bottles
where large volumes of methane were added, the same volume was first removed from the
headspace. The GC calibration curves can be found in the Appendix B.
3.5 Calculations
3.5.1 Statistical Significance
To determine statistical differences between data sets, the single factor experiment
analysis of variance method was used. Unless otherwise stated, statistically significant refers to
a difference with a confidence level of over 95%. A difference that was not considered to be
statistically significant had a confidence level of less than 85%.
3.5.2 Error Calculations
All error bars and error shown in the results of this study were calculated as the standard
error in the mean. The standard error in the mean is a good descriptor for error bars as it can be
used to better show the differences between groups of measurements in experimental biology
46
(Cumming, Fidler, & Vaux, 2007). The standard error (SE) in the mean is calculated by the
following formula:
n
SDSE =
Where SD is the standard deviation and n is the number of replicates.
3.5.3 Extent of Degradation
Theoretically, the amount of COD required to produce a given amount of CH4 during
anaerobic digestion can be predicted. This is based on the number of electrons that must be
transferred from the electron donor during cellular metabolism to produce CH4. Each mole of
methane requires 8 electron equivalents which is equivalent to 64 g of COD. In theory, 0.35 L of
CH4 at STP requires 1 g of COD or BOD (Rittmann & McCarty, 2001). Based on this ratio the
amount of COD removed to produce a measured amount of CH4 can be calculated. In this study,
percent COD removal refers to this calculated amount of COD removed divided by the amount
of COD added to each assay bottle. An example of this calculation can be found in Appendix C.
VSS removal was determined by calculating the VSS of the substrate and granules added
to each bottle at the start of the experiment as well as measuring the total VSS at the end of the
experiment left in each bottle. Percent VSS removal was calculated by dividing the difference
between initial and final amount of VSS by the initial amount of VSS in each assay bottle.
3.5.4 Reaction Rates
The anaerobic digestion of many substrates can be described as having a first-order
reaction kinetics rate law (Chynoweth et al., 1993). Although there is some deviation from this
with complex substrates, it is usually applicable. The production of biogas and methane is
proportional to the amount of COD being utilized during the anaerobic digestion. The data was
normalized by dividing the amount of COD utilized to produce the measured amount of methane
by the amount of substrate COD added to each bottle. The rate of utilization was represented by
the following equation:
rCOD = dYCOD / dt = -k * YCOD
47
This equation can be integrated to give:
YCOD = Y0 * (1 – e-k * t)
Where rCOD is the normalized utilization rate of COD, YCOD is the cumulative amount of
COD used at time t, Y0 is the total amount of COD used at t equal to infinity, t is the time in days,
and k is the reaction rate in days-1.
To fit the data to this equation, the Microsoft Excel solver tool was used. The solver tool
was set to use an iterative method to change the Y0 and k values until the R2 value of the error
was closest to 1. In this case the R2 value was set to the coefficient of determination which was
defined as:
( )
( )
−
−
−=
∑
∑n
Meani
n
ied
YY
YY
R
1
2
1
2
.Pr2 1
Where Yi refers to the normalized measured value i, Ypred. is the YCOD calculated using
certain Yo, k, and t values, YMean is the mean of all the measured values, and R2 is the coefficient
of determination.
48
4.0 Results and Discussion
4.1 Biomass Properties
4.1.1 Waste Aerobic Wastewater Treatment Sludge
The bacterial sludge samples used in this study were characterized in terms of sludge
properties, biological macromolecule content, and elemental composition (Table 4.1.1, Table
4.1.2, and Table 4.1.3).
Table 4.1.1 Summary of waste activated sludge properties.
S WAS #1 S WAS #2 K WAS
Source Mill A – sulphite
pulp mill Mill A – sulphite
pulp mill Mill B – Kraft
pulp mill
Date Retrieved 2006.12.21 2007.10.10 2007.09.07
Total Solids [mg/mL] n.d. 11.1* 24.4*
Total Suspended Solids [mg/mL] 11.4 ± 0.1 8.7 ± 0.2 17.9 ± 0.2
Volatile Suspended Solids [mg/mL] 10.5 ± 0.1 7.3 ± 0.1 13.9 ± 0.1
COD [mg/mL] 13.5 ± 0.2 11.7 ± 0.5 27 ± 1
Soluble COD [mg/mL] 2.8 ± 0.1 1.4 ± 0.1 0.3 ± 0.1
pH n.d. 7.44# 6.98#
* These values were measured by SGS # Insufficient replicates were performed to determine error in values n.d. = not determined
The suspended solids were lower than total solids because of the different techniques
used for measurement. Suspended solids were calculated after the soluble portion of the sample
has passed through a filter. The organic content of the solids, which is estimated by VSS, was
higher in sulphite WAS than in the Kraft WAS. Approximately 22% of the solids in the K WAS
were inorganic ash and were not likely biodegradable. S WAS was less concentrated and had
lower solids and COD than K WAS. Also, more of the organic matter was soluble in S WAS as
indicated by the higher amount of soluble COD than in K WAS.
Protein accounts for about 54% of the VSS of S WAS and 33% of the VSS of K WAS
(Table 4.1.2). This may suggest a larger portion of K WAS was non-bacterial matter because
protein content was lower than 50%, the usual protein fraction associated with bacteria. This
non-bacterial matter could have been cellulose or lignin. Previous studies have found that
49
mainly high molecular weight fractions of lignin adsorb to activated sludge flocs and that more
Kraft lignin adsorbed compared with sulphite mill lignin and lignosulphonates (Ganczarczyk &
Obiaga, 1974). If the volatile solids of the sulphite mill sludge contained a smaller fraction of
lignin, it could be more readily digested by the anaerobic bacteria.
Table 4.1.2 Waste activated sludge biological macromolecule content.
S WAS #2 K WAS
Total Carbohydrates [mg/mL] 0.81 ± 0.01 2.01 ± 0.06
Soluble Carbohydrates [mg/mL] 0.06 ± 0.00 0.01 ± 0.00
Total Protein [mg/mL] 3.94* 4.58*
Soluble Protein [µg/mL] 17 ± 4 6 ± 4
Total Organic Carbon [mg/mL] 5.9 ± 0.1 6.6 ± 0.1
* These values were measured by SGS n.d. = not determined
Carbohydrates accounted for about 11% of the VSS of S WAS and 14% of the VSS of K
WAS. These values are within the ranges found in previous studies of WAS from pulp and
paper mills and other sources (Kyllönen et al., 1988; Tanaka et al., 1997). Both WAS samples
had low soluble biological macromolecule content, especially soluble protein.
The iron content was much higher in S WAS than K WAS (Table 4.1.3). This was likely
due to a nutrient mixture added to the WAS at Mill A where it was produced. The iron-rich
mixture was added to improve settling properties of the WAS by encouraging strong floc
formation. Similar carbon content was found between S WAS and K WAS, but organic carbon
as percent of solids was much lower in K WAS. This suggested that there was more inorganic
carbon that would not be digestible in K WAS. There was also a higher sulphate content in K
WAS than in S WAS which may lead to higher H2S formation during anaerobic digestion.
Carbon to nitrogen ratio for the wastes was about 11 and 8 for K WAS and S WAS
respectively. According to previous studies, the digestion of municipal solid waste requires a
ratio of at least 76 (Chynoweth & Isaacson, 1987). This shows that nitrogen will not be limiting
in the case of the digestion of the sludges in this study if all measured nitrogen is accessible to
anaerobic degradation. The carbon to reactive phosphorous ratio was 707 and 225 for K WAS
and S WAS, respectively. These conditions may be nutrient limiting and phosphorous addition
would be required for anaerobic digestion (Chynoweth & Isaacson, 1987). For the following
50
experiments, both nitrogen and phosphorus were added to the medium to make sure that there
would be no nutrient limitations.
Table 4.1.3 Elemental analysis of waste activated sludge samples.
S WAS #2 K WAS
C [wt% dry solids] 44.0% 45.5%
H [wt% dry solids] 5.5% 5.3%
N [wt% dry solids] 5.8% 3.9%
Total Reactive Phosphorus [mg/mL] 0.022 0.016
Sulphate [mg/mL] 0.200 0.600
Calcium [mg/mL] 0.190 0.890
Iron [mg/mL] 0.180 0.037
Potassium [mg/mL] 0.054 0.054
Sodium [mg/mL] 0.600 0.600
Phosphorous [mg/mL] 0.081 0.140
CHN analysis was performed by the ANALEST labs at the University of Toronto. All other measurements performed by SGS.
4.1.2 Anaerobic Granules
The anaerobic granule samples used in the BMP assays performed in this study were
analysed in a similar manner to the WAS samples (Table 4.1.4). There was a large difference in
the suspended solids of the granules collected in 2006 and 2007. This may have been due to
differences in operation at the different times the samples were taken.
51
Table 4.1.4 Physical and chemical properties of anaerobic granule samples used in anaerobic digestion
experiments in this study.
Granule Sample #1 Granule Sample #2
Date Sampled Oct. 2006 Sept. 2007
Sampling Port Height [m] 6.2 6.2
Total Solids [mg/mL] 68.5*
COD [mg/mL] 68 ± 9 76 ± 10
Soluble COD [mg/mL] 4.4 ± 0.5
Volatile Suspended Solids [mg/mL] 29.1 ± 0.4 61.8 ± 1.8
Total Suspended Solids [mg/mL] 36.6 ± 0.9 77.4 ± 1.9
Total Carbohydrates [mg/mL] 4.1 ± 0.3
Soluble Carbohydrates [mg/mL] 0.17 ± 0.01
Total Protein [mg/mL] 22.3*
Soluble Protein [mg/mL] 0.18 ± 0.01
Calcium [mg/mL] 1.8*
Iron [mg/mL] 1.4*
Sodium [mg/mL] 1.7*
* These values were measured by SGS
4.2 BMP Assays Performed
In this study five BMP assay experiments were performed. These experiments will be
referenced throughout this document by a number from 1 through 5 (Table 4.2.1). A full
description of each assay setup can be found in Appendix A. Briefly, experiment 1 was
performed to investigate the digestibility of S WAS without pretreatment. The digestibility was
compared to samples of the feed normally sent to the IC reactor that the granules were sampled
from. Along with these substrates, the soluble fraction of S WAS was tested to determine the
degradability of only the soluble components of the untreated WAS. The substrates were also
placed in bottles without granules to determine if the samples produced any gases on their own in
an anaerobic environment. There was also a concern about the presence of H2 in the headspace
of the bottles as a source of energy for the anaerobic bacteria, so a triplicate set of positive
control samples was run with a headspace that only contained N2 and CO2 in an 80/20 volume
ratio.
Experiment 2 was performed to test for the possible toxicity of S WAS. This experiment
used three sets of substrates: S WAS alone, the positive control of glucose and acetate, and the
52
toxicity test substrate which consisted of a second set of positive controls containing an
additional 25% of substrate COD from S WAS. If S WAS was toxic, it should reduce the
amount of biogas produced by the toxicity test substrate compared with the positive control
bottles that only contained glucose and acetate. Another notable aspect of this experiment was
that FeS in the medium was completely allowed to settle out of solution before the medium was
added to the substrate bottles. In experiment 1, FeS particles were present in the assay bottles
and may have contributed negatively to biogas production.
Table 4.2.1 Description of BMP assay experiments performed in this study.
Exp. #
# of Bottles
Length of BMP
assay [days]
Substrate Added to
Each Bottle [mg COD]
Granules Added to
Each Bottle [mg VSS] Purpose
1 27 27 34.3 83.7 S WAS #1 digestibility, soluble S WAS #1 digestibility, gas production without granules
2 11 42 38.9+ 57.7 S WAS #1 toxicity to granules
3 30 44 3.5 4.67 Pretreatment effect on K WAS and S WAS #2
4 24 34 35.2^ 46.6 Pretreatment effect on K WAS
5 24 34 35.2^ 50.0 Pretreatment effect on S WAS #2
+ some bottles contained more COD in this assay because some bottles both contained the positive control of glucose and acetate along with an extra 25% of COD in the form of S WAS ^ some bottles contained more COD in this assay because they were filled with substrate on the basis of VSS rather than COD
Experiment 3 was performed to test the effect of pretreatment on K WAS and S WAS.
All three pretreatments were performed on samples of S WAS and K WAS before the BMP
assay was started. Due to a calculation error in the preparation of this experiment, the amount
granules and COD added to the assay bottles in this experiment was very low. Due to this major
error the results are not reported in this document.
Experiments 4 and 5 were performed to test the effect of pretreatment on K WAS and S
WAS. Experiment 4 focussed on K WAS and experiment 5 focussed on S WAS. This
experiment was run because of the error introduced in experiment 3. In addition to adding each
substrate to the assay bottles in terms of equal amounts of COD, samples of K WAS and S WAS
pretreated by thermal and caustic methods were added on a VSS basis. The pretreated WAS
samples were added so that the assay bottles contained the same amount of VSS as untreated
WAS samples and were labelled “thermal #2” and “caustic #2”. This was done to determine if
solids had an effect on WAS digestion.
53
4.3 Untreated WAS Anaerobic Digestibility
4.3.1 Kraft Mill and Sulphite Mill WAS
In experiment 1, the anaerobic digestibility of S WAS and the soluble fraction of S WAS
were investigated. A single factor experiment analysis of variance revealed that the difference in
cumulative biogas production by test and control samples was not statistically significant (Figure
4.3.1). This suggests that the soluble fraction of untreated S WAS is mainly composed of
recalcitrant compounds.
0
0.02
0.04
0.06
0.08
0.1
0.12
0 5 10 15 20 25 30
Time [days]
Cu
mu
lati
ve
Bio
ga
s P
rod
uct
ion
[mL/
mg
su
bst
rate
CO
D]
Exp. 1, S WAS
Exp. 1, Sol. S WAS
Figure 4.3.1 Cumulative biogas production subtracting blank (H2O as substrate) values for S WAS and the
soluble fraction of S WAS in experiment 1.
There was a large error in the values associated with the amount of biogas being
produced. This suggests that the method of measurement of biogas in this experiment was not
very precise. When small amounts of biogas were produced between measurements, the liquid
displacement method did not properly measure the biogas production, introducing an error. It is
also important to note that some bottles developed a vacuum during this experiment, possibly
due to the combination of a lowering of headspace gas temperature during measurement and
small amounts of oxygen making it into the bottle and being used up by bacteria present in the
bottle.
54
In experiments 2, 4, and 5, anaerobic digestion of K WAS and S WAS produced more
biogas than the controls (Figure 4.3.2). S WAS produced a similar amount of biogas in both
experiments 2 and 5. K WAS produced a much lower amount of biogas over the course of the
assays.
0
0.05
0.1
0.15
0.2
0.25
0 5 10 15 20 25 30 35 40 45
Time [days]
Cu
mu
lati
ve
Bio
ga
s P
rod
uct
ion
[mL/
mg
su
bst
rate
CO
D]
Exp. 2, S WAS
Exp. 4, K WAS
Exp. 5, S WAS
Figure 4.3.2 Cumulative biogas production subtracting blank (H2O as substrate) values for K WAS and S
WAS in experiments 2, 4, and 5.
The cumulative biogas production measured in experiments 2, 4, and 5 were different
from those of experiment 1 for S WAS. This was the result of several factors. The first was the
reduced accuracy of the liquid-displacement method used to measure biogas production in
experiment 1. This method provided less control for temperature effects and was not able to
measure vacuum formation. The second contributing factor was the state of the medium used to
dilute the granules. FeS was used to reduce the medium and was added in excess. The FeS
existed as fine particles that settled to the bottom of the bottle the medium was prepared in.
Ideally, only reduced medium without any FeS particles would be used to dilute the granules. In
experiment 1, the medium was added to the granules without allowing the particles to completely
settle. Although this did not affect activity of the granules in the positive controls, the FeS had a
noticeable impact on the WAS. The WAS turned black and settled to the bottom of the bottles.
The values from experiments 2, 4, and 5 were in the range of previously reported biogas
production values for municipal and pulp mill WAS with K WAS producing biogas at the lower
55
end of the range (Table 4.3.1). For comparison, biogas production in experiment 4 and 5 was
0.09 mL/mg VSS added for K WAS and 0.32 mL/mg VSS added for S WAS. Municipal WAS
typically produces 0.146 to 0.217 mL CH4/mg VS added in anaerobic digesters (Bougrier et al.,
2006; Bougrier et al., 2007). The digestion of untreated pulp mill WAS was 0.09mL/mg VSS for
BCTMP mill WAS and 0.220 mL/mg VS for Kraft mill WAS (Puhakka et al., 1988; Puhakka,
1992a). Previous studies show a wide range of values emphasizing the importance of testing
WAS characteristics before implementation of anaerobic digestion.
Table 4.3.1 Total biogas production from S WAS and K WAS. Each substrate was digested in triplicate
during the BMP assay.
Exp. # Sample
Total biogas produced
subtracting blank [mL/mg
substrate COD] vol% CH4 in
biogas % COD removed
by digestion % VSS reduction
by digestion
1 S WAS #1 0.003 ± 0.034
1 Soluble S WAS #1 -0.037 ± 0.023
2 S WAS #1 0.196 ± 0.017
4 K WAS 0.045 ± 0.005 65% ± 10% 6% ± 0.5% 27% ± 2%
5 S WAS #2 0.197 ± 0.009 85% ± 4% 42% ± 2% 15% ± 7%
There was a large difference in the digestibility of S WAS and K WAS used in this study.
COD removal was calculated based on the theoretical amount of COD required to account for
the observed methane production. 6% of the total COD was removed in the case of K WAS and
42% in the case of S WAS. This along with the amount of biogas production suggests the S
WAS samples were much more readily degraded than the K WAS samples.
Typical design values for anaerobic digesters dealing with WAS are volatile solids
reduction of 40% to 60% with retention times between 20 and 40 days at full scale (Task Force
on Wastewater Residuals Stabilization, 1995). In the studies looking at pulp and paper mill
WAS the values found were approximately 40% VS removal or 41% VSS removal (Puhakka et
al., 1988; Puhakka, 1992a). In this project, VSS removal was 27% for K WAS and 15% for S
WAS. Several factors might explain these lower values. The tests were run on a batch scale and
there were large errors associated with VSS measurements at the end of the BMP assays. The
granules in the assay bottles settled quickly so achieving good mixing while taking samples for
suspended solids measurement was difficult. The WAS used in the assays was stored for 4 or 5
months in cold storage so there was a possibility that more easily digestible organic solids may
56
have already been digested. There was also the possibility these wastes contained larger
proportions of recalcitrant matter such as lignin than WAS investigated in other studies.
