monitoring the role of aceticlasts in anaerobic digestion: activity and capacity

10
Monitoring the role of aceticlasts in anaerobic digestion: Activity and capacity A.S. Conklin a, *, T. Chapman b , J.D. Zahller c , H.D. Stensel d , J.F. Ferguson d a Carollo Engineers, 1218 Third Avenue, Suite 1600, Seattle, WA 98101, USA b Brown and Caldwell, 701 Pike Street, Suite 1200, Seattle, WA 98101, USA c HDR Inc., 500 108th Avenue NE, Suite 1200, Bellevue, WA 98004, USA d Department of Civil and Environmental Engineering, University of Washington, 201 More Hall, Box 352700, Seattle, WA 98195, USA article info Article history: Received 9 February 2008 Received in revised form 31 August 2008 Accepted 1 September 2008 Published online 1 October 2008 Keywords: Anaerobic digester stability V max,ac Digester capacity abstract Aceticlastic methanogens are seen as a key to digester capacity and stability. This paper develops and applies an assay to measure digester stability by measuring the maximum aceticlastic methane production rate (V max,ac ). The V max,ac in combination with acetate concentrations was found to be an effective digestion monitoring tool to indicate process upsets. At steady state, thermophilic, first stage and short SRT digesters generally had a greater V max,ac than mesophilic, second stage or long SRT digesters. The ratio of the V max,ac to the plant aceticlastic methane production rate, termed the Acetate Capacity Number (ACN), is a measure of the excess capacity of the digester. Either V max,ac or ACN can be used to estimate the capability to handle higher organic loading rates. Monod modeling was used to predict V max,ac , ACN and maximum VS loading rates for mesophilic and thermophilic digestion and for staged digesters to better understand expected diges- tion capacity and stability. ª 2008 Elsevier Ltd. All rights reserved. 1. Introduction The degradation of organic matter in anaerobic digesters occurs through three basic steps: hydrolysis, fermentation and methanogenesis. Previous research has found that around 70% of the methane produced in the digestion of domestic sludge comes from the transformation of acetate to methane usually by the aceticlastic methanogens (Jeris and McCarty, 1965; Smith and Mah, 1966). The role of these methanogens is important in maintaining the carbon flow through the system via conversion of acetic acid to methane and carbon dioxide. When methanogenesis is not rapid enough, volatile fatty acids (VFA) accumulate, which may lead to lower pH and digester upsets. The research presented in this paper focuses on the production of methane from acetate by acetotrophic methanogenesis and on the hypothesis that aceticlastic methanogenesis is a key step in digestion of municipal wastewater sludge which may determine the capacity of the system to convert organic matter to methane and avoid system upsets caused by VFA accumulation. In studies in our group, Zahller (2004) and Bucher (2003) found that after batch feeding of wastewater sludges that the gas production rate increased and acetate accumulated, indicating that hydrolysis and acidogenesis proceeded faster than methanogenesis. These findings support those of Kaspar and Wuhrmann (1978) who showed that aceticlastic methanogens operate at nearly 50% of their maximum capacity in anaerobic digestion and have limited ability to handle high production of acetate. Noike et al. (1985) found that aceticlastic methanogenesis proceeded more slowly than hydrolysis of starches but more rapidly than hydrolysis of cellulose. In the reactors of Bucher (2003) and * Corresponding author. Tel.: þ1 206 684 6532; fax: þ1 206 903 0419. E-mail address: [email protected] (A.S. Conklin). Available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/watres 0043-1354/$ – see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2008.09.024 water research 42 (2008) 4895–4904

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Page 1: Monitoring the role of aceticlasts in anaerobic digestion: Activity and capacity

w a t e r r e s e a r c h 4 2 ( 2 0 0 8 ) 4 8 9 5 – 4 9 0 4

Avai lab le a t www.sc iencedi rec t .com

journa l homepage : www.e lsev ie r . com/ loca te /wat res

Monitoring the role of aceticlasts in anaerobic digestion:Activity and capacity

A.S. Conklina,*, T. Chapmanb, J.D. Zahllerc, H.D. Stenseld, J.F. Fergusond

aCarollo Engineers, 1218 Third Avenue, Suite 1600, Seattle, WA 98101, USAbBrown and Caldwell, 701 Pike Street, Suite 1200, Seattle, WA 98101, USAcHDR Inc., 500 108th Avenue NE, Suite 1200, Bellevue, WA 98004, USAdDepartment of Civil and Environmental Engineering, University of Washington, 201 More Hall,