Comparing experiments 4 and 5, there was a large difference in VSS removal between K
WAS and S WAS after anaerobic digestion. One reason for this might have been the large
amount of soluble organic matter present in S WAS. 12% of the COD in S WAS was soluble
and could potentially be more easily digested. Experiment 1 showed that the soluble fraction
was not more easily digestible the total amount of S WAS, but these compounds may still be
preferentially digested over insoluble organic matter. This may explain why less VSS was
removed. More easily digestible soluble organic matter may have been used as a carbon source
first instead of hydrolysis of more complex compounds.
A main reason for the difference in digestibility between S WAS and K WAS was the
source of the aerobic sludges. K WAS was sourced from an ASB system which handles sludge
differently from an activated sludge system. In an ASB system, after sufficient BOD is removed,
excess aerobic sludge is allowed to settle near the exit of the aerated pond and is stored there for
anywhere from months to years. During this time the sludge is in an anoxic zone of the pond
where it slowly undergoes anaerobic digestion. Over time, any readily anaerobically digestible
fraction of the sludge would be digested. Activated sludge systems, such as the one where S
WAS was from, produce a continuous stream of excess sludge that is not stored for extended
periods of time and would likely not undergo any anaerobic digestion prior to sampling.
Another possible reason for the higher overall biodegradability of S WAS over K WAS
was likely the higher iron content of S WAS. In one study examining the anaerobic and aerobic
digestibility of WAS samples from various sources found a very strong positive correlation
between anaerobic biodegradability and iron content (Park, Abu-Orf, & Novak, 2006). Iron
addition to activated sludge systems is often used to improve the settling quality of the bacterial
flocs. In the presence of reducing conditions and sulphide in an anaerobic environment, the iron
is reduced and precipitates out of solution. Since trivalent iron is one of main components that
holds bacterial flocs together, it precipitating out of solution can lead to the breakdown of flocs
(Nielsen & Keiding, 1998). This could also explain why S WAS samples also contained larger
amounts of soluble COD. During storage, the sludge samples may have become anaerobic and
sulphate reduction would have led to the production of sulphide which would cause the
precipitation of reduced iron. The sludge itself appeared black which indicated the presence
precipitated iron sulphide.
57
4.3.2 WAS Digestion Compared to High-Rate Anaerobic Digester Feed
In experiment 1, the anaerobic digestion potential of S WAS was compared to the
digestion potential of the wastewater that was fed to the reactor where the anaerobic granules
used in these experiments were collected (Table 4.3.2). The reactor feed produced considerably
more biogas than S WAS and the soluble fraction of S WAS in this experiment. The reactor feed
was more easily digested than S WAS, which was an expected result since this feed is what the
granules were adapted to digesting.
Table 4.3.2 Biogas yield in experiment 1 subtracting blank control values for S WAS, the soluble fraction of S
WAS, and feed sent to the reactor granules were sampled from.
Exp. # Substrate
Total biogas produced
subtracting blank [mL/mg
substrate COD]
1 S WAS 0.003 ± 0.034
1 Soluble S WAS -0.037 ± 0.023
1 Reactor Feed 0.118 ± 0.034
4.3.3 WAS Toxicity
The toxicity of S WAS to the anaerobic granules was tested in experiment 2. This was
done in order to determine whether the lack of digestion of S WAS in experiment 1 was due to
inherent toxicity or was possibly due to other experimental factors. In experiment 2, the FeS
particles used to reduce the medium were allowed to settle and the medium used to dilute the
granules was free of FeS particles. To test for possible toxicity, two triplicate sets of granules
were fed equal amounts of positive control substrate. To one of those sets, S WAS was also
added, increasing the amount of COD in those bottles by 25%. In essence this added about one
quarter of the S WAS added in experiment 1. If the S WAS was toxic, it would negatively affect
the biogas production when compared with the other positive control.
A single factor experiment analysis of variance found that the difference in the amount of
biogas produced between the positive control and the sample containing S WAS was not
statistically significant (Table 4.3.3). This suggests that the S WAS was not toxic at the
concentration used in this assay. It also suggests that the S WAS was not digested at all as no
58
additional biogas was produced even though more COD was added. The presence of a more
easily degraded substrate seemed to prevent the S WAS from being degraded. This result might
also explain the low biogas production observed in experiment 1 and the lower VSS reduction
found with S WAS when compared to K WAS in experiments 4 and 5. When the granules were
sampled from the IC reactor they were a mixture of anaerobic biomass, nutrients, and the feed
entering the reactor. The feed collected along with the granules remained in each bottle during
storage at 4oC. Over time, the feed could be degraded slowly even at this lower temperature.
Experiment 1 was performed close to the day the granules were sampled and likely contained
more easily digestible reactor feed. This would explain why adding S WAS gave no
improvement in biogas production compared with the blank control: the reactor feed was
digested with preference over the S WAS. The lower VSS reduction of S WAS compared to
KWAS in experiments 4 and 5 could be explained by the same principle. S WAS contained a
larger fraction of soluble organic matter than K WAS. During the digestion of S WAS, the
soluble organic matter was digested preferentially to the VSS present in the waste leading to a
smaller reduction in solids during the digestion.
Table 4.3.3 Total biogas produced during the WAS toxicity BMP assay in experiment 2.
Glucose, Acetate, Propionate mix added
[mg COD] S WAS Added
[mg COD]
Total biogas produced subtracting blank
[mL]
38.9 18.8 ± 0.9
38.9 9.70 20.3 ± 1.3
4.3.4 WAS Digestion without Anaerobic Granules – Self Digestion
Experiment 1 was also performed to test if activated sludge on its own would produce
any biogas. Samples of S WAS, the soluble fraction of S WAS, and reactor feed were added to
medium without any granules present (Table 4.3.4). When compared to the blank value, it is
clear that the WAS samples and reactor feed samples produced negligible amounts of biogas.
This was an expected result as part of the difficulty of dealing with WAS is that it does not
breakdown on its own.
59
Table 4.3.4 Total biogas production for samples containing no granules and the blank control which
contained anaerobic granules but no substrate in experiment 1.
Exp. # Substrate Total biogas
produced [mL]
1 Blank$ 10.2 ± 0.8
1 S WAS 1.6 ± 0.8
1 Soluble S WAS 0.5 ± 0.5
1 Reactor Feed 0.9 ± 0.5
$This sample contained anaerobic granules and H2O as the substrate.
4.4 Pretreatment Effects
4.4.1 NaOH Requirements to Bring WAS to pH 12
Caustic pretreatment required the pH of WAS samples to be at 12. Each sample required
a different amount of NaOH to reach that pH (Table 4.4.1).
Table 4.4.1 NaOH requirements of WAS for caustic pretreatment.
Exp. # Substrate
Amount of NaOH added to make pH 12
[mg/mL]
Amount NaOH added to make pH 12
[mg/mg VSS]
3 K WAS 1.98 0.142
S WAS 1.58 0.218
4 K WAS 1.88 0.134
5 S WAS 2.30 0.317
4.4.2 Physical and Chemical Changes
In experiment 4 and 5 the effects of thermal, caustic, and sonication pretreatment on K
WAS and S WAS were compared. The physical and chemical changes in the WAS after
pretreatment were determined through measuring the changes in properties of the soluble
fraction of WAS. COD, suspended solids, carbohydrates, and proteins were measured (Figure
4.4.1 to Figure 4.4.4). The error bars shown in the graphs represent standard error in the mean of
the data collected.
60
K WAS
0
5
10
15
20
25
30
Untreated Thermal Caustic Sonication
Pretreatment
To
tal
CO
D [
mg
/mL]
Soluble
S WAS
0
5
10
15
20
25
30
Untreated Thermal Caustic Sonication
PretreatmentT
ota
l C
OD
[m
g/m
L]
Soluble
Figure 4.4.1 These graphs show the total and soluble COD before and after pretreatment. This data is from
two experiments (4 and 5) with a total of 5 replicates.
The change in total COD was in the range of the error of measurement (Figure 4.4.1).
There were some small changes in total COD after pretreatments that could have been caused by
oxidation during pretreatment. Large changes in soluble COD were realized suggesting that
solubilisation of the organic matter was achieved. The largest increases in soluble COD
occurred with thermal and caustic pretreatment. The pretreatments had different effects with K
WAS and S WAS. Thermal was less effective than caustic pretreatment at solubilising COD
with K WAS, but with S WAS thermal and caustic pretreatment performed similarly. Sonication
was the least effective pretreatment at solubilising COD with both WAS samples. All
pretreatments solubilised COD of S WAS more effectively than K WAS.
In the case of K WAS, the fraction of the total COD that was soluble increased from
1.3% (untreated) to 25% for thermal pretreatment and 60% for caustic pretreatment. In the case
of S WAS, the fraction of the total COD that was soluble increased from 11% to 67% and 70%
for thermal and caustic pretreatment, respectively. These findings were in the same range of
values as those reported by previous studies examining pretreatment of municipal WAS. One
study comparing thermal and caustic pretreatment found the soluble COD of municipal WAS
increased from 3% (untreated) to 60% with thermal pretreatment (170oC) and to 63% with
61
caustic pretreatment (pH 12 NaOH, 130oC) (Valo et al., 2004). Another study found that the
fraction of soluble COD of a municipal WAS increased from 8% to 17.6% with thermal (121oC)
pretreatment and 85% with caustic (7g/L NaOH, 121oC) pretreatment (Kim et al., 2003). A
study investigating the pretreatment of Kraft mill WAS with caustic pretreatment (2.4g/L NaOH,
ambient temp.) found the soluble fraction of COD increased from 7% to 32% (Navia et al., 2002).
Sonication resulted in the smallest increase in the fraction of soluble COD, but there was
a large difference in solubilisation degree between K WAS and S WAS. The soluble fraction of
COD went up to 5 % in K WAS and 22% in S WAS. These values are in the range of what has
been found in previous studies comparing this pretreatment on municipal WAS with others. One
study looking at pretreatment of municipal WAS found the soluble fraction increased to 45%
with thermal pretreatment (170oC) and 15% for sonication pretreatment (20kHz, 0.45W/mL)
(Bougrier et al., 2006).
K WAS
0
2
4
6
8
10
12
14
16
18
20
Untreated Thermal Caustic Sonication
Pretreatment
Su
spe
nd
ed
So
lid
s [m
g/m
L]
Ash Volatile
S WAS
0
2
4
6
8
10
12
14
16
18
20
Untreated Thermal Caustic Sonication
Pretreatment
Su
spe
nd
ed
So
lid
s [m
g/m
L]
Ash Volatile
Figure 4.4.2 These graphs show the change in total and volatile suspended solids before and after
pretreatment. This data is from two experiments (4 and 5) with a total of 5 replicates.
All pretreatments decreased the total suspended solids in the waste with the exception of
sonication of K WAS (Figure 4.4.2). Thermal and caustic decreased the total amount of
suspended solids the most. In the case of K WAS, VSS was reduced by 28% (thermal) and 40%
(caustic) after pretreatment. In the case of S WAS, VSS was reduced 64% for both thermal and
62
caustic pretreatment. One study comparing thermal and caustic pretreatment of a mixture of
municipal and industrial WAS found VSS decreased by 30% for thermal (180oC) and 45% for
caustic (0.3mg/mL NaOH, pH 12, 130oC) (Tanaka et al., 1997).
Sonication decreased the amount of suspended solids the least out of all the pretreatments.
With K WAS there was no significant decrease in VSS and in the case of S WAS, VSS was
reduced by 32%. This large difference in results between K WAS and S WAS can be explained
by the differences in solid content of the two wastes. The effectiveness of sonication varied
depending on the solids content of a waste (Grönroos et al., 2005; Khanal et al., 2007) . K WAS
had twice as high a concentration of suspended solids than S WAS. The higher concentration of
solids would decrease the propagation of ultrasonic waves and therefore more energy would be
required to solubilise them. The amount of solubilisation of S WAS, however, was in the range
of solubilisation found by previous studies. Sonication of municipal WAS can decrease VS in
the range of 12% to 39% with similar intensity and frequency as used in this study (Kim et al.,
2003).
In all cases, the ash portion of the suspended solids, representing the inorganic fraction,
did not change indicating that mainly organic matter was solubilised. It also appeared that the
pretreatments tested were more effective at reducing solids in S WAS than K WAS. The COD
solubilisation and suspended solids reduction results indicate that insoluble matter in K WAS
was very resistant to the pretreatments.
The total amount of carbohydrates was reduced after thermal and caustic pretreatment
(Figure 4.4.3). This was likely due to the Maillard reactions where carbohydrates and proteins
react in the presence of water (Penaud et al., 1999). Although a significant portion of the
carbohydrates remained insoluble with all the pretreatments, thermal and caustic pretreatments
were the most effective at solubilising the carbohydrates, and sonication was least effective. As
was found with COD solubilisation and suspended solids reduction, the pretreatments were more
effective at solubilising S WAS than K WAS. The solubilisation of carbohydrates indicates the
hydrolysis of large insoluble carbohydrates like cellulose and the destruction of EPS holding
bacterial flocs together.
63
K WAS
0
0.5
1
1.5
2
2.5
Untreated Thermal Caustic Sonication
Pretreatment
Ca
rbo
hy
dra
tes
[mg
/mL]
Soluble
S WAS
0
0.5
1
1.5
2
2.5
Untreated Thermal Caustic Sonication
Pretreatment
Ca
rbo
hy
dra
tes
[mg
/mL]
Soluble
Figure 4.4.3 These graphs show the total and soluble carbohydrates before and after pretreatment. This data
is from two experiments (4 and 5) with a total of 6 replicates.
K WAS
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Untreated Thermal Caustic Sonication
Pretreatment
So
lub
e P
rote
in [
mg
/mL]
S WAS
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Untreated Thermal Caustic Sonication
Pretreatment
So
lub
e P
rote
in [
mg
/mL]
Figure 4.4.4 These graphs show the amount of soluble protein before and after pretreatment. This data is
from two experiments (4 and 5) with a total of 8 or 16 replicates.
64
All pretreatments resulted in large increases in soluble protein concentrations (Figure
4.4.4). Soluble protein has been found to be a good measure of the solubilisation of organic
matter in biological sludges (Khanal et al., 2007), and is correlated to dissociation of EPS and
bacterial cells. The largest increases in soluble protein were found with thermal and caustic
pretreatment with the least improvement seen with sonication. Both S WAS and K WAS
contained similar amounts of total protein, but much more of that protein was made soluble with
S WAS with all pretreatments.
Overall S WAS was solubilised to a greater extent than K WAS. This may be correlated
to why S WAS was more biodegradable than K WAS. K WAS likely contained a higher degree
of non-bacterial matter such as lignin which may be resistant to solubilisation. The reduction of
iron in S WAS during storage may have cause bacterial flocs in the WAS to break down leading
to solids that were more easily broken down.
4.4.3 Anaerobic Digestion
4.4.3.1 Extent of Digestion
The VSS and TSS were measured at the end of experiments 4 and 5 in an attempt to
estimate total suspended solids reduction after anaerobic digestion (Figure 4.4.5).
In experiment 4 it was found there was no statistically significant difference in VSS
removal by anaerobic digestion after pretreatment. This suggests that solids remaining after
pretreatment of K WAS were digestible to the same extent as the solids of the untreated waste.
The improvements in digestibility of VSS may also have been so small as to be within the range
of error of the measurement. In experiment 5, thermal and caustic pretreatment, but not
sonication, led to an increase in VSS digestibility. The pretreatments likely partially hydrolysed
the solids or disrupted their structure to the extent that they were not solubilised, but were made
more accessible to hydrolytic enzymes. The fraction of VSS left undigested in both experiments
likely represented the fraction of suspended solids that either required longer residence times for
digestion or were not biodegradable.
In experiment 5, thermal #2 and caustic #2 both had lower average VSS reductions than
the thermal and caustic samples suggesting a lower amount of hydrolysis occurred in these
samples. This is consistent with the previously mentioned possibility that in the presence of an
easily digestible substrate, the hydrolysis and anaerobic digestion of more complex substrates is
65
greatly reduced. These samples had almost three times the amount of COD as the other samples
in the assay due to the addition of substrate based on VSS content. The presence of large
amounts of soluble COD could have inhibited the hydrolysis of volatile suspended solids. This
could have occurred by substrate inhibition of hydrolytic enzymes. The bacteria producing the
enzymes may also have been discouraged from producing hydrolytic enzymes because of the
presence of readily available substrates for their metabolism.
0%
10%
20%
30%
40%
50%
60%
Untreated Thermal Caustic Sonicated Thermal #2 Caustic #2
Pretreatment
% T
ota
l V
SS
Re
mo
ve
d
K WAS, Experiment 4
S WAS, Experiment 5
Figure 4.4.5 Percent of total VSS removed after anaerobic digestion of untreated and pretreated WAS
samples.
Thermal and caustic pretreatments resulted in more COD being removed during
anaerobic digestion than with untreated samples (Figure 4.4.6). Pretreatment increased the
amount of COD removed during K WAS and S WAS digestion from 7% to 30% and 42% to
over 60%, respectively. This suggests that pretreatment increased the bioavailability of the
organic matter in K WAS and S WAS. However, although caustic pretreatment was more
effective at solubilising COD in K WAS than thermal pretreatment, it did not lead to a
corresponding increase in COD removal. This suggests that some of the oxidizable matter
solubilised by caustic pretreatment was not biodegradable.
66
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
Untreated Thermal Caustic Sonicated Thermal #2 Caustic #2
Pretreatment
% C
OD
Re
mo
ve
d
K WAS, Experiment 4
S WAS, Experiment 5
Figure 4.4.6 Percent of substrate COD removed after anaerobic digestion. Values were calculated based on
the total amount COD required to produce measured methane production. Sample calculations can be found
in Appendix C.
Sonication only slightly improved the overall COD removal. The amount of COD
removed during KWAS digestion increased to just over 10% while in S WAS digestion it was
increased to 45%. These values were consistent with solubilisation results found with K WAS,
but not in the case of S WAS. Although sonication was effective at reducing suspended solids
and solubilising COD in S WAS, the overall amount of COD removed during anaerobic
digestion was not significantly improved. This suggests that the solubilised matter was not
biodegradable.
4.4.3.2 Total Biogas Production and CH4 Content
The effect of pretreatments on biogas production was investigated in the BMP assays
performed in experiment 4 and 5.
Thermal and caustic pretreatment equally improved the biogas production from K WAS
(Figure 4.4.7). A single factor experiment analysis of variance confirmed that there was no
67
statistically significant difference between the improvements resulting from the pretreatments.
Total biogas production was improved by about 280% compared to untreated samples. Even
though caustic pretreatment solubilised a higher fraction of protein and COD, it increased biogas
production to the same extent as thermal pretreatment. This suggests that the caustic
pretreatment produced digestible as well as recalcitrant soluble compounds.
0
0.05
0.1
0.15
0.2
0.25
0 5 10 15 20 25 30 35 40
Time [days]
Cu
mu
lati
ve
Bio
ga
s P
rod
uct
ion
[mL/
mg
su
bst
rate
CO
D]
Untreated K WAS
Thermal
Caustic
Sonication
Thermal #2
Caustic #2
Figure 4.4.7 Cumulative biogas production during experiment 4 (K WAS) subtracting blank control values.
Data points represent the mean of triplicate values and error bars represent the standard error in the mean.
The pretreated K WAS samples compared on the basis of VSS (thermal #2 and caustic
#2) produced the same biogas yield as thermal and caustic samples added on the basis of COD.
These bottles contained 50% more COD. Sonication was the least effective pretreatment and
increased biogas production from K WAS by 65%. Although biogas yield was increased by
pretreatment, VSS reduction was the same in all samples. This suggests the solubilised
components of the WAS were the main source for the measured increases in biogas production.
In experiment 5, thermal pretreatment increased the total biogas yield from S WAS by
50% (Figure 4.4.8). Thermal #2, added on the basis of VSS, contained 3 times as much COD as
the other thermally pretreated sample, but only increased biogas yield by 21%. The higher
68
concentration of substrate in thermal #2 would have resulted in a lower anaerobic biomass to
substrate ratio, which can lead to decreases in biogas yield in BMP assays (Chynoweth et al.,
1993). There was also the possibility that there was substrate inhibition as described previously
or that with high concentrations, some components of the waste were toxic to the anaerobic
microorganisms.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 5 10 15 20 25 30 35 40
Time [days]
Cu
mu
lati
ve
Bio
ga
s P
rod
uct
ion
[mL/
mg
su
bst
rate
CO
D]
Untreated S WAS
Thermal
Sonication
Thermal #2
Figure 4.4.8 Cumulative biogas production during experiment 5 (S WAS) subtracting blank control values.
Data points represent the mean of triplicate values and error bars represent standard error in the mean.
The improvement in biogas yield with caustic pretreatment of S WAS was only 18%,
which was much less than the increase with thermal pretreatment (Figure 4.4.9). Caustic
pretreatment of S WAS produced similar solubilisation results in terms of COD and solids
reduction as thermal pretreatment, but biogas yield was not improved to the same degree.
Caustic #2, added on the basis of VSS, did not produce a statistically different amount of biogas
compared with the untreated sample. This was likely due to same reasons that thermal #2 did not
have as high a biogas yield. However, biogas production rate was higher with thermal #2 and
caustic #2 samples than the untreated S WAS samples, suggesting the pretreatments improved
the reaction rate more than the overall biogas yield.
69
0
0.05
0.1
0.15
0.2
0.25
0.3
0 5 10 15 20 25 30 35 40
Time [days]
Cu
mu
lati
ve
Bio
ga
s P
rod
uct
ion
[mL/
mg
su
bst
rate
CO
D]
Untreated S WAS
Caustic
Sonication
Caustic #2
Figure 4.4.9 Cumulative biogas production during experiment 5 (S WAS) subtracting blank values. Each
data point represents the mean value of a triplicate and error bars represent standard error in the mean.
In experiment 5, sonication showed no statistically significant improvement in overall
total biogas yield (Figure 4.4.8 and Figure 4.4.9). Sonication did appear to improve the rate at
which the biogas was produced at the beginning of the BMP assay. The solubilisation caused by
sonication improved rate rather than increasing the overall biodegradability of S WAS. This is
consistent with some studies of sonication as a pretreatment (Elliott & Mahmood, 2007).
Biogas yield was generally higher for S WAS than K WAS in both untreated and
pretreated samples (Table 4.4.2 and Table 4.4.3). Pretreatment caused a more drastic increase in
biodegradability of K WAS, but overall yields were higher with S WAS. This is consistent with
the physical and chemical characteristics of the WAS samples tested. S WAS contained more
soluble COD, carbohydrates, and proteins. Pretreatment also was more effective at solubilising
organic matter and disrupting solids in S WAS than in K WAS.
The volume fraction of methane was approximately the same for all the biogas produced
within each experiment (Table 4.4.2 and Table 4.4.3). Table 4.4.3 shows values that exceed
100% methane content which is impossible. The high concentrations were likely due to error in
70
measurements of biogas volume. These errors could have occurred due to several reasons
including temperature changes in the bottle during measurement, CO2 equilibrium in the bottle
changing, or other issues.
Table 4.4.2 Total biogas produced, volume fraction of CH4 in the biogas, and substrate COD and VSS added
in experiment #4 where K WAS was digested. Each value represents an average of a triplicate.
Pretreatment
COD Added
[mg]
VSS Added
[mg]
Biogas produced subtracting blank [mL/mg substrate
COD]
Increase in biogas yield compared to untreated sample
vol% CH4 in biogas
Untreated 35.2 18.3 0.045 ± 0.005 – 64% ± 10%
Thermal 35.2 14.0 0.183 ± 0.012 305% 72% ± 9%
Thermal #2 45.9 18.3 0.163 ± 0.007 260% 68% ± 2%
Caustic 35.2 14.6 0.171 ± 0.009 278% 76% ± 8%
Caustic #2 44.3 18.3 0.170 ± 0.005 276% 73% ± 8%
Sonication 35.2 20.6 0.067 ± 0.009 48% 72% ± 3%
Table 4.4.3 Total biogas produced, volume fraction of CH4 in the biogas, and substrate COD and VSS added
in experiment #5 where S WAS was digested. Each value represents and average of a triplicate.
Pretreatment
COD Added
[mg]
VSS Added
[mg]
Biogas produced subtracting blank [mL/mg substrate
COD]
Increase in biogas yield compared to untreated sample
vol% CH4 in biogas
Untreated 35.2 21.9 0.197 ± 0.009 – 85% ± 4%
Thermal 35.2 7.2 0.295 ± 0.008 50% 89% ± 3%
Thermal #2 106.7 21.9 0.238 ± 0.004 21% 109% ± 3%
Caustic 35.2 7.2 0.232 ± 0.011 18% 104% ± 3%
Caustic #2 107.1 21.9 0.203 ± 0.003 3% 107% ± 2%
Sonication 35.2 13.9 0.188 ± 0.019 -4% 98% ± 8%
It is difficult to compare biogas production values with literature as a variety of different
units and measures are reported and no studies have examined digestion of pretreated pulp and
paper mill WAS. For instance, after 7 days of anaerobic treatment, the yield of biogas from
municipal WAS increased from 0.132 mL/mg COD for untreated WAS to 0.175 mL/mg COD
(thermal at 121oC), 0.182 mL/mg COD (caustic, 7 g/L NaOH, 121oC), and 0.159 mL/mg COD
(sonication at 42 kHz) (Kim et al., 2003). Another study found that after a 23 day anaerobic
digestion, biogas yield for pretreated municipal WAS increased 45% for a thermal pretreatment
(170oC) and similarly for caustic pretreatment (130oC at pH 12) (Valo et al., 2004). Finally, a
71
study of a rapid thermal pretreatment of municipal WAS at a full-scale anaerobic digester
increased methane production by over 40% (Dohányos et al., 2004). It appears that with
pretreatment of pulp and paper mill WAS, similar or better improvements in biogas and methane
yield can be realized compared to pretreatment of municipal WAS.
Sonication pretreatment was much less effective in this study compared with other
studies. One study on sonicated municipal sludge reported increases in biogas production from
150% to 300% (Mao & Show, 2006). Another study comparing several pretreatments found
methane yield to improve from 0.221 mL CH4/mg COD for untreated sludge to 0.325 mL/mg
COD with thermal pretreatment (170oC) and 0.333 mL/mg COD with sonication (20kHz,
0.45W/mL) (Bougrier et al., 2006). This corresponds to an increase of around 50%. It appears
that pulp and paper mill WAS may be less amenable to sonication pretreatment than municipal
WAS. This result may be due to inherent differences in the content of these two wastes. The
cellulose and lignin contained within K WAS and S WAS may not have been affected by
sonication and likely would have remained intact after pretreatment. The difference in
pretreatment effectiveness between sludge samples may also be due to limitations in equipment
used in this study. Although the sonicating reactor used in this study provided similar conditions
to lab-scale experiments described in previous studies, it could not achieve the high intensities
that commercialized ultrasonic systems are capable of.
4.4.3.3 Biogas Production Rate
As described in the methodologies section, the COD consumption during the BMP assays
was fit to first-order reaction kinetics. The following equation was used to describe the kinetics
of COD utilization:
YCOD = Y0 * (1 – e-k * t
)
Where YCOD represents the cumulative amount of COD used at time t, Y0 is the total
amount of COD used at t equal to infinity, t is the time in days, and k is the reaction rate.
72
Table 4.4.4 Summary of first-order reaction kinetics regressions. R2 represents the coefficient of
determination and goodness of fit of the regressions. Y0 represents the use of COD at time at infinity and k
represents the reaction rate constant.
Exp. # Substrate Pretreatment k [days-1]
Y0 [mg COD utilized / mg substrate COD
added] R2
4 Glucose, Acetate, and Propionate
1.023 1.260 0.95
K WAS Untreated 0.0006 2.519 0.77
Thermal 0.169 0.287 0.93
Thermal #2 0.194 0.249 0.96
Caustic 0.200 0.264 0.94
Caustic #2 0.227 0.255 0.94
Sonication 0.098 0.103 0.80
5 Glucose, Acetate, and Propionate
0.807 1.257 0.89
S WAS Untreated 0.010 1.477 0.98
Thermal 0.113 0.649 0.99
Thermal #2 0.098 0.680 0.98
Caustic 0.091 0.613 0.98
Caustic #2 0.073 0.605 0.98
Sonication 0.088 0.449 0.97
All regressions show good agreement with the data except in the case of untreated K
WAS and sonicated K WAS in experiment 4 (Table 4.4.4). This was likely due to a large
amount of variation in methane production measured between replicates. It could also be due to
the complexity of the waste, but only those two samples out of all tested had poor correlations.
In experiment 4 the methane production rate for K WAS exposed to thermal or caustic
pretreatment was increased over 30 times. This brought the reaction rate to within one order of
magnitude of the positive control. Sonication also increased the methane production rate, but not
as significantly as the other pretreatments.
In experiment 5 the methane production rates with S WAS were increased about 10 times
with all pretreatments. In this experiment sonication seemed much more effective at improving
the reaction rate with S WAS than with K WAS. Untreated S WAS had a higher methane
production rate than untreated K WAS again suggesting that the S WAS tested was a much more
digestible substrate.
73
Upon inspection of the data, there appeared to be two phases of digestion for untreated as
well as thermal and caustic pretreated S WAS. This was especially prominent under conditions
labelled thermal #2 and caustic #2 in experiment 5, which were prepared on the basis of equal
VSS (Figure 4.4.10). The larger amount of COD added to these bottles likely amplified the
differences between these phases of digestion.
0
0.1
0.2
0.3
0.4
0.5
0.6
0 5 10 15 20 25 30 35 40
Time [days]
CO
D C
on
sum
ed
[mg
CO
D/m
g s
ub
stra
te C
OD
at
sta
rt]
BMP Data
Regression 1
Regression 2
Figure 4.4.10 COD consumption for caustic #2 pretreated S WAS in experiment 5. The lines represent two
first-order kinetics regressions performed on different parts of the data to account for the different kinetics.
COD consumption was calculated based on the theoretical amount of COD required to produced measured
amounts of methane.
The regressions were performed again on each of these phases of digestion separately
with first-order reaction kinetics (Table 4.4.5). In all cases the first phase of digestion had a
slower reaction rate than the second phase. In the case of S WAS, after thermal and caustic
pretreatment, the reaction rates of each phase individually were much higher than that of all the
data regressed together. The reaction rates were of the same order of magnitude as the positive
control.
74
Table 4.4.5 Summary of regressions performed on different portions of the data from experiment 5 on S
WAS. Range of regression refers to the portion of the data to which the regression was applied.
Exp # Substrate Pretreatment
Range of Regression
[days] k [days-1]
Y0
[mg COD utilized / mg substrate COD
added] R2
5 S WAS Untreated 0 - 5.4 0.008 1.087 0.91
Untreated 5.4 - 34 0.015 1.061 0.96
Untreated 0 - 34 0.010 1.477 0.98
Thermal #2 0 - 4.4 0.281 0.255 0.97
Thermal #2 4.4 - 34 0.765 0.597 0.93
Thermal #2 0 - 34 0.098 0.680 0.98
Caustic #2 0 - 4.4 0.432 0.134 0.95
Caustic #2 4.4 - 34 0.491 0.511 0.97
Caustic #2 0 - 34 0.073 0.605 0.98
4.5 Comparison of WAS Properties to Pretreatment Performance
The potential for methane production from the soluble COD in each sample was
calculated to determine how much the solubilised COD contributed to the increases in methane
production resulting from pretreatment (Figure 4.5.1). This calculation was based on the amount
of methane that could theoretically be produced if all soluble COD was consumed. This
calculation is similar to that used to determine COD consumption in the previous section and is
described in further detail in Appendix C.
The methane potential of the soluble COD varied in comparison to the actual amount of
methane produced in experiment 4 (K WAS) and experiment 5 (S WAS). In untreated samples,
soluble COD accounted for about 20% of the total methane production. Thermal and caustic
samples had much higher soluble COD content and could potentially produce much more
methane than was actually produced during the experiments. In the case of caustic pretreatment
in experiment 4, the methane potential of the soluble COD was nearly double the actual amount
of methane that was produced. In this case, caustic pretreatment resulted in compounds that
were not anaerobically digestible. In the case of the sonicated samples, soluble COD accounted
for approximately 40% and 60% of actual methane produced for K WAS and S WAS,
respectively.
75
K WAS
0
50
100
150
200
250
300
350
400
450
Untreated Thermal Caustic Sonicated
Pretreatment
To
tal
Me
tha
ne
Pro
du
ced
[µ
mo
l]Actually Produced
Potentially from Soluble COD
S WAS
0
50
100
150
200
250
300
350
400
450
Untreated Thermal Caustic Sonicated
PretreatmentT
ota
l M
eth
an
e P
rod
uce
d [
µm
ol]
Actually Produced
Potentially from Soluble COD
Figure 4.5.1 Cumulative methane production in experiment 4 (K WAS) and 5 (S WAS) compared with the
potential for methane production based on soluble COD content.
The methane potential of soluble carbohydrates and proteins was also determined. The
methane potential of each fraction was determined based on COD content. Carbohydrates were
assumed to have the same COD content as glucose: 1.07g COD/g. Proteins were assumed to
have the same COD content as bovine serum albumin: 1.22g COD/g. The COD content of
bovine serum albumin was calculated based on its amino acid content (Hirayama, Akashi,
Furuya, & Fukuhara, 1990) and resulting carbon, hydrogen, nitrogen, and oxygen ratio:
C3.8H7.4NO1.9.
A portion of the net increase in methane production after pretreatment could be
associated with the increase in soluble carbohydrates and soluble proteins in pretreated samples
(). In experiment 4 (K WAS), soluble protein could have accounted for 20% to 30% of the
increase in methane production for pretreated samples. Soluble carbohydrates could have
accounted for approximately 10% of the increase in methane production. In experiment 5 (S
WAS), soluble protein of thermal and caustic samples could have accounted for up to 60% of the
increase in methane production and soluble carbohydrates could have accounted for up to 15% of
the increase. Sonication produced less methane than would be expected from soluble proteins
and soluble carbohydrates.
76
The increases in methane production were not completely accounted for by increases in
soluble protein and carbohydrate content. The remaining increases in methane production could
have come from solubilised lipid matter, nucleic acids, or volatile fatty acids.
K WAS
0
20
40
60
80
100
120
140
160
180
Thermal Caustic SonicatedPretreatment
Me
tha
ne
Pro
du
ced
[µ
mo
l]
Soluble Carbohydrates
Soluble Protein
Other compounds
S WAS
0
20
40
60
80
100
120
140
160
180
1 2 3
Pretreatment
Me
tha
ne
Pro
du
ced
[µ
mo
l]
Total Produced After
Sonication
Figure 4.5.2 Net increase in methane produced compared with untreated samples during anaerobic digestion
showing the methane potential of soluble carbohydrate and protein content. Sonicated S WAS produced less
methane than predicted by methane potential of soluble.
Upon inspection of the data, linear relationships were observed in some cases when
biogas produced by assay bottles was plotted versus the physical and chemical components of
samples added to each bottle (Figure 4.5.3 and Figure 4.5.4). All measured values were
inspected separately with biogas production in this manner to determine if other linear
relationships existed. Each component of WAS had a different degree of correlation to a linear
regression (Table 4.5.1).
77
y = 6.02x + 3.63
R2 = 0.96
y = 4.52x + 1.49
R2 = 0.90
0
5
10
15
20
25
30
0 0.5 1 1.5 2 2.5 3 3.5 4
Soluble Carbohydrates Content [mg]
To
tal
Bio
ga
s P
rod
uct
ion
[m
L]
K WAS
S WAS
Figure 4.5.3 Regression of total biogas production versus soluble carbohydrate content of assay bottles in
experiment 4 and 5.
y = 0.25x + 4.12
R2 = 0.95
y = 0.20x + 2.64
R2 = 0.63
0
5
10
15
20
25
30
0 20 40 60 80 100
Soluble COD Content [mg]
To
tal
Bio
ga
s P
rod
uct
ion
[m
L]
K WAS
S WAS
Figure 4.5.4 Regression of total biogas production versus soluble COD content of assay bottles in experiment
4 and 5.