Box 352700, Seattle, WA 98195, USA

a r t i c l e i n f o

Article history:

Received 9 February 2008

Received in revised form

31 August 2008

Accepted 1 September 2008

Published online 1 October 2008

Keywords:

Anaerobic digester stability

Vmax,ac

Digester capacity

* Corresponding author. Tel.: þ1 206 684 653E-mail address: [email protected] (A

0043-1354/$ – see front matter ª 2008 Elsevidoi:10.1016/j.watres.2008.09.024

a b s t r a c t

Aceticlastic methanogens are seen as a key to digester capacity and stability. This paper

develops and applies an assay to measure digester stability by measuring the maximum

aceticlastic methane production rate (Vmax,ac). The Vmax,ac in combination with acetate

concentrations was found to be an effective digestion monitoring tool to indicate process

upsets. At steady state, thermophilic, first stage and short SRT digesters generally had

a greater Vmax,ac than mesophilic, second stage or long SRT digesters. The ratio of the

Vmax,ac to the plant aceticlastic methane production rate, termed the Acetate Capacity

Number (ACN), is a measure of the excess capacity of the digester. Either Vmax,ac or ACN

can be used to estimate the capability to handle higher organic loading rates. Monod

modeling was used to predict Vmax,ac, ACN and maximum VS loading rates for mesophilic

and thermophilic digestion and for staged digesters to better understand expected diges-

tion capacity and stability.

ª 2008 Elsevier Ltd. All rights reserved.

1. Introduction methanogenesis and on the hypothesis that aceticlastic

The degradation of organic matter in anaerobic digesters

occurs through three basic steps: hydrolysis, fermentation

and methanogenesis. Previous research has found that

around 70% of the methane produced in the digestion of

domestic sludge comes from the transformation of acetate to

methane usually by the aceticlastic methanogens (Jeris and

McCarty, 1965; Smith and Mah, 1966). The role of these

methanogens is important in maintaining the carbon flow

through the system via conversion of acetic acid to methane

and carbon dioxide. When methanogenesis is not rapid

enough, volatile fatty acids (VFA) accumulate, which may lead

to lower pH and digester upsets.

The research presented in this paper focuses on the

production of methane from acetate by acetotrophic

2; fax: þ1 206 903 0419..S. Conklin).er Ltd. All rights reserved

methanogenesis is a key step in digestion of municipal

wastewater sludge which may determine the capacity of the

system to convert organic matter to methane and avoid system

upsets caused by VFA accumulation. In studies in our group,

Zahller (2004) and Bucher (2003) found that after batch feeding

of wastewater sludges that the gas production rate increased

and acetate accumulated, indicating that hydrolysis and

acidogenesis proceeded faster than methanogenesis. These

findings support those of Kaspar and Wuhrmann (1978) who

showed that aceticlastic methanogens operate at nearly 50% of

their maximum capacity in anaerobic digestion and have

limited ability to handle high production of acetate. Noike et al.

(1985) found that aceticlastic methanogenesis proceeded more

slowly than hydrolysis of starches but more rapidly than

hydrolysis of cellulose. In the reactors of Bucher (2003) and

.

Page 2: Monitoring the role of aceticlasts in anaerobic digestion: Activity and capacity

w a t e r r e s e a r c h 4 2 ( 2 0 0 8 ) 4 8 9 5 – 4 9 0 44896

Zahller (2004) the initial burst of activity was likely from the

rapid hydrolysis of easily degradable substrates such as

starches and the slow degradation rates at the end of the

feeding cycle were from the hydrolysis of materials such as

cellulose.

Finding a method to monitor for digester capacity is

important because digester failure can be costly. There is

considerable debate in the literature as to the best way to

monitor digester stability. The parameters most commonly

discussed are: methane and carbon dioxide concentrations in

biogas (Callaghan et al., 1997), gas production or methane

production rates (Chynoweth et al., 1994), pH (Killilea et al.,

2000), alkalinity (Denac et al., 1988; Hawkes et al., 1992), gas

phase hydrogen concentration (Cord-Ruwisch et al., 1997) and

VFAs (Ahring et al., 1995). None of these methods can deter-

mine how close a digester is to failure. Methods that monitor

the activity of the aceticlasts, however, may measure the

capacity of the microbial community to use a key interme-

diate and thus indicate digester capacity and stability.

This research developed a batch bottle test aimed at

determining the activity of the aceticlastic methanogens

(Vmax,ac). The batch bottle test method used in this research is

a modification of batch bottle test assays developed and used

previously (James et al., 1990; Owen et al., 1979; Shelton and

Tiedje, 1984). This method uses replicate unfed and acetate-

fed bottles, monitoring methane produced over time and

determining Vmax,ac by subtracting 30% of the methane

production rate of the unfed bottles from the methane

production rate of the acetate-fed bottles. This parameter

directly indicates the maximum activity of the aceticlastic

methanogens and thus is a useful indicator of digester

capacity. The Vmax,ac parameter used in this research differs

from the aceticlastic capacity parameters developed previ-

ously both in how the parameter is calculated and in how the

parameter is measured. The excess digestion capacity can be

determined by comparing the Vmax,ac of a digesting sludge to

the plant aceticlastic methane production rate (Vplant,ac).

The objectives of this study were to develop a method to

measure digester capacity and aceticlastic stability in anaerobic

digestion, to apply the method to different anaerobic digesters,

and to investigate if variations can be related to different

digester operating conditions. This paper describes the devel-

opment of the Vmax,ac method. It then validates the use of the

Vmax,ac method by comparing it to traditional stability param-

eters during a forced upset of a bench-scale digester and an

unplanned upset of a full-scale digester. Finally the Vmax,ac and

Vplant,ac parameters were determined for full- and pilot-scale

digesters to gain understanding of digester capacity and

stability. Monod and Herbert (Roels, 1983) based kinetic equa-

tions were adapted to predict the acetate utilization capacity of

anaerobic digesters at different operating conditions.

2. Methods

2.1. Acetate utilization capacity (Vmax,ac) activity test

From the Monod adaptation of the Michaelis–Menten equa-

tion, the acetate utilization rate is a function of substrate

concentration (S ) and active biomass (Xa). When S [ the half

saturation coefficient (KS) the Michaelis–Menten equation

reduces to Eq. (1) and the initial substrate utilization rate

measures Vmax.

�dSdt¼ kXa ¼ Vmax (1)

where k equals the specific acetate growth rate.

The Vmax,ac test was conducted by adding 20 mL of digester

sludge to 55-mL serum bottles purged with an 80/20% mixture

of N2/CO2, bubbled through a 0.2% solution of titanium citrate.

One set of triplicate serum bottles was fed between 70 and

100 mM sodium acetate, while the other set of triplicate

bottles received an equal volume of water. Vmax,ac tests con-

ducted on mesophilic and thermophilic sludges were insen-

sitive to acetate concentrations above 50 mM sodium acetate.

Both sets were capped and shaken at 150 rpm in a 35 �C water

bath. The methane production rate was determined by the

change in headspace methane concentration versus time,

typically for several hours.

In digesting sludge approximately 70% of the methane is

often produced by aceticlastic methanogens (Vac), and the

remaining 30% of the methane is produced by hydro-

genotrophic methanogens ðVH2 Þ (Jeris and McCarty, 1965;

Smith and Mah, 1966). Using this percentage, 30% of the

methane produced in the unfed bottles is assumed to be

produced by hydrogenotrophic methanogens ðVH2 ;uÞ. The

methane produced from the aceticlastic methanogens in the

fed bottles (Vmax,ac) was then calculated as the gross methane

production rate of the fed bottles (Vtotal,f) less 30% of the unfed

methane production rate ðVH2 ;uÞ. The total aceticlastic

methanogen biomass can be calculated to increase slightly

during the test as the acetate was consumed; however, this

biomass increase was not significant as indicated by linear

slopes of methane production versus time.

The error associated with the Vmax,ac values was deter-

mined using the linear mixed effects model (Pinheiro and

Bates, 2000) in order to account for multiple measurements

from three replicate bottles. The mixed effect model combines

both fixed effects (parameters associated with an entire pop-

ulation) with random effects (parameters associated with the

individual units). The linear mixed effect model is a function

in the data analysis program R version 1.9.0 (The R Foundation

for Statistical Computing, ISBN 3-900051-00-3). The program

uses the restricted maximum likelihood (REML) method to

estimate the errors associated with the linear fits. The stan-

dard error of the Vmax,ac value was then determined by the

linear combination of the standard errors for the slopes of the

unfed and fed bottles.