78
The parameter most highly correlated with biogas yield in a linear model for both
experiments was soluble carbohydrates. The poorest was suspended solids. Experiment 4
showed poor correlations with soluble COD and soluble protein, while experiment 5 had strong
correlations with COD, soluble COD, and soluble protein. This suggests that the soluble COD in
K WAS was not as biodegradable as soluble COD in S WAS. When all the data from both
experiments were combined, soluble COD and soluble carbohydrates were strongly correlated
with total biogas yield.
Table 4.5.1 Summary of the slope, intercept, and correlation coefficient of regressions of total biogas
production versus the contents of each assay bottle. “All Data” refers to the data set from both experiment 4
and experiment 5.
Substrate Measurement Correlated with Biogas Production
Data
Linear Regression Parameter
Total COD [mg]
Soluble COD [mg]
VSS [mg]
TSS [mg]
Total Carb. [mg]
Soluble Carb. [mg]
Total Protein
[mg]
Soluble Protein
[mg]
Slope 0.34 0.20 -0.44 -0.07 2.63 4.52 1.84 2.58
Intercept -8.04 2.64 12.62 6.76 -2.43 1.49 -8.19 1.75
Exp. 4, K WAS
R2 0.45 0.63 0.18 0.01 0.13 0.90 0.64 0.80
Slope 0.22 0.25 0.64 0.61 3.69 6.02 0.72 1.68
Intercept 0.39 4.12 3.17 -0.12 0.29 3.63 0.12 3.95
Exp. 5, S WAS
R2 0.95 0.95 0.32 0.58 0.84 0.96 0.96 0.94
Slope 0.25 0.27 0.37 0.48 4.15 6.48 0.73 1.78
Intercept -2.92 2.71 2.99 -1.79 -4.04 1.42 -0.17 3.08
All Data
R2 0.87 0.93 0.07 0.24 0.69 0.91 0.94 0.94
The solubilisation of biological macromolecules is theoretically the best indicator for
biogas yield. COD only provides a general measurement of oxidizable matter with no indication
of biodegradability. Soluble carbohydrates are known to be readily biodegradable by anaerobic
bacteria and should be well correlated to biogas production. Few other studies have looked at
the correlation of measured parameters with biogas or methane production. In (Bougrier et al.,
2006), it was found that the percent soluble COD had a linear correlation with biogas production,
but that correlation was very different for thermal and sonicated municipal WAS. In a study
looking at municipal landfill solids (Y. S. Wang, Byrd, & Barlaz, 1994), it was found that total
carbohydrates did not correlate to biogas production, which is consistent with the findings in this
study. In studies looking at biogas production from marine and terrestrial biomass, it has been
79
found that soluble carbohydrates and soluble protein are highly correlated to methane yields
(Chynoweth & Isaacson, 1987).
4.6 Energy Balance
Simple energy balances were performed to compare improvement in energy production
in the form of biogas with energy requirements of each pretreatment. For thermal and caustic
pretreatments, WAS was considered to have the same specific heat capacity as water over the
temperature range 5oC to 170oC: 4.22 kJ kg-1 K-1 (Perry & Green, 1997). The only energy
requirement for these pretreatments was assumed to be the heat required to increase the
temperature of the WAS from 5oC to the temperature used in the pretreatment.
The energy required for sonication was calculated based on the energy input and the time
required for sonication. Two different values were used for the energy input required for
sonication. One of the values was based on the total energy applied to the sonication reactor
during the course of the pretreatment. The other value was based on previous measurements
performed with the same sonication reactor to determine the actual energy transferred to the
WAS in the form of ultrasonic waves.
The total energy produced was based on the total biogas produced from the BMP assays.
The energy from biogas was based on an average heating value for biogas from sewage:
25.2 J/mL biogas at STP (Deublein & Steinhauser, 2008). The equations and sample
calculations for the energy balance can be found in Appendix C.
Thermal and caustic showed the most improvement in energy production versus the
amount of energy required (Table 4.6.1 and Table 4.6.2). In the case of S WAS, 270% to 295%
net increases in energy in the form of biogas were observed. However, the heat that could be
recovered during the thermal and caustic pretreatment was not taken into account in this energy
balance. Nor was the need to cool the WAS before it is sent to anaerobic digestion. This cooling
can be applied by incoming WAS on its way to the pretreatment, greatly decreasing the overall
energy requirements.
Sonication appeared to use a lot of energy, even when taking into account only the
amount of energy that was actually applied to WAS. Pretreatment with sonication would appear
to run at a net energy loss. This result is likely not representative of full-scale installations of
sonication systems. Previous studies of a full-scale SonixTM sonicating pretreatment found that
the system was effective at pretreating WAS before anaerobic digestion (Hogan et al., 2004).
80
The manufacturer of the SonixTM system reports an energy consumption of 4.5MJ/m3 (Wong,
2005), which is significantly lower than the sonication requirements of the lab-scale reactor used
in the experiments conducted in this study.
Table 4.6.1 Energy requirements and increase in energy from biogas as a result of pretreatment of K WAS in
experiment 4. Sonication (actually applied to WAS) is based on the actual amount of energy passed to the
WAS through sonication.
Pretreatment
Energy required for pretreatment
[MJ / m3 WAS]
Total energy from biogas
produced [MJ/m3 WAS digested]
Net increase in energy
from biogas [MJ/m3 WAS
digested]
Net percent change in energy output
after pretreatment
None 0 25.4
Thermal 0.624 100.8 75.5 295%
Caustic 0.496 94.3 69.0 270%
Sonication (input electricity)
2520 38.1 12.7 -9891%
Sonication (actually applied to WAS)
343 38.1 12.7 -1304%
Table 4.6.2 Energy requirements and increase in energy from biogas as a result of pretreatment of S WAS in
experiment 5. Sonication (actually applied to WAS) is based on the actual amount of energy passed to the
WAS through sonication.
Pretreatment
Energy required for pretreatment
[MJ / m3 WAS]
Total energy from biogas
produced [MJ/m3 WAS digested]
Net increase in energy
from biogas [MJ/m3 WAS
digested]
Net percent change in energy output
after pretreatment
None 0 54.8
Thermal 0.624 82.4 27.6 49%
Caustic 0.496 74.5 19.7 35%
Sonication (input electricity)
2100 58.7 3.95 -3826%
Sonication (actually applied to WAS)
286 58.7 3.95 -515%
4.7 Economic Analysis of Pretreatments
An economic analysis was performed for thermal and caustic pretreatment in terms of
operating costs versus potential savings associated with biogas production and decreased
81
amounts of sludge requiring disposal. Sonication was excluded from this analysis since the
experimental data showed it produced very little improvement in biogas production and energy
consumption by the sonication reactor used in this study was much higher than most commercial
systems.
The analysis was performed assuming a plant with an existing high-rate anaerobic
digester on site followed by an aerobic biological wastewater treatment plant (Figure 4.7.1).
WAS would be first sent to pretreatment then dewatered. During dewatering, solids would be
separated and removed for disposal while the solubilised organic matter would be sent to the
high-rate anaerobic digester. This arrangement was chosen since: i) the methane potential assays
in this study were performed on sludge before dewatering so the data is most applicable in this
scenario; ii) the pretreatments tested were previously reported to improve dewatering of sludge
which would lead to the main savings associated with sludge disposal; and iii) UASB and other
types of high-rate digesters require low solids concentrations for effective operation so
dewatering after pretreatment could separate solids from solubilised organic matter. This is only
one possible arrangement for pretreatment, but is applicable for industrial or municipal
installations of high-rate anaerobic digesters.
Figure 4.7.1 Assumed process arrangement of pretreatment and dewatering used for economic analysis of
operating costs.
Wastewater
Secondary
Sludge (WAS)
Sludge Recycle
Aeration Tank
Effluent
High-Rate
Anaerobic
Digester
Pretreatment
Secondary
Settling
Dewatering
Biogas
Sludge solids
for disposal
Soluble fraction of
sludge for digestion
82
The economic analysis was based on two scenarios for the disposal of WAS. The first
scenario was that the WAS would be dewatered then sent to a boiler where it would be burned
along with other solid residues. The second scenario was that the WAS would be dewatered and
sent to a landfill site directly. Mill A provided some data associated with the costs of disposal of
WAS and plant operations. Many of the costs associated with the disposal of WAS are highly
variable and can differ greatly depending on sludge properties and methods of disposal.
Table 4.7.1 Assumed values used to calculate biogas production from soluble fraction of S WAS. These
values were based on the results obtained from experiment 5 in this study.
Variable Thermal Caustic
Soluble COD Content [kg COD/m3 WAS] 8.46 9.68
Solids Reduction 54.8% 54.3%
Biogas yield after pretreatment [m3/kg COD] 0.295 0.232
NaOH Added [kg/m3 WAS] 2.30
Table 4.7.2 Assumed values for variables used in the economic analysis. Values were based on estimates
provided by Mill A. All values in Canadian dollars.
Variable Assumed Value
Total dewatering cost [tonne solids-1] $146.00
Natural gas cost [GJ-1] $14.00
Biogas worth [m-3] $0.29
Steam cost [GJ-1] $17.50
NaOH [kg-1] $0.23
Pretreatments were assumed to be as effective as those performed on S WAS in this study
(Table 4.7.1). It was assumed that only the soluble COD was sent to the anaerobic digester and
that it had the same overall biogas yield as the whole waste. Energy requirements for
pretreatment were based on the values calculated in the energy balance for this study. Two
scenarios were used to calculate the energy requirements for pretreatment. One scenario
involved no heat recovery after pretreatment and the second assumed that 75% of the heat used
to heat the incoming WAS was provided by the WAS leaving the pretreatment. It was also
assumed that all heating requirements were satisfied by 190oC steam produced at the mill. Steam
cost was based on the amount of natural gas required to produce it. The savings associated with
83
dewatering were calculated by assuming the cost of dewatering was proportional to the solids
content of the waste. In the case of the thermal and caustic pretreatment, a reduction in solids of
55% was realized, leading to a 55% reduction in dewatering costs. Biogas savings were
calculated based on the biogas yields found for pretreated S WAS determined in this study. The
worth of biogas was based on methane content in comparison with natural gas Assuming biogas
contained 74% methane, its heating value would be 74% that of natural gas, which would make
it worth 74% the price of natural gas (Table 4.7.2).
4.7.1 WAS Disposal through Combustion
The addition of WAS to the boiler results in the requirement of supplementary fuel to be
added to maintain proper boiler operation. This is mainly due to the water content of dewatered
sludge. The cost associated with sending WAS to a boiler was based on the amount of added
fuel that was not going to generate steam because it was required to burn the WAS. This loss in
steam production was estimated at Mill A to be 3 tonnes of steam per tonne of dewatered WAS
solids sent to the boiler per day.
Savings associated with boiler operation were calculated in two scenarios. The first
assumed that dewatering efficiency was not improved by pretreatment meaning the solids
content of the dewatered sludge was around 30%. Any savings associated with the boiler would
then be based on the decrease in WAS solids requiring disposal. The second scenario assumed
that dewatering efficiency was improved by the pretreatment to the point where the addition of
WAS to the boiler would not lead to any loses in steam production. This scenario may be
possible as both thermal and caustic treatment have been found to greatly enhance the
dewatering of sludges (Gurjar, 2001). In this scenario there was no cost associated with sending
the WAS to the boiler for disposal.
Thermal and caustic pretreatment both produced savings with respect to WAS disposal
(Table 4.7.3). In the case where no heat was recovered and dewatering of solids was not made
more efficient the cost of dewatering was decreased by 77% and 50% by thermal and caustic
pretreatment, respectively. Heat recovery did not change the net savings for the pretreatments.
This was likely due to relatively low amount of energy required. Improvement in dewatering
efficiency, however, greatly improved the net savings. In the case of thermal pretreatment, the
cost of disposal of WAS decreased by 99%. Caustic pretreatment seemed to be substantially
more expensive in terms of chemical costs. The lower temperature allowed by caustic
84
pretreatment did not affect the costs of operation significantly. The lower temperatures, however,
may be more significant for capital cost as the system would be able to run at a lower pressure.
If it is assumed that 2 000 m3 of WAS is produced per day, net annual savings associated
with thermal and caustic pretreatment with no improvement in dewatering would be $1.7 million
and $1.1 million, respectively. The net savings would be compared to WAS disposal without
any anaerobic digestion and with all the WAS being sent to the boiler. The CambiTM thermal
pretreatment system costs around $3 million for a turn-key installation (Elliott & Mahmood,
2007). This would lead to a 2 or 3 year payback on investment. The savings presented here may
be an overestimation as operational costs associated with equipment maintenance, pumping, and
any added personnel required to run the process were not included.
Table 4.7.3 Economic analysis of thermal and caustic pretreatment in the case sludge solids were disposed of
in a boiler. All values represent Canadian dollars per m3 of WAS produced requiring pretreatment. Heat
recovery refers to 75% of the energy used during pretreatment being recycled. Improved dewatering refers
to the scenario where pretreatment improves the solid content of dewatered sludge to the point where the
solids do not require supplemental fuel for burning.
Improved Dewatering
Pretreatment None Thermal Caustic Thermal Caustic
WAS solids dewatering cost $1.62 $0.73 $0.74 $0.73 $0.74
WAS disposal cost in boiler $1.48 $0.67 $0.68 $0.00 $0.00
Steam cost without heat recovery
$0.011 $0.009 $0.011 $0.009
Steam cost with heat recovery
$0.003 $0.002 $0.003 $0.002
Caustic cost $0.76 $0.76
Biogas production $0.72 $0.64 $0.72 $0.64
Net cost of disposal without heat recovery
-$3.10 -$0.70 -$1.54 -$0.03 -$0.87
Net cost of disposal with heat recovery
-$3.10 -$0.69 -$1.54 -$0.02 -$0.86
4.7.2 WAS Disposal by Landfill
Land filling is a frequently used option for the disposal of WAS solids. Landfill costs
can range from $4 to over $20 per cubic metre of dewatered sludge depending on the mill and
location. Mill A reports that their land fill fees will be increasing to over $12 per cubic metre in
the coming years. Two scenarios for the economic analysis were calculated because of the wide
85
range in landfill costs. The first scenario used the lower value for landfill costs of $4 per cubic
metre of dewatered sludge and the second scenario used the higher value of $20 per cubic metre
of dewatered sludge.
It was further assumed that dewatering led to an increase in solids concentration from 3%
to 30% leading to a 10 times reduction in volume of sludge requiring land filling. Savings
associated with each pretreatment were based on the percent reduction in solids found in this
study with S WAS in experiment 5. A 55% decrease in solids content after pretreatment led to a
55% decrease in total volume of WAS sent to landfill.
Table 4.7.4 Economic analysis of thermal and caustic pretreatment in the case sludge solids were disposed of
by land filling. All values represent Canadian dollars per m3 of WAS produced requiring pretreatment.
Heat recovery refers to 75% of the energy used during pretreatment being recycled.
Landfill Cost:
$4 m-3 dewatered WAS Landfill Cost:
$20 m-3 dewatered WAS
Pretreatment None Thermal Caustic None Thermal Caustic
WAS solids dewatering cost $1.621 $0.733 $0.741 $1.621 $0.733 $0.741
Landfill costs $0.400 $0.180 $0.18 $2.000 $0.90 $0.91
Energy costs without heat recovery
$0.011 $0.009 $0.011 $0.011 $0.009
Energy costs with heat recovery
$0.003 $0.002 $0.003 $0.003 $0.002
Caustic cost $0.000 $0.759 $0.000 $0.000 $0.759
Biogas production $0.716 $0.643 $0.716 $0.716 $0.643
Net cost of disposal without heat recovery
-$2.02 -$0.21 -$1.048 -$2.92 -$0.932 -$1.780
Net cost of disposal with heat recovery
-$2.02 -$0.20 -$1.042 -$2.91 -$0.923 -$1.773
Thermal and caustic pretreatment both resulted in substantial savings in disposal and
dewatering costs (Table 4.7.4). The cost associated with the energy required for pretreatment
was very low compared with the costs associated with caustic addition and dewatering and
disposal of WAS. This resulted in a higher savings associated with thermal pretreatment than
caustic pretreatment. A larger savings was calculated when assuming the lower range of landfill
cost compared to the higher range. This is due to the fact that when landfill costs are high, they
make up a larger proportion of the total cost of disposal of WAS. With the lower landfill cost the
cost of disposal of WAS was decreased by 90% and 48% for thermal and caustic pretreatment,
86
respectively. With the higher landfill cost the cost of disposal of WAS decreased by 55% and
12% after thermal and caustic pretreatment, respectively.
If it is assumed that 2 000 m3 of WAS is produced per day and the landfill cost is in the
high range, then the net annual savings associated with thermal and caustic pretreatment would
be $1.4 million and $0.8 million, respectively. These values are relative to WAS disposal
without any anaerobic digestion and all dewatered material being disposed of in a landfill. This
savings is lower than that associated with the savings associated with burning the WAS in the
boiler.
87
5.0 Conclusion
5.1 Summary
This study investigated the potential to increase the rate and extent of anaerobic
bioconversion of pulp mill waste aerobic biological treatment sludge (WAS) to biogas. Three
pretreatment technologies were tested in these experiments: i) thermal pretreatment performed at
170oC; ii) thermochemical (caustic) pretreatment performed at pH 12 and at a temperature of
140oC; and iii) sonication performed at 20kHz at an energy density of 1W/mL.
Anaerobic digestion of Sulphite (S) WAS and Kraft (K) WAS was performed using
microbial granules obtained from a high-rate anaerobic digester operating at a pulp and paper
mill. The yield of biogas from pulp mill WAS that was not pretreated was similar to that
obtained from municipal and industrial sludges. Specifically, biogas production was
approximately 0.05 mL/mg COD for K WAS and 0.20 mL/mg COD for S WAS.