2.2. Vplant,ac determination and acetate capacity number

Since Vmax,ac depends on the acetate using biomass, Xa, which

in turn depends on digester loading, it is important to

compare the maximum rate of acetate use to the actual rate

occurring in the digester. The plant methane production rate

from acetate was calculated from volatile solids (VS) removal

data as shown in Eq. (2).

Vplant;ac

�LCH4

=Ldigester d�¼�VSfeed$Rfeed � VSdigester$Rsludge

�QV

�0:395

LCH4

gCOD

�0:7 ð2Þ

Page 3: Monitoring the role of aceticlasts in anaerobic digestion: Activity and capacity

w a t e r r e s e a r c h 4 2 ( 2 0 0 8 ) 4 8 9 5 – 4 9 0 4 4897

where Q is digester feed rate (L/d), V is digester volume (L),

Rfeed and Rdigester are chemical oxygen demand (COD) to VS

ratios of the feed and digesting sludge, respectively, VSfeed and

VSdigester are the VS concentrations (g/L) in the feed and

digester, respectively, 0:395 LCH4=gCOD at 35 �C represents the

stoichiometry of COD conversion to methane, and 0.7 is the

fraction of COD degraded via acetate.

The Vplant,ac rate can also be determined based on the

product of the gas production rate and the digester gas

methane content. However, gas production data were not

thought to be accurate at several plants, and we chose to use

the VS-based calculation for all plants surveyed. Since the

digesters in this study were completely mixed, the effective

Vplant,ac depended to some extent on loading for the previous

days. To account for this, the Vplant,ac for a given day was

determined by averaging values for the 5–10 days prior to

sampling.

The ratio of Vmax,ac to Vplant,ac, termed the acetate capacity

number (ACN) or the excess capacity of the digester for acetate

use, indicates the capacity of aceticlastic methanogens to

handle shock loads. A value close to one indicates that the

aceticlastic methanogens are operating near their maximum

rate and any additional feed will result in an upset condition,

and a value below one indicates acetate accumulation and

a condition approaching failure (Zahller et al., 2007).

2.3. Wastewater treatment plant samples

Anaerobic digesting sludge samples were obtained from five

municipal wastewater treatment plants for this study: West

Point Treatment Plant (WP) in Seattle, WA; South Treatment

Plant (SP) in Renton, WA; Chambers Creek Wastewater

Treatment Plant (CC) in University Place, WA; the Annacis

Island Treatment Plant (AI) in Vancouver, BC; and the Central

Treatment Plant in Tacoma, WA (Tac). All digesters treated

mixed primary and secondary waste sludges with VS

concentrations that ranged from 3.0 to 5.6%. Temperature-

phased anaerobic digestion pilot plants were sampled at WP

and SP. Three temperature-phased digesters are used at Tac,

which has an initial autothermal aerobic digester. Annacis

Island digesters are a two-stage thermophilic system.

Samples were collected and immediately transported to labs

at the University of Washington where Vmax,ac tests were

performed.

2.4. Lab-scale digester operation

One bench-scale digesters was operated for this study and

was used to measure the effects of digester upset. The digester

was completely mixed, maintained in a 35 �C constant

temperature chamber, and seeded with 4 L of digesting sludge

from the WP. The digester had an operating volume of 4-L,

a total volume of around 6-L and was manually fed thickened

raw sludge (50% primary sludge and 50% waste activated

sludge) daily to maintain a 10 day SRT. An upset condition was

induced by daily dosing with CuCl2. The digester received

40 mg/Lreactor CuCl2 on day 0, followed by 20 mg/L on days 1–4,

30 mg/L on day 5, 40 mg/L on day 6, 30 mg/L on day 7, 60 mg/L

on day 8 and 50 mg/L on days 9–14.