The three pretreatments tested solubilised organic matter and reduced the solids content
of the WAS to different extents. Thermal and caustic pretreatments were the most effective at
solubilising COD and reducing suspended solids. In the case of K WAS, soluble COD was
increased from 1% to 25% and 60% for thermal and caustic pretreatment, respectively. In the
case of S WAS, soluble COD was increased from 11% to just under 70% for both pretreatments.
The VSS in K WAS were reduced by 28% and 40% by thermal and caustic pretreatment,
respectively. Both pretreatments reduced the amount of VSS in S WAS by 64%. Thermal and
caustic pretreatments also produced corresponding increases in soluble carbohydrate and protein
content indicating the destruction of bacterial flocs in the WAS. Sonication was the least
effective pretreatment. The amount of COD solubilised by sonication was 5% with K WAS and
22% with S WAS. Sonication did not reduce the VSS content of K WAS, but reduced it by 32%
with S WAS.
Thermal and caustic pretreatment also performed the best at increasing biogas yield and
biogas production rate. The yield of biogas from K WAS increased by 280% with thermal and
caustic pretreatment, while for S WAS the yield of biogas increased by 50% and 18% for
thermal and caustic pretreatment, respectively. Both pretreatments increased the biogas
production rate by approximately 300 times for K WAS and 10 times for S WAS. Sonication
resulted in much less improvement to overall biogas yield: 65% for K WAS and 0% for S WAS.
88
Sonication increased biogas production rate by 150 times for K WAS and 8 times for S WAS.
Overall, S WAS was a much more readily biodegradable waste than K WAS.
The increase in biogas yield corresponded to increases in COD removal by anaerobic
digestion. The digestion of thermal and caustic pretreated WAS led to COD reductions of 30%
for K WAS and over 60% for S WAS. The digestion of sonicated K WAS and S WAS led to the
removal of 10% and 30% of the COD, respectively.
The improvements in methane produced could be partially accounted for by increases in
soluble carbohydrate and soluble protein content based on calculated methane potential.
Methane potential of the soluble COD in some pretreated samples was larger than that actually
produced suggesting non-digestible soluble matter was produced as a result of the pretreatment.
A linear relationship was also found between soluble carbohydrate content of the sludges and
total biogas production.
Simplistic energy balances and economic analyses were performed on the pretreatments
to determine the overall benefits of pretreatment based on data from this study. Thermal and
caustic pretreatment were both found to be net energy positive processes and show potential to
significantly reduce the cost associated with WAS disposal.
5.2 Implications
In this study, thermal pretreatment was the most effective at improving anaerobic
digestion performance. For both S WAS and K WAS, thermal pretreatment resulted in the
greatest increase in biogas yield and production rate. Thermal pretreatment has also been proven
to be a good method to improve the dewatering of biological wastewater solids. It has also been
commercialized as a pretreatment for municipal WAS. These characteristics suggest thermal
pretreatment is an attractive option for an anaerobic digestion system.
Caustic showed similar results to thermal pretreatment and had the added benefit of
requiring lower temperatures. Lower temperatures mean that corresponding full-scale equipment
would not need to withstand as high pressures as those required for thermal pretreatment. One
negative aspect to caustic pretreatment, however, was that it led to a larger amount of soluble
recalcitrant compounds in the pretreated WAS. In an industrial application, this would mean
soluble COD passing through an anaerobic digester remaining undigested.
Sonication was the least effective preatreatment; it improved the rate of biogas
production but not the overall yield. This result does not completely preclude sonication as a
possible pretreatment since the effectiveness of this technology varies greatly between conditions
89
and reactor configurations. Commercialized sonication equipment has been shown to be
effective at improving biogas yield and rate with lower power requirements than other
pretreatments.
S WAS was found to be much more anaerobically biodegradable than K WAS. K WAS
likely contained a larger fraction of non-biodegradable organic matter such as lignin. During
long periods of storage in an ASB system, a large fraction of anaerobically biodegradable
material in K WAS would have already been digested. Iron content was also much higher in S
WAS. Iron content has been found to increase the anaerobic biodegradability of sludges because
the reduction of iron by sulphide under anaerobic conditions leads to destruction of sludge flocs.
The addition of iron in activated sludge systems to improve flocculation may also improve
pretreatment and anaerobic digestion of resulting sludge.
5.3 Recommendations
To verify and expand on the conclusions presented in this study it is recommended that
the following studies be undertaken:
1. Dewaterability of pretreated WAS should be investigated as any improvements in
dewaterability would be an added benefit to the pretreatment process.
2. Anaerobic digestion tests should be run on the soluble fractions of pretreated WAS
samples. This would simulate the digestion potential of filtrates produced after the
dewatering of WAS.
3. Pretreatments should be run on dewatered sludge samples. Previous studies have
found some pretreatments are more effective when wastes are more highly
concentrated. Dewatering sludge before pretreatment would also require smaller
equipment due to smaller volumes of material that would require processing.
4. Pretreated WAS samples should be digested in lab-scale continuous high-rate
anaerobic digesters to examine WAS effects on granule formation and retention. This
would give a better indication of potential biogas yield in a full-scale reactor.
90
5. A more thorough economic analysis should be performed involving sizing of and
capital costs of equipment to better estimate the savings potential of pretreatment and
anaerobic digestion of WAS.
91
6.0 Abbreviations
ASB – aerobic stabilization basin
BCTMP – bleached chemi thermo mechanical pulp
BMP – biochemical methane potential
BOD – biological oxygen demand
COD – chemical oxygen demand
CSTR – continuously-stirred tank reactor
EPS – extracellular polymers
IC – internal circulation
K WAS – Kraft mill waste aerated basin secondary sludge
r.c.f. – relative centrifugal force
STP – standard temperature and pressure
S WAS – sulphite mill waste activated sludge
TKN – total Kjeldahl nitrogen
TS – total solids
TSS – total suspended solids
UASB – uplfow anaerobic sludge blanket
VS – volatile solids
VSS – volatile suspended solids
WAS – waste activated sludge or waste aerobic stabilization basin sludge
92
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98
PRETREATMENT OF PULP MILL WASTEWATER TREATMENT RESIDUES TO
IMPROVE THEIR ANAEROBIC DIGESTION
APPENDICES
by
Nicholas Wood
© Copyright by Nicholas Wood 2008
99
Table of Contents
Table of Contents.......................................................................................................................... 99
List of Tables .............................................................................................................................. 100
List of Figures ............................................................................................................................. 102
1.0 Appendix A: Raw Data................................................................................................... 103
1.1 Biomass Properties...................................................................................................... 104
1.1.1 ANALEST Carbon Hydrogen Nitrogen Analysis .............................................. 104
1.1.2 SGS Metals Analysis .......................................................................................... 104
1.2 Experiment 1............................................................................................................... 107
1.3 Experiment 2............................................................................................................... 109
1.4 Experiment 4............................................................................................................... 110
1.5 Experiment 5............................................................................................................... 113
2.0 Appendix B: Experimental Methods .............................................................................. 116
2.1 Procedure for Preparing Basic Mineral Medium........................................................ 117
2.1.1 Stock Solution Preparation ................................................................................. 117
2.1.2 Procedure for Making Nutrient Medium ............................................................ 120
2.2 Calibration Curves ...................................................................................................... 123
2.2.1 Pressure Transducer Calibration ......................................................................... 123
2.2.2 GC Calibration .................................................................................................... 124
2.3 Lowry Protein Modifications...................................................................................... 126
3.0 Appendix C: Sample Calculations .................................................................................. 128
3.1 COD-Based Calculations ............................................................................................ 129
3.1.1 BMP Assay Substrate Concentration.................................................................. 129
3.1.2 Positive Control Substrate Concentrations ......................................................... 129
3.1.3 COD Removal Based on Methane Production ................................................... 130
3.2 Methane Content Mass Balance.................................................................................. 131
3.3 Protein Concentration ................................................................................................. 133
3.4 Energy Balance ........................................................................................................... 134
3.4.1 Thermal and Caustic Pretreatment...................................................................... 134
3.4.2 Sonication Pretreatment ...................................................................................... 135
3.4.3 Biogas Energy Content ....................................................................................... 135
4.0 References....................................................................................................................... 136
100
List of Tables
Table 1.1.1 Elemental analysis of carbon, hydrogen, and nitrogen content of biomass samples
used in this study. All percentages refer to percent of total dry solids. ..................................... 104
Table 1.2.1 Summary of substrates for assay bottles in experiment 1. Positive control with no
H2 had headspace flushed with N2/CO2 gas mix before being put into the incubator. The test was
performed to see if H2 in headspace of bottle would effect biogas production. ......................... 107
Table 1.2.2 Summary of properties of biomass used in experiment 1........................................ 107
Table 1.2.3 Cumulative biogas production values in mL of biogas for experiment 1................ 108
Table 1.2.4 Cumulative methane production values in µmol of methane for experiment 1....... 108
Table 1.3.1 Summary of substrates for assay bottles in experiment 2. 25% S WAS refers to the
addition of an additional 25% of substrate in terms of COD to a second control. ..................... 109
Table 1.3.2 Summary of properties of biomass used in experiment 2........................................ 109
Table 1.3.3 Cumulative biogas production subtracting blank values in mL of biogas for
experiment 2................................................................................................................................ 109
Table 1.4.1 Summary of BMP assay bottle substrates for experiment 4. ................................... 110
Table 1.4.2 Summary of properties of biomass used in experiment 4. "Amount added to bottle"
refers to the amount of each substrate added to assay bottles during the experiment. ............... 110
Table 1.4.3 VSS and TSS remaining in each assay bottle at the end of the BMP assay in
experiment 4................................................................................................................................ 111
Table 1.4.4 Cumulative biogas production subtracting blank values in mL of biogas for
experiment 4................................................................................................................................ 112
Table 1.4.5 Cumulative methane production subtracting blank values in µmol of methane for
experiment 4................................................................................................................................ 112
Table 1.5.1 Summary of BMP assay bottle substrates for experiment 5. ................................... 113
Table 1.5.2 Summary of properties of biomass used in experiment 5. "Amount added to bottle"
refers to the amount of each substrate added to assay bottles during the experiment. ............... 113
Table 1.5.3 VSS and TSS remaining in each assay bottle at the end of the BMP assay in
experiment 5................................................................................................................................ 114
Table 1.5.4 Cumulative biogas production subtracting blank values in mL of biogas for
experiment 5................................................................................................................................ 115
Table 1.5.5 Cumulative methane production subtracting blank values in µmol of methane for
experiment 5................................................................................................................................ 115
101
Table 2.2.1 Raw data used for pressure transducer calibration. Syringe size refers to the size of
the glass syringe used to add air to the headspace of the serum bottle. ...................................... 123
Table 2.2.2 Bottle setup information about bottles used for methane calibration curve. Bottle 1
was completely filled with 99% methane calibration gas. All other bottles had a certain volume
of gas from bottle 1 added to their headspace............................................................................. 125
102
List of Figures
Figure 2.2.1 Calibration curve for pressure transducer used in biogas measurements............... 123
Figure 2.2.2 Calibration curve of GC peak area versus serum bottle headspace methane
concentration............................................................................................................................... 125
Figure 2.3.1 Absorbance as a function of lignin concentration when Lowry assay reagents are
added in the presence and absence of copper. ............................................................................ 126
103
1.0 Appendix A: Raw Data
104
1.1 Biomass Properties
1.1.1 ANALEST Carbon Hydrogen Nitrogen Analysis
The following is the raw data received for the carbon hydrogen nitrogen elemental
analysis performed by ANALEST labs at the University of Toronto. Anaerobic Granules refers
to the granules sampled on September 2007.
Table 1.1.1 Elemental analysis of carbon, hydrogen, and nitrogen content of biomass samples used in this
study. All percentages refer to percent of total dry solids.
K WAS S WAS Anaerobic Granules
1 2 1 2 1
C 45.54% 45.47% 43.96% 43.94% 46.03%
H 5.37% 5.26% 5.46% 5.49% 5.42%
N 3.93% 3.81% 5.79% 5.85% 7.02%
1.1.2 SGS Metals Analysis
Below are the results received from SGS for the analysis of biomass samples. S WAS
refers to WAS from Mill A, K WAS refers to excess sludge from Mill B, and An Gran refers to
the anaerobic granules samples from Mill A’s IC reactor on September 2007. Data includes a
complete metals analysis, organic and inorganic nitrogen analysis, sulphur content analysis, and
reactive phosphorus analysis.
105
106
107
1.2 Experiment 1
Table 1.2.1 Summary of substrates for assay bottles in experiment 1. Positive control with no H2 had
headspace flushed with N2/CO2 gas mix before being put into the incubator. The test was performed to see if
H2 in headspace of bottle would effect biogas production.
Bottle # Substrate Granules Added
1 – 3 H2O (Blank) Yes
4 – 6 S WAS Yes
7 – 9 S WAS No
10 – 12 Soluble fraction of S WAS Yes
13 – 14 Soluble fraction of S WAS No
16 – 18 IC reactor feed Yes
19 – 21 IC reactor feed No
22 – 24 Glucose + Acetate (Positive Control) Yes
25 – 27 Glucose + Acetate (Positive Control, No H2) Yes
Table 1.2.2 Summary of properties of biomass used in experiment 1.
Substrate
Total COD
[mg/mL]
Soluble COD
[mg/mL]
VSS [mg/mL],
[mg/g]
TSS [mg/mL],
[mg/g]
Amount Added to
Assay Bottles [mL]
H2O 0.00 0.00 0.00 0.00 15.00
Glucose + Acetate 2.98 2.98 0.00 0.00 15.00
S WAS 12.95 2.84 9.78 10.60 2.65
Sol. S WAS 2.84 2.84 0.00 0.00 12.07
Reactor Feed 7.90 7.90 0.00 0.00 4.34
Granules (Bottle 1) 15.83
Granules (Bottle 2) 2.29 0.98 1.24 85.00
Granules (Sample Bottle) 67.66 29.07 36.62 2.88
108
Table 1.2.3 Cumulative biogas production values in mL of biogas for experiment 1. Bottle #, [mL biogas]
Time
Since
Start
[h] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 16 17 18 19 20 21 22 23 24 25 26 27
5.6 0.0 0.0 1.4 0.0 0.0 3.8 0.0 3.1 0.0 3.0 1.4 1.2 0.0 0.9 4.5 1.6 4.5 0.0 1.2 1.3 5.6 5.9 5.9 10.0 8.8 9.8
14.3 0.0 0.0 1.4 0.0 0.0 3.8 0.0 3.1 0.0 3.0 1.4 1.2 0.0 0.9 4.5 1.6 4.9 0.0 1.2 1.5 13.6 14.9 15.3 18.0 17.3 18.4
29.4 0.0 0.0 1.4 0.0 0.0 3.8 0.0 3.1 0.0 3.2 1.4 1.2 0.0 0.9 4.5 2.1 5.5 0.0 1.2 1.5 18.9 19.8 19.4 22.2 21.8 7.0
42.3 0.0 0.0 1.4 0.0 0.0 3.8 0.0 3.1 0.0 3.2 1.4 1.2 0.0 0.9 4.5 2.1 5.5 0.0 1.2 1.5 20.0 20.4 20.4 22.2 22.3 21.9
91.1 1.5 1.5 2.2 1.2 1.6 4.6 0.8 3.2 0.3 4.5 2.4 2.2 0.0 0.9 7.8 5.1 8.0 0.0 1.2 1.5 24.2 25.0 25.0 27.2 27.6 26.9
140.4 3.0 2.7 3.6 2.1 2.2 5.2 0.8 3.2 0.3 4.8 2.4 2.3 0.0 0.9 8.8 5.9 8.5 0.0 1.2 1.5 28.0 27.8 28.1 30.0 30.1 29.6
187.6 3.0 2.7 3.6 2.9 3.4 6.0 0.8 3.2 0.3 4.8 3.2 2.9 0.0 0.9 8.8 6.8 10.0 0.0 1.2 1.5 28.5 27.8 28.1 30.8 30.6 30.1
260.7 3.9 3.7 3.8 5.6 6.1 8.5 0.8 3.2 0.8 8.3 5.7 5.1 0.0 0.9 12.1 9.4 12.9 0.0 1.2 1.5 32.2 30.8 31.1 34.1 33.6 33.1
333.2 5.7 5.6 7.3 6.2 6.3 10.3 0.8 3.2 0.8 8.3 7.1 6.1 0.0 0.9 13.1 10.0 14.1 0.0 1.2 1.6 32.8 31.4 32.1 35.9 34.7 35.0
423.4 7.4 7.8 9.7 8.0 8.8 11.0 0.8 3.2 0.8 9.5 7.8 8.8 0.0 0.9 14.1 11.8 15.0 0.0 1.2 1.6 35.8 32.3 36.3 36.4 36.3 37.0
497.8 7.4 7.8 9.9 8.0 8.8 11.8 0.8 3.2 0.8 9.5 7.8 8.8 0.0 0.9 14.1 11.8 15.0 0.0 1.2 1.6 35.8 33.1 36.3 37.4 37.0 37.0
645.1 10.1 9.0 11.6 9.0 10.0 12.0 0.8 3.2 0.8 9.5 8.6 8.8 0.0 0.9 15.1 12.5 15.2 0.0 1.2 1.6 36.7 35.0 36.9 39.0 38.1 38.7
Table 1.2.4 Cumulative methane production values in µmol of methane for experiment 1. Bottle #, [µmol CH4]
Time
Since
Start
[h] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 16 17 18 19 20 21 22 23 24 25 26 27
5.6 13.5 12.7 13.8 17.1 17.4 15.4 0.0 0.0 0.0 10.6 21.8 10.7 0.0 0.0 106.3 102.1 101.3 0.0 0.0 0.0 203.3 196.5 221.2 271.4 250.1 267.3
14.3 22.4 21.0 22.1 23.6 26.6 24.1 0.0 0.0 0.0 16.8 29.3 16.9 0.0 0.0 129.7 127.1 127.8 0.0 0.0 0.0 416.9 426.2 467.1 489.0 481.9 503.6
29.4 36.1 33.3 35.0 37.4 39.3 35.8 0.0 2.5 0.0 26.8 39.5 25.8 0.0 0.0 159.2 161.0 157.0 0.0 0.0 0.0 528.7 572.2 587.8 592.3 592.0 487.1
42.3 44.9 40.2 47.4 47.8 43.0 51.5 2.5 4.1 0.0 35.5 46.8 33.2 0.0 0.0 182.2 168.9 171.9 0.0 0.0 0.0 567.3 579.6 606.0 633.2 606.2 639.7
91.1 109.6 100.1 103.2 96.3 100.6 90.5 3.0 4.7 1.0 71.0 85.8 69.9 0.7 0.9 134.9 253.1 260.4 0.7 1.5 1.4 716.2 743.5 770.6 819.9 822.4 791.7
140.4 155.4 146.7 152.6 136.6 152.6 140.4 4.6 5.7 1.8 103.7 112.1 99.1 1.2 0.0 313.5 317.3 307.8 1.2 12.6 12.7 812.2 822.4 855.5 859.5 878.6 892.4
187.6 216.8 186.2 202.9 191.8 211.7 178.3 5.8 6.9 2.7 133.0 162.6 120.8 2.5 1.8 361.8 341.1 358.7 12.8 13.0 12.4 902.1 872.6 941.2 978.5 912.1 961.0
260.7 292.4 285.6 297.7 254.4 280.8 253.9 8.9 10.9 5.2 215.5 228.8 202.3 2.7 1.9 456.2 416.9 438.5 12.9 13.5 13.0 979.3 978.0 973.9 1072.9 1057.2 1014.3
333.2 343.8 322.6 332.7 295.2 323.1 293.3 12.9 16.3 9.5 256.8 270.1 253.6 2.9 2.1 498.1 472.3 479.4 12.1 12.4 12.5 986.3 994.7 1026.4 1067.0 1065.6 1040.5
423.4 404.3 374.8 400.2 368.1 396.4 376.5 26.1 29.9 23.9 314.1 336.8 312.6 4.9 4.1 567.4 544.0 524.2 13.7 13.2 14.2 1065.7 1015.6 1152.9 1162.2 1131.3 1121.0
497.8 449.1 415.7 445.2 420.1 439.9 401.8 42.3 49.2 42.1 343.9 369.4 341.9 5.3 9.1 581.7 576.7 546.5 14.6 16.5 14.8 1078.5 1049.4 1166.3 1191.7 1156.5 1126.8
645.1 568.4 523.0 523.0 502.8 517.8 496.3 71.1 94.2 84.0 411.4 448.5 413.7 6.0 13.8 662.2 633.8 616.7 20.0 18.4 17.8 1124.7 1205.5 1254.2 1278.9 1249.2 1222.5
109
1.3 Experiment 2
Table 1.3.1 Summary of substrates for assay bottles in experiment 2. 25% S WAS refers to the addition of an
additional 25% of substrate in terms of COD to a second control.