2.5. Analytical methods

VS were measured according to Standard Methods (APHA

et al., 1995). The COD was determined according to a modifi-

cation of Standard Methods 5220 D by adding 2 mL of diluted

sludge to a HACH high range (0–1500 mg COD/L) COD vial and

heating the vial for 2 h at 150 �C. The COD was measured with

a HACH DR/4000U spectrophotometer (Loveland, CO). The pH

was determined with a Corning general purpose combination

probe and a Beckman B11 pH meter (Beckman Instruments,

Inc., Fullerton, CA). The alkalinity was determined by titrating

a 1:10 dilution of the sludge to pH 4.5 by the addition of

0.2 N H2SO4. This method followed Standard Methods 2320B.

VFAs and gas composition were measured by gas chro-

matography. The VFA concentrations were determined using

a Hewlett–Packard free fatty acid phase (FFAP) capillary

column (Agilent Technologies, Wilmington, DE) attached to

a flame ionization detector (FID) with helium as the carrier

gas. The oven temperature started at 80 �C and after 1 min

increased to 120 �C at 20 �/min and then ramped to 205 �C at

10 �/min. The methane and carbon dioxide were determined

by injecting 0.1 mL of reactor headspace onto a Hayesep

packed column (Supelco, Bellefonte, PA) attached to a thermal

conductivity detector (TCD) with helium as the carrier gas.

The oven temperature for the TCD was 110 �C. Gas phase

hydrogen samples were taken by injecting headspace samples

into a Carle Series 100 AGC Gas Chromatograph (Chandler

Engineering, Tulsa, OK) with a reduction gas detector (RGD)

(SRI Instruments, Torrance, CA). The carrier gas was N2, and

the oven temperature was maintained at 110 �C.

2.6. Acetotroph modeling

A model that combines Monod kinetics for aceticlastic growth

and overall first order conversion of degradable substrates

allows prediction of the effects of SRT, temperature and

staging in completely mixed digesters and with comparison to

Vmax,ac and ACN measurements, helps to interpret the

differences that we observe. The aceticlast growth equation

relates the biomass growth rate of the aceticlasts (rxa) to

biomass yield (Y ), kd, substrate utilization rate (rS) and Xa. The

aceticlastic biomass in digesters can be estimated from a mass

balance equation for a completely mixed reactor with no

influent biomass (Eq. (3)), incorporating biomass growth and

endogenous decay.

VdXdt¼ �QXa þ ½YðrSÞ � kdXa�V (3)

where V¼ volume of the digester (L) and Q¼ volumetric flow

rate (L/d).

Assuming a completely mixed, continuous flow reactor at

steady state, dX=dt equals 0, rS equals DS divided by the SRT,

and Eq. (3) can be solved for Xa, which in turn can be

substituted into Eq. (1) to determine Vmax,ac, assuming that

acetate consumed equals 70% of the COD reduction.

Vmax;ac ¼ kYð0:7DSÞ

1þ kdSRT(4)

The DS (or COD destruction) of a first stage (or single stage)

digester can be modeled by a first order relation (Batstone

Page 4: Monitoring the role of aceticlasts in anaerobic digestion: Activity and capacity

w a t e r r e s e a r c h 4 2 ( 2 0 0 8 ) 4 8 9 5 – 4 9 0 44898

et al., 2002). The mass balance for conversion of degradable

substrate in a completely mixed reactor at steady state is:

QS0 ¼ QSþ K1SV (5)

which can be simplified to solve for the amount of substrate

that is degraded in terms of the influent substrate (S0):

DS ¼ S0

�K1SRT1

1þ K1SRT1

�(6)

where S0¼ influent degradable COD (mg COD/L), DS¼ de-

gradable COD (mg COD/L) removed in a first or single stage

digester and K1¼ first order degradation coefficient (d�1).

The assumption that sludge degrades with a first order rate

equal to K1 is an approximation to describe the overall VS

reduction (Batstone et al., 2002) and simulates the rate-

limiting conversion of particulate degradable COD to soluble

intermediates, which are further stoichiometrically converted

to acetate. A single first order rate probably underestimates

degradation in short SRT systems (Conklin et al., 2004; Straub

et al., 2006) and overestimates degradation in second stage

digesters. An equation for Vmax,ac in a first stage digester can

be developed by substituting Eq. (4) into Eq. (6) (Eq. (7)).

Vmax;ac ¼ k

"0:7Y

�S0

hK1SRT1

1þK1SRT1

i1þ kdSRT1

#(7)

The estimate of DS from Eq. (6) assumes that all acetate

created by degradation of particulate organics will be further

converted to methane by aceticlastic methanogens. In fact,

the methanogens grow relatively slowly and are washed out

at low SRT values. The minimum SRT can be estimated from

Eq. (8):

1SRTmin

¼ Yk� kd (8)

In the following modeling of 1st stage digesters, curves for

Vmax,ac and ACN are truncated at calculated SRTmin values for

the aceticlasts. The ACN of first stage digesters can be calcu-

lated by assuming that the Vplant,ac equals 0.7DS/SRT. With

this assumption the ACN equals (Eq. (9)):