Bottle # Substrate
1 – 3 H2O (Blank)
4 – 6 Glucose + Acetate (Positive Control)
10 – 12 S WAS
14 – 15 Glucose + Acetate (Positive Control, + 25% S WAS)
Table 1.3.2 Summary of properties of biomass used in experiment 2.
Substrate
Total COD
[mg/mL]
VSS [mg/mL],
[mg/g]
TSS [mg/mL],
[mg/g]
Amount Added to
Assay Bottles [mL]
H2O 0 0 0 15
Glucose + Acetate 7.77 0 0 5
S WAS 15.12 10.51 11.41 2.57
S WAS (25%) 0.64
Granules (Bottle 1) 10.02
Granules (Bottle 2) 85
Granules (Sample Bottle) 37.47 49.05 1.54
Table 1.3.3 Cumulative biogas production subtracting blank values in mL of biogas for experiment 2. Bottle #, [mL biogas]
Time Since
Start [h] 1 2 3 4 5 6 10 11 12 14 15
11.2 1.5 1.7 1 9 10 9.2 1.1 1.6 1.6 11.2 9.3
23.0 2.1 2.3 2 14.2 14.8 14.8 2.4 2.4 2.5 14.8 13.6
35.0 2.1 2.3 2 15.3 15.6 15.4 2.4 2.4 2.5 14.8 14.7
81.5 2.1 3.1 2.5 16.1 17.4 17.2 3.6 3.2 3.2 16.7 16.1
131.2 4.2 4.2 4 18.9 20.4 20.7 5.3 4.8 4.9 19.5 20.8
179.0 4.8 5 4.4 21.4 22.2 22.2 6.3 6.3 5.9 20.3 22.5
298.9 5.9 6.3 5.1 24 24.3 24.6 8.3 8.1 7.8 22.7 24.6
442.8 5.9 6.3 5.9 24 25 25.2 9.5 9.6 9.9 22.7 25.1
635.3 6.5 7.8 6.7 25 26.7 27 11.7 12.4 12.6 24.9 27.6
803.3 8.7 10.3 9.2 26.8 28.5 29.3 14.7 15.5 16.4 27.9 30.2
1016.7 8.7 10.3 9.2 26.8 28.5 29.3 16.3 17.9 16.8 28.4 30.9
110
1.4 Experiment 4
Table 1.4.1 Summary of BMP assay bottle substrates for experiment 4.
Bottle # Substrate
Basis for Amount of
Substrate Added
1 – 3 H2O (Blank) Blank
4 – 6 Glucose + Acetate (Positive Control) COD
7 – 9 K WAS COD
10 – 12 Thermal K WAS COD
13 – 15 Caustic K WAS COD
16 – 18 Sonicated K WAS COD
19 – 21 Thermal K WAS (Thermal #2) VSS Equal to K WAS
22 – 24 Caustic K WAS (Caustic #2) VSS Equal to K WAS
Table 1.4.2 Summary of properties of biomass used in experiment 4. "Amount added to bottle" refers to the
amount of each substrate added to assay bottles during the experiment.
Substrate
Total
COD
[mg/mL]
Soluble
COD
[mg/mL]
VSS
[mg/mL],
[mg/g]
TSS
[mg/mL],
[mg/g]
Total
Carbs.
[mg/mL]
Soluble
Carbs.
[mg/mL]
Total
Protein
[mg/mL]
Soluble
Protein
[mg/mL]
Amount
Added
to Bottle
[mL]
H2O 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 10.00
Glucose +
Acetate 2.94 2.94 0.00 0.00 1.65 1.65 0.00 0.00 10.00
K WAS 26.76 0.26 13.93 17.85 2.01 0.01 4.58 0.01 1.32
Thermal K
WAS 24.03 6.44 9.59 12.69 1.78 0.59 4.58 0.84 1.47
Thermal K
WAS (#2) 1.91
Caustic K
WAS 24.19 14.10 10.01 14.82 1.65 0.76 4.58 1.32 1.46
Caustic K
WAS (#2) 1.83
Sonicated K
WAS 25.11 1.13 14.66 18.33 2.07 0.15 4.58 0.33 1.40
Granules
(Bottle 1) 29.97 2.43 28.62 35.98 1.62 0.08 10.96 0.09 1.63
Granules
(Bottle 2) 0.72 0.03 0.41 0.54 0.04 0.00 90.00
Granules
(Sample
Bottle )
61.04 4.95 58.28 73.28 3.29 0.16 22.33 0.17 0.80
111
Table 1.4.3 VSS and TSS remaining in each assay bottle at the end of the BMP assay in experiment 4.
Bottle # VSS Remaining [mg] TSS Remaining [mg]
1 17.5 19.1
2 26.1 30.9
3 32.6 35.1
4 31.7 41.7
5 36.7 44.0
6 34.0 33.7
7 45.0 59.5
8 49.5 50.0
9 47.5 20.5
10 49.0 59.7
11 42.0 56.4
12 25.2 51.2
13 37.2 49.2
14 43.6 56.0
15 36.0 45.0
16 40.0 37.3
17 44.7 45.3
18 35.3 14.0
19 52.0 60.0
20 45.2 34.4
21 48.0 56.0
22 40.5 42.0
23 40.5 36.0
24 37.0 35.5
112
Table 1.4.4 Cumulative biogas production subtracting blank values in mL of biogas for experiment 4. Bottle #, [mL biogas]
Time Since
Start [h] 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
6.98 2.07 2.52 1.90 -0.18 -0.08 0.11 0.13 0.04 -0.14 -0.04 -0.10 -0.09 -0.21 -0.21 0.00 -0.06 0.44 -0.72 -0.63 -0.41 -0.22
15.92 11.37 9.70 8.84 -0.08 0.07 0.18 0.68 0.66 0.50 0.56 0.64 0.72 0.36 0.30 0.78 0.90 1.45 0.23 0.45 0.77 0.89
30.17 17.48 14.95 14.42 0.00 0.08 0.27 1.20 1.16 0.93 1.13 1.19 1.22 0.50 0.58 0.96 1.48 2.09 0.90 1.15 1.40 1.47
54.17 19.18 16.55 16.01 -0.04 0.01 0.21 1.54 1.42 1.23 1.46 1.49 1.51 0.43 0.53 1.23 1.83 2.53 1.35 2.11 2.31 2.36
79.17 19.63 16.97 16.45 -0.06 0.04 0.19 1.72 1.52 1.37 1.65 1.63 1.65 0.36 0.56 1.22 2.01 2.40 1.42 2.25 2.59 2.55
103.67 20.12 17.41 16.96 0.00 0.12 0.21 2.11 1.99 1.74 2.02 2.25 2.19 0.33 0.55 1.25 2.69 2.84 2.12 2.84 3.13 3.39
127.75 20.42 17.64 17.33 0.01 0.09 0.25 2.38 2.20 1.99 2.21 2.42 2.44 0.47 0.67 1.38 2.98 3.26 2.30 3.07 3.43 3.53
175.42 20.93 18.08 17.86 0.05 0.11 0.15 2.81 2.73 2.42 2.67 3.12 3.03 0.34 0.60 1.33 3.68 3.71 3.11 3.69 4.02 4.55
222.92 21.34 18.42 18.32 0.18 0.44 0.37 3.07 3.47 2.94 2.92 3.42 3.27 0.66 0.88 1.65 4.25 4.50 3.51 4.19 4.55 4.65
296.08 21.95 18.87 18.86 0.29 0.40 0.26 3.60 4.01 3.41 3.39 4.04 3.84 0.65 0.89 1.53 4.97 4.95 4.47 4.66 5.02 5.72
367.25 22.45 19.43 19.40 0.49 0.86 0.61 4.07 5.00 3.99 3.72 4.46 4.24 1.08 1.25 2.08 5.70 5.93 4.85 5.31 5.67 5.95
464.67 23.01 19.87 19.99 0.70 0.86 0.48 4.71 5.66 4.57 4.26 5.15 4.88 1.11 1.26 1.90 6.41 6.26 5.75 5.76 6.14 6.94
571.33 23.54 20.48 20.57 0.99 1.39 0.95 5.33 6.72 5.30 4.67 5.59 5.49 1.58 1.76 2.49 7.23 7.23 6.25 6.45 6.91 7.16
658.50 24.03 20.98 20.99 1.19 1.71 1.30 5.85 6.93 5.78 5.17 6.13 5.99 1.89 2.06 2.77 7.65 7.68 6.82 6.68 7.27 7.62
826.42 24.50 21.12 21.14 1.40 1.91 1.47 6.08 7.30 5.99 5.42 6.49 6.17 1.99 2.08 3.01 7.77 7.83 6.84 7.08 7.82 7.75
Table 1.4.5 Cumulative methane production subtracting blank values in µmol of methane for experiment 4. Bottle #, [µmol CH4]
Time Since
Start [h] 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
6.98 75.43 65.88 97.47 7.56 9.19 9.35 22.71 20.15 16.48 18.30 19.30 15.42 13.56 13.91 9.91 22.92 21.07 20.33 20.30 21.77 16.99
15.92 335.31 345.41 340.26 0.87 1.35 -1.91 25.26 26.97 26.75 22.11 23.73 22.74 9.16 8.31 5.85 31.97 34.79 25.81 28.82 30.64 26.22
30.17 585.23 545.52 573.75 1.50 1.97 -1.12 45.34 45.44 38.87 44.89 43.87 30.05 8.35 14.20 -5.96 38.88 56.55 54.13 49.83 53.43 44.40
54.17 631.46 638.19 634.44 4.03 6.23 4.40 66.98 62.69 59.65 63.55 58.00 64.22 25.61 24.65 18.30 78.14 76.74 65.57 74.67 84.01 79.15
79.17 656.31 653.90 650.63 1.73 0.76 -6.13 70.91 68.89 70.10 65.56 74.97 71.20 24.89 17.66 20.79 88.54 89.19 78.05 94.39 97.98 80.86
103.67 675.52 628.52 663.04 1.32 -0.74 -2.15 88.36 83.70 80.27 89.03 86.27 88.30 28.32 24.45 18.85 105.76 107.36 95.02 117.06 116.47 112.23
127.75 687.71 664.13 685.55 1.39 1.09 -2.18 100.92 96.40 93.49 100.61 102.81 98.38 28.35 25.55 22.86 118.82 123.17 110.06 129.15 131.49 124.02
175.42 686.20 661.39 686.02 3.39 1.34 -3.82 108.03 105.67 107.28 103.76 111.73 105.82 30.97 28.35 18.08 132.46 132.61 116.23 137.95 137.68 131.55
222.92 713.78 695.37 676.89 3.89 6.81 3.62 122.89 117.52 103.65 115.29 121.12 112.98 33.58 36.30 31.18 147.80 150.68 133.32 143.05 160.65 146.81
296.08 694.63 691.50 708.92 11.94 8.08 -1.19 130.67 120.05 125.52 119.24 116.55 118.14 37.04 31.82 32.01 160.07 155.01 149.29 158.33 153.13 144.09
367.25 720.48 707.05 690.78 16.08 13.23 1.55 144.45 137.09 132.29 128.76 129.68 116.76 42.22 33.64 35.74 161.28 170.94 147.77 159.66 171.71 143.42
464.67 705.42 678.45 711.49 23.97 15.04 10.93 151.39 156.90 133.60 129.63 133.76 131.27 48.40 45.66 35.27 167.55 167.99 165.25 166.92 173.93 163.92
571.33 701.82 706.21 656.81 29.96 20.93 14.36 168.01 156.52 142.36 135.24 151.83 144.24 54.98 52.90 50.85 180.65 189.47 166.40 183.36 176.94 165.41
658.50 638.79 715.00 696.19 24.66 23.63 28.34 179.38 167.16 140.76 142.23 150.76 164.77 62.71 51.42 39.93 182.12 173.28 193.40 192.11 184.83 161.34
826.42 708.60 753.68 703.66 32.63 30.72 39.84 202.59 156.36 161.04 185.08 154.40 169.97 56.30 60.38 72.20 199.42 216.95 174.10 224.77 219.46 170.98
113
1.5 Experiment 5
Table 1.5.1 Summary of BMP assay bottle substrates for experiment 5.
Bottle # Substrate
Basis for Amount of
Substrate Added
1 – 3 H2O (Blank) Blank
4 – 6 Glucose + Acetate (Positive Control) COD
7 – 9 S WAS COD
10 – 12 Thermal S WAS COD
13 – 15 Caustic S WAS COD
16 – 18 Sonicated S WAS COD
19 – 21 Thermal S WAS (Thermal #2) VSS Equal to S WAS
22 – 24 Caustic S WAS (Caustic #2) VSS Equal to S WAS
Table 1.5.2 Summary of properties of biomass used in experiment 5. "Amount added to bottle" refers to the
amount of each substrate added to assay bottles during the experiment.
Substrate
Total
COD
[mg/mL]
Soluble
COD
[mg/mL]
VSS
[mg/mL],
[mg/g]
TSS
[mg/mL],
[mg/g]
Total
Carbs.
[mg/mL]
Soluble
Carbs.
[mg/mL]
Total
Protein
[mg/mL]
Soluble
Protein
[mg/mL]
Amount
Added
to Bottle
[mL]
H2O 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 10.00
Glucose +
Acetate 2.50 2.50 0.00 0.00 1.71 1.65 0.00 0.00 10.00
S WAS 11.69 1.36 7.26 8.67 0.81 0.06 3.94 0.02 3.01
Thermal S
WAS 12.20 8.71 2.50 3.80 0.64 0.39 3.94 1.34 2.89
Thermal S
WAS (#2) 8.75
Caustic S
WAS 13.51 9.57 2.76 4.16 0.84 0.40 3.94 1.39 2.61
Caustic S
WAS (#2) 7.93
Sonicated S
WAS 13.10 3.91 5.19 6.42 0.87 0.25 3.94 1.02 2.69
Granules
(Bottle 1) 39.95 1.58 27.06 35.11 2.17 0.08 10.28 0.08 1.85
Granules
(Bottle 2) 0.11 -0.03 0.56 0.69 0.04 0.00 90.00
Granules
(Sample
Bottle )
86.77 3.43 58.77 76.25 4.71 0.18 22.33 0.17 0.85
114
Table 1.5.3 VSS and TSS remaining in each assay bottle at the end of the BMP assay in experiment 5.