ACN ¼ kYSRT1þ kdSRT

(9)

In staged digestion, the majority of the biodegradable

substrate available for digestion is consumed in the first stage,

resulting in decreased biomass in the second stage due to little

growth and significant endogenous decay. The decrease in

Vmax,ac from the first to the second stage can be estimated

from mass balance equations on both digesters. The Vmax,ac of

the second stage is estimated by conducting a mass balance

for second stage digestion, which includes aceticlastic

biomass in the influent Xa1 (Eq. (10))

Xa1 ¼Yð0:7� S0

hK1SRT1

1þK1SRT1

i1þ kdSRT1

(10)

and assumes further first order degradation of the degradable

COD. The substrate utilization rate of DS2 can be represented

by Eq. (11).

DS2 ¼S0

1þ KISRT1��

K1SRT2

1þ K1SRT2

�(11)

By substituting Eqs. (4), (10) and (11), the Vmax,ac of the second

stage digesters can be estimated as described in Eq. (12).

Vmax;ac ¼ k

2640:7Y

�S0

hK1SRT1

1þK1SRT1

i1þkdSRT1

þ 0:7Y�

S01þK1SRT1

hK1SRT2

1þK1SRT2

i1þ kdSRT2

375 (12)

3. Results

3.1. Developing the Vmax,ac test

In order to develop a method to measure the Vmax,ac, it was

important to verify that the method was in fact measuring the

maximum acetate utilization rate and that test conditions

were not inhibiting methanogenesis. The effects of three

parameters were tested: Naþ toxicity, shaking during incu-

bation and refrigerated storage.

Previous research found that high concentrations of

cations were toxic to aceticlastic methanogens and that the

toxicity of the specific cations varied (Kugleman and McCarty,

1965; McCarty and McKinney, 1961). The effects of Naþ and

Ca2þ cation toxicity on digester sludge from WP were deter-

mined by adding digested sludge to serum bottles along with

either 75 mM of sodium acetate (NaCH3COO) or 37.5 mM

calcium acetate (Ca(CH3COO)2). This test found that sodium

acetate was degraded more rapidly ð1:05 LCH4=Lsludge dÞ than

calcium acetate ð0:76 LCH4=Lsludge dÞ. The inhibitory concen-

tration of Naþ for aceticlastic methanogens in digester sludge

from WP was determined by adding digester sludge and

75 mM sodium acetate to serum bottles along with various

concentrations of NaCl (0–304 mM). The results indicated

that 100 mM Naþ was only slightly inhibitory and caused

a 4% decrease in the acetate utilization rate, while Naþ

concentrations in excess of 200 mM resulted in greater than

90% decrease in acetate utilization rates. The results of these

two experiments lead to the conclusion that sodium acetate

was suitable for the test method at concentrations below

100 mM.

The degree of shaking during incubation may affect the

ability of aceticlastic methanogens to use the added acetate

and thus affect the Vmax,ac. This effect was determined by

measuring the Vmax,ac for sludge from WP at three different

shaking rates. This test found that sludge incubated with

shaking intensities of 0, 75 and 200 rpm had Vmax,ac values of

1.18� 0.05, 1.29� 0.05 and 1:34� 0:03 LCH4=Lsludge d, respec-

tively. These results showed that shaking did increase the

Vmax,ac of the digester sludge, but that an insignificant

increase was gained by increasing the shaking intensity from

75 rpm to 200 rpm. A shaking intensity of 150 rpm was chosen

for further tests.

The effects of refrigerated storage were determined by

performing a Vmax,ac test on sludge samples from WP digesters

and then refrigerating the sample for a day and repeating the

test. Refrigerated storage decreased the Vmax,ac by 11 to 17%

(differences significant with 95% confidence). From this data,

it was concluded that refrigerated sludge storage should be

avoided, and when possible, the Vmax,ac test should be

conducted immediately after sampling.

Page 5: Monitoring the role of aceticlasts in anaerobic digestion: Activity and capacity

w a t e r r e s e a r c h 4 2 ( 2 0 0 8 ) 4 8 9 5 – 4 9 0 4 4899

3.2. Application of Vmax,ac test to monitoringdigester upsets

3.2.1. Induced failure of a lab-scale digesterThe effectiveness of the Vmax,ac test in monitoring digester

capacity during failure was tested by inducing a digester upset

condition in a 10-day SRT, bench-scale digester. During the

course of the upset, the Vmax,ac along with other stability

parameters were monitored (acetate, headspace methane

fraction, headspace hydrogen concentration, biogas flow rate,

alkalinity and pH).