Bottle # VSS Remaining [mg] TSS Remaining [mg]
1 34.0 46.0
2 34.0 46.0
3 31.3 48.7
4 44.3 62.7
5 30.7 39.3
6 40.3 53.0
7 51.3 72.0
8 66.0 89.3
9 66.0 82.7
10 44.8 59.2
11 32.4 52.8
12 36.4 38.4
13 34.4 41.2
14 49.0 63.5
15 36.5 46.0
16 60.0 73.0
17 48.0 58.0
18 52.0 66.5
19 73.3 92.7
20 61.3 80.0
21 66.7 85.3
22 57.3 71.3
23 66.0 83.3
24 67.3 86.0
115
Table 1.5.4 Cumulative biogas production subtracting blank values in mL of biogas for experiment 5. Bottle #, [mL biogas]
Time Since
Start [h] 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
6.25 2.19 1.64 1.73 0.30 0.18 0.11 0.57 0.27 0.44 -0.07 -0.04 -0.35 0.26 0.25 0.14 0.60 0.11 -0.01 0.82 0.75 0.48
15.00 8.21 6.97 7.28 0.47 0.35 0.14 1.17 0.86 1.01 0.25 0.34 -0.01 0.85 0.71 0.75 1.63 0.95 0.88 1.43 1.21 0.87
31.42 14.49 13.71 13.68 0.69 0.53 0.26 1.89 1.58 1.72 0.78 0.88 0.47 1.27 1.10 0.70 3.86 2.58 2.66 2.94 3.02 2.64
55.50 15.61 15.04 14.75 0.95 0.85 0.44 2.53 2.16 2.39 1.12 1.44 0.88 1.82 1.51 1.14 4.79 3.78 3.84 3.58 3.89 3.51
80.75 16.05 15.44 15.13 1.00 1.03 0.50 3.31 2.87 3.11 1.42 2.04 1.34 2.26 1.68 1.38 6.00 4.75 4.97 4.08 4.39 3.97
104.75 17.00 16.29 15.93 1.17 1.29 0.67 4.04 3.62 3.94 2.01 2.79 2.11 2.66 1.73 1.77 7.94 6.65 6.96 4.94 5.18 4.74
129.00 17.30 16.77 16.48 1.35 1.35 0.77 4.60 4.10 4.34 2.34 3.11 2.44 2.83 1.95 1.94 9.59 8.23 8.75 5.64 6.23 5.58
178.58 17.87 17.32 17.15 2.10 2.17 1.50 6.03 5.51 5.75 3.55 4.34 3.67 3.46 2.30 2.58 14.45 13.11 13.64 9.69 10.22 9.49
235.50 18.44 17.70 17.52 2.70 2.66 1.91 7.38 6.40 6.62 4.37 5.10 4.43 4.00 2.76 3.12 18.42 17.25 17.47 13.70 13.40 13.43
297.75 19.03 18.19 18.04 3.52 3.40 2.68 8.14 7.15 7.50 5.01 5.84 5.08 4.75 3.43 3.93 20.51 19.38 19.52 16.22 15.98 16.05
370.75 19.69 18.66 18.47 4.15 4.10 3.30 8.69 7.66 8.19 5.60 6.45 5.61 5.40 4.00 4.42 21.83 20.65 20.88 17.81 17.55 17.44
465.17 20.05 19.08 19.02 4.82 4.67 3.85 9.35 8.28 8.71 6.10 6.99 6.06 5.80 4.47 5.01 22.79 21.60 21.77 18.50 18.03 18.14
567.42 20.97 19.42 19.36 5.73 5.54 4.64 9.87 8.87 9.43 6.89 7.87 6.77 6.63 4.65 5.71 24.50 23.26 23.45 20.27 19.85 19.74
660.33 21.68 19.87 19.98 6.68 6.37 5.44 10.51 9.61 9.81 7.35 8.54 7.23 7.15 5.06 6.23 25.52 24.04 24.19 21.34 20.86 20.59
824.25 22.08 20.09 20.25 7.46 7.00 6.32 10.91 10.00 10.25 7.86 8.95 7.64 7.77 5.42 6.69 26.34 24.89 25.05 22.36 21.61 21.23
Table 1.5.5 Cumulative methane production subtracting blank values in µmol of methane for experiment 5. Bottle #, [µmol CH4]
Time Since
Start [h] 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
6.25 84.75 52.59 70.93 3.10 2.72 1.59 13.76 10.17 7.62 6.09 10.49 2.84 9.56 8.60 5.96 12.93 6.78 12.95 0.87 1.97 1.38
15.00 290.11 250.18 265.08 4.60 0.38 0.61 35.52 32.34 33.78 27.47 31.88 23.48 32.10 28.24 31.92 64.81 61.34 63.36 40.25 41.63 33.41
31.42 580.53 560.84 524.49 7.62 5.40 6.00 60.23 58.27 57.17 48.52 47.96 46.23 45.20 46.06 45.08 162.21 156.00 155.91 118.74 125.23 107.07
55.50 589.71 552.90 549.55 14.85 10.72 7.27 83.86 78.25 74.89 59.75 67.56 56.53 60.08 57.49 61.10 193.65 185.26 199.21 145.34 134.87 142.57
80.75 623.14 578.55 569.87 18.92 18.77 10.15 113.86 111.32 104.45 79.51 97.89 55.18 79.18 78.53 71.18 245.73 238.20 254.07 173.54 172.21 165.00
104.75 620.73 590.69 604.65 19.22 18.51 18.33 145.02 122.64 123.89 91.85 121.03 92.42 83.43 79.74 87.53 311.45 315.39 302.20 191.26 201.55 173.38
129.00 662.90 643.31 611.94 27.56 26.85 20.54 168.93 153.95 155.67 119.68 138.75 123.13 97.14 98.29 94.37 412.65 404.39 415.02 243.89 247.82 233.88
178.58 667.51 582.04 600.44 63.91 61.43 53.44 238.99 215.86 225.19 187.94 202.73 179.96 123.13 121.04 116.80 657.52 625.46 650.62 427.13 445.18 430.07
235.50 658.91 656.47 660.21 87.33 68.42 70.13 252.04 230.69 242.99 207.67 209.83 196.23 135.79 136.22 130.13 772.60 768.34 808.64 582.92 599.60 555.25
297.75 727.51 660.90 661.48 111.50 108.50 97.63 270.17 266.22 258.20 230.31 237.26 218.12 156.37 153.59 162.55 866.99 840.80 840.57 655.38 702.14 653.83
370.75 709.54 693.59 647.97 138.15 122.58 118.88 289.06 278.12 267.88 242.02 257.43 232.18 182.36 170.37 177.08 895.59 872.30 867.71 708.48 690.44 702.21
465.17 749.80 752.63 699.47 173.93 136.84 139.80 321.96 291.51 324.99 282.08 287.61 257.89 197.12 174.03 196.86 966.84 937.06 1012.35 786.27 821.85 749.36
567.42 784.49 711.25 733.79 180.63 166.03 160.04 335.69 327.31 319.44 297.77 305.86 278.67 224.42 206.97 218.49 1020.15 1008.62 962.07 843.81 810.29 807.92
660.33 813.76 745.89 714.94 213.34 186.00 185.64 350.85 335.40 336.68 305.60 316.61 295.73 234.95 209.60 225.66 1028.61 1051.60 1032.44 884.19 842.06 829.61
824.25 830.23 740.67 742.85 255.07 212.62 226.48 368.82 368.18 347.85 340.12 355.77 300.40 260.03 236.65 259.71 1079.81 1115.02 1066.49 915.83 936.17 875.09
116
2.0 Appendix B: Experimental Methods
117
2.1 Procedure for Preparing Basic Mineral Medium
The following instructions describe how to produce the nutrient medium used in
this study. The nutrient was developed for methanogenic microorganisms for a previous
study. (Edwards & Grbić-Galić, 1994)
2.1.1 Stock Solution Preparation
MM1: Phosphate buffer (100x)
KH2PO4 27.2 g
K2HPO4 34.8 g
Adjust pH to 7.0. Make up to 1 litre with distilled H2O (MilliQ H2O).
MM2: Salt solution (100x)
NH4Cl 53.5 g
CaCl2.6H2O 7.0 g (or 4.79 g CaCl2.2H2O)
FeCl2.4H2O 2.0 g
Make up to 1 Litre with Milli Q H2O.
N.B.: the FeCl2 tends to oxidize and precipitate. However, this has not been a problem
with the methanogenic cultures. I just shake the stock solution before using it. The
precipitation problem can be minimized by preparing this solution anaerobically.
MM3: Trace Minerals (500x)
H3BO3 0.3 g
ZnCl 0.1
Na2MoO4.2H2O 0.1 g
118
NiCl2.6H2O 0.75 g
MnCl2.4H2O 1.0 g
CuCl2.2H2O 0.1 g
CoCl2.6H2O 1.5 g
Na2SeO3 0.02 g
Al2(SO4)3.18H2O 0.1 g
Add 1 ml concentrated H2SO4 per litre to dissolve all components. Make up to 1 litre.
MM4: Magnesium sulfate solution (source of sulfate) (500x)
MgSO4.7H2O 62.5 g/L
OR MgCl2.6H2O 50.8 g/L
Use this to minimize sulphate reductions. This is normally used for our standard media.
MM5: Redox indicator (1000x)
Resazurin 1 g/L
MM6: Saturated bicarbonate
Mix ca. 20 g NaHCO3 in 100 ml MilliQ H2O. Pour slurry into 160-ml serum bottle,
cover with foil and autoclave. After autoclaving, sparge with O2-free N2 for a least 15
minutes while cooling. Seal with sterile black butyl rubber stopper and crimp. The
preparation will have undissolved NaHCO3 in the bottom.
Solubility of NaHCO3 (from CRC handbook)
Cold water: 6.9 g/100 ml
119
Hot water: 16.4 g/100 ml
MM7: Vitamins (10,000x and 100x)
Biotin 0.02 g
Folic acid 0.02 g
Pyridoxine HCl 0.1 g
Riboflavin 0.05 g
Thiamine 0.05 g
Nicotinic acid 0.05 g
Pantothenic acid 0.05 g
PABA 0.05 g
Cyanocobalamin
(vitamin B12) 0.05 g
Thioctic (lipoic) acid 0.05 g
Coenzyme M 1.0 g
Adjust pH to 7.0 with 2N NaOH. Make up to 1 Litre. Store in one or two ml aliquots
frozen. Dilute the stock 1/100 to get 100x stock. Filter sterilize 100x stock into sterile
160-ml serum bottle and sparge with sterile O2-free N2 for 15 minutes. Seal with sterile
black butyl rubber stopper and crimp.
MM8: Amorphous Ferrous Sulfide
This procedure is based on (Brock & O'Dea, 1977).
(NH4)2Fe(SO4)2.6H2O 19.6 g/500 ml
Na2S.9H2O 12.0 g/500 ml
Procedure to make 500 ml: Deoxygenate 2.5 Litres of MilliQ H2O with O2-free N2 for >
1 hour.
120
Weigh out the ferrous ammonium sulfate in a small beaker.
Weigh out the sodium sulfide in a separate small beaker.
Bring a 1-L Erlenmeyer flask with stopper and the chemicals (as powders) into an
anaerobic glove box.
After gassing the 2.5 L of MilliQ H2O, seal the bottle, and bring it into the glove box.
Inside the glove box:
Put 500 ml of MilliQ H2O into the 1-L Erlenmeyer flask. Add the Na2S and mix until
dissolved. Add the (NH4)2Fe(SO4)2. A black precipitate forms immediately. Put the
Erlenmeyer into the glove box antechamber and cycle three times to evacuate the H2S
being formed. Return flask to glove box. Allow precipitate to settle for 24 hours. Wash
by removing (decanting or siphoning) as much as possible of the clear supernatant, and
replacing with about 500 ml of O2-free water. Repeat 3 more times. The rate of settling
decreases as the precipitate is washed and sometimes more than 24 hours is required.
The purpose of washing is to remove any free sulfide in the water. The iron and the
sulfide in the reactants combine in equimolar proportions to form FeS (ferrous sulfide).
Make sure that on the last wash, you resuspend the precipitate such that the total volume
is 500 ml to get the right concentration. Dispense the final 500 ml of slurry into five 160-
ml serum bottles. Seal and crimp in the glove box. Remove and autoclave. The
amorphous ferrous sulfide prepared this way is sterile and anaerobic. The approximate
concentration of FeS in the slurry is 2 g/L (as Sulfide).
2.1.2 Procedure for Making Nutrient Medium
In a 1L screw cap flask, add: 500 ml MilliQ H2O
10 ml MM1
10 ml MM2
2 ml MM3
121
2 ml MM4
1ml MM5
Make up to about 970 ml. Add a magnetic stir bar. Seal with a cap punctured with 2
holes for gas sparging, e.g. with appropriate tubing equipped with an in-line filter for
sterile sparging. Cover stopper and tubing with foil and autoclave.
Remove from autoclave and place in ice bath to cool. While cooling stir and sparge with
O2-free N2/CO2 for about 1/2 hour. Once cool, clamp or seal inlet and outlet tubing and
transport immediately into a glove box.
Also take into glove box: Anaerobic vitamin stock (MM7, 100x, in serum bottle)
MM6 (also in serum bottle)
MM8 (also in serum bottle)
1 disposable sterile filter (0.2 µm)
3 plastic disposable sterile 10-ml syringes with needles
1 extra sterile needle.
Add 10 ml of each MM7, MM8 and MM6 in that order as described below (these three
solutions are kept anaerobic and sterile in 160-ml serum bottles with crimped black butyl
rubber stoppers).
In order to withdraw 10 ml from these serum bottles without creating a large vacuum
inside the bottle, the pressure must be equilibrated. Equilibrate the bottles by inserting a
sterile needle connected to a sterile filter through the stopper. This will filter sterilize the
glove box atmosphere as it passes into the bottle and preserve the sterility of the
preparation. After inserting the needle and filter, use the 10-ml syringe and needle to
withdraw 10 ml to add to the medium.
122
Mix all the ingredients and check the pH (with pH paper or by removing a small aliquot
out of the glove box and measuring with a pH meter). The pH should be around 7. The
methanogenic culture actually prefer the pH slightly acidic, or about 6.5 to 7.0.
An observation:The medium will be purplish after sparging with N2 (before bringing it
into the glove box). Once the FeS is added, the medium will be black. As the black
precipitate settles, the medium should be clear. It may be a little pink if some oxygen got
in. If your glove box is truly anaerobic, just leaving the medium in the glove box for a
few days will further reduce it. Alternatively, you can add a few more millilitres of FeS
(MM8).
123
2.2 Calibration Curves
2.2.1 Pressure Transducer Calibration
A calibration curve for the pressure transducer used in this study in order to determine the
volume of biogas production during BMP assays. Known volumes of air were added to a
stoppered serum bottle containing 100mL of water simulating a bottle used during a BMP assay.
Air was added using a gas tight syringe. The pressure in the headspace of the bottle was
measured in the same manner that was used for the BMP assays. The readings given by the
transducer were recorded and the data was linearly regressed.
y = 9.207x - 0.028
R2 = 1.000
0
2
4
6
8
10
12
0 0.2 0.4 0.6 0.8 1 1.2Transducer Reading
Vo
lum
e o
f A
ir A
dd
ed
to
Se
rum
Bo
ttle
[mL]
Figure 2.2.1 Calibration curve for pressure transducer used in biogas measurements.
Table 2.2.1 Raw data used for pressure transducer calibration. Syringe size refers to the size of the glass
syringe used to add air to the headspace of the serum bottle.
Gas Added [mL] Syringe Size [mL] Reading
1 0 0.004
2 0 -0.001
3 0 0
4 0.1 0.5 0.017
5 0 0.005
6 0.1 0.5 0.017
124
7 0 0.003
8 0.1 0.5 0.016
9 0 0.003
10 0.3 0.5 0.035
11 0 0.001
12 0.3 0.5 0.034
13 0 0.001
14 0.3 0.5 0.033
15 0.5 0.5 0.058
16 0.5 0.5 0.059
17 0.5 0.5 0.055
18 0 0.002
19 1 2 0.11
20 1 2 0.11
21 1 2 0.112
22 0 0.003
23 2 2 0.218
24 2 2 0.221
25 2 2 0.219
26 0 0.004
27 5 5 0.551
28 5 5 0.552
29 5 5 0.553
30 0 0.005
31 10 5 1.081
2.2.2 GC Calibration
A calibration curve was developed to correlated GC peak area with concentration of
methane in the headspace of serum bottles. Bottle 1 contained a 99% methane reference gas.
Specific volumes of bottle 1 were added to 4 other bottles filled with 100mL of water to simulate
serum bottles to be used during BMP assays. Headspace volume was estimated by bottle weight
when completely filled with water versus being filled with 100mL of water. The difference in
weight was taken as the volume of headspace.
125
Table 2.2.2 Bottle setup information about bottles used for methane calibration curve. Bottle 1 was
completely filled with 99% methane calibration gas. All other bottles had a certain volume of gas from bottle
1 added to their headspace.
Bottle #
Empty
Bottle
Weight [g]
Weight
Full
Bottle [g]
Weight of
Bottle
with
100mL
H2O or
after
filled
with CH4
(bottle 1)
[g]
Calculated
Headspace
Volume
[mL]
Vol. Gas
Removed
before
Bottle 1
Gas Added
[mL]
Vol.
Bottle 1
Added
[mL]
Mole
Fraction
CH4 in
Headspace
Vol. of
Sample
Added to
GC [mL]
1 105.04 263.08 105.31 158.1226 – – 0.99 0.1
2 104.86 263.31 203.92 59.52274 0 0.1 0.001677 0.3
3 105.05 262.92 204 59.05169 0 0.5 0.008391 0.3
4 105 262.68 204.05 58.76104 2 2 0.033911 0.3
5 104.82 262.9 204.41 58.62072 5 5 0.084013 0.3
y = 19211x + 3829.7
R2 = 0.9878
0
50000
100000
150000
200000
250000
300000
350000
0 2 4 6 8 10 12 14 16
Methane Conc. In Headspace [umol/mL]
Pe
ak
Are
a
Figure 2.2.2 Calibration curve of GC peak area versus serum bottle headspace methane concentration.
126
2.3 Lowry Protein Modifications
In this study, the Lowry assay was used to measure protein. The Lowry method is a
colorimetric method and has been found in previous studies to find better results in detecting
protein in activated sludge samples then the Bradford method (Frølund, Griebe, & Nielsen, 1995;
Raunkjaer, Hvitved-Jacobsen, & Nielsen, 1994) . All colorimetric protein assays are affected by
the presence of interfering compounds, but there were several modifications to the Lowry assay
that could be performed to overcome WAS specific interferences. These were tested in various
combinations in this study until a satisfactory results in terms of variability in data was achieved.
The basic method used in this study was based on the method described in (). This
method modifies the original method through the addition of sodium dodecyl sulphate (SDS)
which eliminates interferences caused by various carbohydrates and lipids (Peterson, 1979).
Another modification that was used in this study was that presented in (Frølund et al.,
1995) . This modification was based on the assumption that the copper sulphate in the assay
reagents only enhanced light absorption caused by protein and not by other interfering
compounds. By comparing the absorbances of identical samples exposed to the Lowry reagents
in the presence and absences of copper, the amount of true protein could be determined. This
method was used to remove interferences caused by humic acids. In pulp and paper mill WAS
lignin was likely present which also interacted with the Lowry reagent. To test the effect of
copper the Lowry reagent was added to samples of sulphite mill lignin with and without lignin.
The result was that the presence of copper did not affect the absorbance resulting from lignin
indicating this modification could be used to overcome this interference (Figure 2.3.1).
y = 7.55x + 0.02
R2 = 1.00
y = 7.59x + 0.02
R2 = 1.00
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 0.02 0.04 0.06 0.08 0.1 0.12
Lignin Conc. [mg/mL]
Ab
sorb
an
ce
With Copper
Without Copper
Figure 2.3.1 Absorbance as a function of lignin concentration when Lowry assay reagents are added in the
presence and absence of copper.
After application of these modifications there was still substantial interference and
variability in the results when the total protein content of wastes was measured. Soluble protein
was, however, was measured by this method had low variability implying that something
contained within the solids of the WAS was causing interference.
127
Various methods were tested to separate protein from the solids in WAS and thereby
removing the interfering compounds. The first method attempted was the overnight incubation
with NaOH to solubilise protein followed by filtration. This did not reduce interferences and
resulted in only about half the amount of protein detected compared with results with NaOH.
The second method tested was precipitation using trichloroacetic acid (TCA) in the presence of
deoxycholate (DOC) which has been suggested as a method to separate proteins from other
interfering compounds (Peterson, 1977; Peterson, 1979). It has been tested previously for
determining protein content of microorganisms grown on lignocellulosic substrates with some
success (Tan, L. U. L., Chan, & Saddler, 1984). In this study it was found that even after
precipitation, interferences lingered in precipitated solutions even after performing the
precipitation procedure twice on the same samples, so the modification using the affect of copper
still needed to be performed. Precipitation increased variability in results where differences
between replicates were more then 100% and interferences were not appreciably reduced.
128
3.0 Appendix C: Sample Calculations
129
3.1 COD-Based Calculations
3.1.1 BMP Assay Substrate Concentration
Substrates were added to assay bottles based upon the amount of potential biogas
production in terms of COD. This potential was based upon the amount of methane potential of
a substrate’s COD content. The production of methane can be described by the following half
reaction (Rittmann & McCarty, 2001):
1/8 CO2 + H
+ + e
- =
1/8 CH4 +
1/4 H2O
This equation suggests it takes eight electron equivalents to produce one mole of methane.
COD can be directly related to the required number of electron equivalents available to be
metabolised to form methane. According to (Rittmann & McCarty, 2001), the following
relationship exists between COD and electron equivalents: 8g OD/e- eq. This information
combined with the equation above can calculate the amount of COD required to produce a mole
of methane:
8g OD/e- eq * 8 e
- eq/mol CH4 = 64g OD/mol CH4
In the BMP assays in this study, it was set that 20mL of biogas would be produced over
the course of the assay. It was assumed 70% of that biogas would be methane so a total of 14mL
of methane would be produced over the course of the assay. Using the ideal gas law the moles of
CH4 produced can be calculated…
P * V = n * R * T
Where P is the pressure in Pa and was set to atmospheric pressure: 101 325 Pa, V
represents the volume of methane produced in m3: 14 x 10
-6 m
3, n is the moles of CH4 produced
to be calculated, R is the ideal gas constant: 8.3145, and T is the temperature used during the
incubation of the assay in K: 308.15K.
n = (101 325Pa) * (14 x 10-6
m3) / (8.3145) / (308.15K) = 0.000550 mol = 550 µmol CH4
Based on the amount of methane, the amount of COD required in each assay bottle can
be calculated…
0.000550 mol CH4 * 64g OD / mol CH4 = 0.0352g OD = 35.2mg OD / assay bottle
This calculation was performed with slight variations for each BMP assay.
3.1.2 Positive Control Substrate Concentrations
The positive control in every BMP assay performed in this study was a mixture of
glucose, sodium acetate, and sodium propionate. These components were added so that half the
COD of the substrate would come from glucose and the other half from acetate and propionate.
The half of the substrate COD made of acetate and propionate would be 90% acetate in terms of
COD.
130
The electron equivalents required to reduce glucose, acetate, and propionate can be
estimated using the following half reactions (Rittmann & McCarty, 2001):
(Acetate) 1/8 CO2 +
1/8 HCO
-3 + H
+ + e
- =
1/8 CH3COO
- +
3/8 H2O
(Glucose) 1/4 CO2 + H
+ + e
- =
1/24 C6H12O6 +
1/4 H2O
(Propionate) 1/7 CO2 +
1/14 HCO
-3 + H
+ + e
- =
1/14 CH3CH2COO
- +
5/14 H2O
Based on ratio 8g OD/e- eq, the COD potential of glucose, propionate, and acetate can be
calculated…
8 e- eq/mol CH3COO
- * 8g OD/e
- eq = 64g OD/mol CH3COO
- (acetate)
192g OD/mol C6H12O6 (glucose)
112g OD/mol CH3CH2COO- (propionate)
The moles of each component can be converted to values per weight using the molecular
mass of each compound…
[64g OD/mol CH3COO- * 1 mol CH3COO
- / mol Na
+CH3COO
-] / 82 g/mol Na
+CH3COO
-
= 0.78g OD /g Na+CH3COO
-
1.07g OD/g C6H12O6
0.67g OD/g Na+CH3CH2COO
-
Using these values and the amount of COD substrate required in each assay bottle, the
concentrations of glucose, sodium acetate, and sodium propionate required can be calculated.
For example, to calculate the concentration of glucose in the substrate stock solution to be added
to positive control assay bottles in the case of 35.2mg COD is required per bottle and 10mL
substrate volume added to each bottle…
[35.2mg COD * 50% glucose] / 1.07mg OD/mg C6H12O6 / 10mL substrate volume per bottle =
1.65mg C6H12O6 / mL substrate stock
This was done similarly for sodium acetate and sodium propionate with 45% of the
remaining COD being from acetate and 5% from propionate:
2.03mg Na+C3COO
- / mL substrate stock
0.26mg Na+CH3CH2COO
- / mL substrate stock
3.1.3 COD Removal Based on Methane Production
The amount of substrate COD removed from each bottle was estimated based on the total
amount of methane produced over the course of the BMP assay. Each mole of CH4 produced
requires 64g of OD. For example, if 200µmol CH4 was produced…
200 µmol CH4 = 0.0002 mol CH4 * 64g OD/mol CH4 = 0.0128g OD
= 12.8mg COD removed
131
3.2 Methane Content Mass Balance
Over the course of BMP assays both biogas production and methane concentration of the
headspace were measured. Based upon these two measurements and the use of a mass balance,
the amount of methane produced between measurements was calculated. The produced involved
measuring biogas, bringing the bottle’s headspace to atmospheric pressure using a needle open to
the atmosphere, and then sampling the headspace to measure the methane content. If no biogas
was produced or there was a slight vacuum in the bottle the headspace was not opened to
atmospheric pressure. Based on if the bottle was opened to the atmosphere or not, different
scenarios for the methane production mass balance, although all mass balances are based on the
same basic formula:
nCH4 t0,t1 = nCH4, t1 – nCH4, headspace, t0
Where nCH4 t0,t1 represents the moles of methane produced between the previous
measurement at t0 and the current measurement t1, nCH4, t1 is the total amount of methane
measured at the current measurement, and nCH4, headspace, t0 is the amount of methane measured in
the headspace at the time of the previous measurement.
The amount of methane measured during the current measurement, nCH4, t1, was
calculated differently depending on whether or not if the bottle was brought to atmospheric
pressure before methane measurement. In the case the bottle was brought to atmospheric
pressure:
nCH4, t1 = CCH4 headspace, t1 * (Vbiogas + Vheadspace)
Where CCH4 headspace, t1 is the concentration of methane measured in the headspace of the
bottle during the current measurement t1, Vbiogas is the volume of biogas produced measured by
the transducer, and Vheadspace is the volume of the bottle headspace. In the case the bottle was not
brought to atmospheric pressure:
nCH4, t1 = CCH4 headspace, t1 * (Vheadspace)
This is assuming none of the biogas produced, when compared to the blank, is not
released from the bottle headspace, so all methane remains in the headspace. The amount of
methane measured in the headspace during previous measurement, nCH4, headspace, t0, is calculated
by:
nCH4 headspace, t0 = CCH4 headspace, t0 * (Vheadspace)
Where CCH4 headspace, t0 is the concentration of methane measured during the previous
measurement and Vheadspace is the total volume of the headspace.
For example, during one measurement the transducer reading was 0.700 and after the
bottle was brought to atmospheric pressure, the methane content was measured by GC to give a
peak area of 22515. The previous GC measurement showed a methane peak area of 20351.
Using the previously prepared calibration curves, described in Appendix B, the biogas produced
and methane concentrations can be calculated:
Vbiogas = 9.207 * (0.029) - 0.028 = 0.24mL biogas
132
CCH4 headspace, t0 = [(20351) – 3830] / 19211 = 0.86 µmol CH4 / mL
CCH4 headspace, t1 = [(22515) – 3830] / 19211= 0.97 µmol CH4 / mL
Using these values, the mass balance above, and assuming a headspace volume of 60mL,
the amount of biogas produced between these two measurements can be calculated:
nCH4, t1 = CCH4 headspace, t1 * (Vbiogas + Vheadspace)
nCH4, t1 = 0.97 µmol CH4 / mL * (0.24mL + 60mL) = 58.4 µmol CH4
nCH4 headspace, t0 = CCH4 headspace, t0 * (Vheadspace)
nCH4 headspace, t0 = 0.86 µmol CH4 / mL * (60mL) = 51.6 µmol CH4
nCH4 t0,t1 = nCH4, t1 – nCH4, headspace, t0
nCH4 t0,t1 = 58.4 µmol CH4 – 51.6 µmol CH4 = 6.8 µmol CH4
The volume of methane produced can be calculated using the ideal gas law:
P * V = n * R * T
Where P is the pressure in Pa and was set to atmospheric pressure: 101 325 Pa, V
represents the volume of methane produced in m3, n is the moles of CH4 produced, R is the ideal
gas constant: 8.3145, and T is the temperature used during the incubation of the assay in K:
308.15K.
VCH4 = (6.8 x 10-6
mol CH4) * (8.3145) * (308.15K) / (101 325Pa)
VCH4 = 1.7 x 10-7
m3 = 0.17mL CH4
This suggests the biogas produced had a methane content of 71% on a volume basis.
133
3.3 Protein Concentration
Soluble protein content of biomass samples was determined using a modified version of
the Lowry method (Peterson, 1977) with an added modification (Frølund et al., 1995) . The
added modification involved exposing the same sample to two different reagent solutions, one
containing copper sulphate, the other without any copper. The idea is that the addition of copper
will only enhance the absorbance of proteins, while other compounds that interact with the Folin-
Ciocalteu reagent will have a similar absorbance. Using this difference, the interference caused
by compounds other then proteins can be eliminated. This assumes that in the case copper
sulphate is present, the total absorbancem Atotal, is the sum of the absorbance of protein. Aprotein,
and the absorbance of other compounds, Aother:
Atotal = Aprotein + Aother
When copper sulphate is omitted from the mixture, the resulting absorbance, Ablind, is the
sum of a fraction, X, of the absorbance of protein when copper is present and the absorbance of
other compounds which remains the same:
Ablind = X * Aprotein + Aother
Combining the two equations and solving for the absorbance of protein:
Acorr = Aprotein = 1 / (1 – X) * [Atotal – Ablind]
Where X is determined by finding the average fraction of the absorbance of the standard
curve solutions run without copper compared to the standard solutions run with copper. For
example, take the following set of data for a standard curve of bovine serum albumin (BSA):
BSA Conc. [mg/mL] Atotal Ablind Ablind / Atotal
0.101 0.702 0.138 0.197
0.066 0.428 0.079 0.185
0.005 0.046 0.011 0.245
X (average) = 0.209
Note that these absorbances have had blank absorbances (with MilliQ H2O in the place of
sample) subtracted from all values. Using this X value as well as the calibration curve for protein
with copper added, the concentration of protein, Cprotein, in a sample could be calculated. For
example, given the following set of data:
Atotal = 0.405 and Ablind = 0.375
And with the following calibration curve made with standard solutions of BSA and using
the copper reagent:
Log10(A) = 0.896 * Log10(Cprotein) + 0.715
134
The concentration of protein can be calculated. First, the corrected absorbance of protein
must be determined:
Acorr = Aprotein = 1 / (1 – X) * [Atotal – Ablind]
Acorr = 1 / (1 – 0.209) * [0.455 – 0.375]
Acorr = 0.101
Note that special care was taken so that the corrected value of absorbance was in the
range of the standard curve. If the corrected absorbance is lower then the absorbance of the
lowest concentration in the standard curve or any of the absorbances (total, blind, or corrected)
are higher then the highest absorbance in the standard curve, the data cannot be evaluated
properly. To calculate the concentration the calibration curve is used:
Log10(Cprotein) = [Log10(A) – 0.715] / 0.896
Log10(Cprotein) = [Log10(0.101) – 0.715] / 0.896
Log10(Cprotein) = -1.910
Cprotein = 0.012 mg/mL protein
Note all absorbances had the blank values subtracted from them before calculating the
corrected absorbance. Samples also had to be diluted to ensure the protein content was in the
range of the standard curves. The concentration measured would be the protein content of the
diluted sample.
3.4 Energy Balance
3.4.1 Thermal and Caustic Pretreatment
The energy required for thermal and caustic pretreatment was estimated by determining
the energy required to heat the WAS from 5oC to the temperature of the pretreatment. The heat
capacity of WAS was assumed to be equal to that of water. To determine the heat capacity, the
average of the heat capacities of saturated water from 5oC to 170
oC was determined. From the
data found in Perry’s Chemical Engineer’ Handbook the specific heat capacity of water, Cp, was
found to be: 4.22 kJ kg-1
K-1
. (Perry & Green, 1997) The energy required to heat a liquid can be
determined by the following equation:
H = Cp * (T2 – T1)
Where H is the heat energy required to heat a fluid from T2 to T1 under constant pressure.
In the example of thermal pretreatment where temperature is increased to 170oC:
H = 4.22 kJ kg-1
K-1
* (443.15 K – 278.15 K)
H = 624.4 kJ kg-1
Assuming WAS has a density similar to water this becomes:
H = 624.4 kJ kg-1
* 1 kg / m3 = 624.4 kJ m
-3 WAS = 0.624 J / mL WAS
135
3.4.2 Sonication Pretreatment
The energy for sonication pretreatment was based on the length of sonication and the
power applied. Two values for total energy use were calculated: one based upon the power
applied to the ultrasonic reactor, the other based upon an experimentally derived value for the
actual ultrasonic power applied to the WAS sample. In both cases the total energy requirement,
E, was calculated with the same formula:
E = Papp * t / V
Where Papp refers to the power applied in Watts, t refers to the length of time for
sonication, and V refers to the volume of WAS sonicated. For example, in the case of K WAS a
total power of 400 W was applied for 2100 s (35 minutes) to 400mL of WAS. The total energy
requirement would be:
E = 400 J/s * 2100 s / 400mL
E = 2100J/mL
3.4.3 Biogas Energy Content
The energy contained in biogas was assumed to be 25.2 J / mL biogas at STP which is a
standard biogas heating value used in engineering calculations. (Deublein & Steinhauser, 2008)
This value was then converted to an energy value in terms of Joules of energy in terms of biogas
produced per mL of WAS digested. To calculate the total volume of biogas produced by an
assay bottle subtracting blank values was divided by the volume of substrate added to that bottle.
This value combined with the heating value of biogas was used to determine the amount of
biogas energy potential per mL WAS, Ebiogas, using the following equation.
Ebiogas = Hbiogas * Vtotal biogas / Vsubstrate added
Where Hbiogas is the heating value of biogas, Vtotal biogas is the total amount of biogas
produced by a substrate in a serum bottle in one of the BMP assays, and Vsubstrate added is the
volume of substrate added to that bottle. For example, in the case of thermally treated K WAS,
the average biogas production from a substrate bottle was 5.8mL (STP) and the volume of
substrate added was 1.5mL. The total biogas potential of the WAS sample in terms of biogas
energy would be:
Ebiogas = 25.2 J / mL biogas * 5.8mL biogas produced / 1.5mL WAS added
Ebiogas = 100 J biogas energy / mL WAS digested
x
136
4.0 References
Brock, T. D., & O'Dea, K. (1977). Amorphous ferrous sulfide as a reducing agent for culture of
anaerobes. Applied and Environmental Microbiology, 33(2), 254-256.
Deublein, D., & Steinhauser, A. (Eds.). (2008). Biogas from waste and renewable resources: An
introduction. Germany: Wiley-VCH Verlag GmbH & Co.
Edwards, E. A., & Grbić-Galić, D. (1994). Anaerobic degradation of toluene and o-xylene by a
methanogenic consortium. Applied and Environmental Microbiology, 60(1), 313-322.
Frølund, B., Griebe, T., & Nielsen, P. H. (1995). Enzymatic activity in the activated-sludge floc
matrix. Applied Microbiology and Biotechnology, 43, 755-761.
Perry, R. H., & Green, D. W. (Eds.). (1997). Perry's chemical engineers' handbook (7th ed.)
McGraw-Hill.
Peterson, G. L. (1977). A simplification of the protein assay method of lowry et al. which is
more generally applicable. Analytical Biochemistry, 83, 346-356.
Peterson, G. L. (1979). Review of the folin phenol protein quantification method of lowry,
rosebrough, farr and randall. Analytical Biochemistry, 100, 201-220.
Raunkjaer, K., Hvitved-Jacobsen, T., & Nielsen, H. (1994). Measurement of pools of protein,
carbohydrate and lipid in domestic wastewater. Water Research, 28(2), 251-262.
Rittmann, B. E., & McCarty, P. L. (2001). Environmental biotechnology: Principles and
applications. New York, NY: McGraw-Hill.
Tan, L. U. L., Chan, M. K. -., & Saddler, J. N. (1984). A modification of the lowry method for
detecting protein in media containing lignocellulosic substrates. Biotechnology Letters, 6(3)