In Fig. 1 the progression of each measured parameter over

the 14 day CuCl2 addition is shown. The first noticeable effect

of inhibition was seen after 9 days of CuCl2 additions when the

Vmax,ac began to decline and the acetate concentration rose

above 100 mg/L. During the first 8 days, the Vmax,ac averaged

2:20 LCH4=Lsludge d and ranged from 2:15 LCH4=Lsludge d to

2:29 LCH4=Lsludge d. On day 9, the Vmax,ac dropped by

10% to 2:0 LCH4=Lsludge d (a difference that was significant with

95% confidence), followed by failure on day 12 with Vmax,ac of

1:3 LCH4=Lsludge d and ACNs less than 1. The steady state

0

0.5

1

1.5

2

2.5

0 2 4 6 8 10 12 14

Time(days)

Vm

ax

,a

c (L

/L

-d

), A

CN

0

300

600

900

1,200

1,500

Acetate (m

g/L

)

Vmax,acACNAcetate

0

10

20

30

40

50

60

70

0 2 4 6 8 10 12 14

Time(days)

CH

4 (%

), H

2 (p

pm

)

0

2

4

6

8

10

12

14

Gas P

ro

du

ctio

n (L

/d

ay)

CH4H2Gas Production

5.5

6

6.5

7

7.5

8

8.5

0 2 4 6 8 10 12 14

Time(days)

pH

0

2,000

4,000

6,000

8,000

10,000

12,000 Alk

alin

ity

(m

gC

aC

O3/L

)

pHAlkalinity

a

b

c

Fig. 1 – Response of traditional stability measurements to

an induced pilot-scale digester failure. Note: X-axis shows

time from the start of the induced CuCl2 digester failure

test. (a) Shows the response of Vmax,ac, ACN and acetate

concentrations. (b) Shows the response of Gas Production,

headspace methane and hydrogen concentrations. (c)

Shows the response of pH and alkalinity (Conklin et al.,

2005).

Vplant,ac was determined from steady state COD destruction

data and equaled 1:4 LCH4=Lsludge d. The ACN equaled 1.5 for

the first 8 days of the CuCl2 addition and began to decrease (as

the Vmax,ac decreased) on day 9. The alkalinity, headspace

methane content, biogas production rate and pH were within

normal operating ranges after 9 days of CuCl2 addition and

began to decrease after 11 days of CuCl2 addition. Headspace

hydrogen concentrations showed no pattern that would be

useful with regard to process control. The most useful indi-

cators of the initial stages of inhibition were increased acetate

concentrations coupled with decreased Vmax,ac values.

3.2.2. Full-scale digester upsetThe Vmax,ac test was also used to monitor the recovery of

a full-scale digester from an upset condition. After March 6,

2002, the volatile acids in WP Digester 4 increased from stable

values around 60 mg/L to a peak concentration of 1200 mg/L

(20 mM as acetic acid). At this point the digester operators

decreased feeding, and the volatile acids gradually decreased

(Fig. 2a). Once the volatile acids were back to steady state

levels, feeding was gradually increased to the steady state rate

of around 60 kgal/d, corresponding to a 30 day SRT. However,

after 20 days of normal operation, the volatile acids again

increased and peaked at 1800 mg/L (30 mM/L as acetic acid).

Again the digester operators decreased the feeding rate and

the volatile acids gradually dropped (Fig. 2a).

0

20

40

60

80

100

0 50 100 150

Time from 3/1/02 (days)

Feed

in

g R

ate (kg

al/d

ay),

Vo

latile A

cid

s as A

cetic A

cid

(m

M/L

)

0

0.2

0.4

0.6

0.8

1

Vm

ax

,a

c (L

CH

4/L

slu

dg

e-d

)FeedRateVolatileAcidsVmax,ac

0

0.2

0.4

0.6

0.8

1

1.2

30 130 230 330 430

Time from 3/1/02 (days)

Vp

la

nt,a

c an

d V

ma

x,a

c

(L

CH

4/L

slu

dg

e-d

)

Control Digester Vmax,ac Dig 4 Vmax,ac

Control Digester Vplant,ac Dig 4 Vplant,ac

a

b

Fig. 2 – Vmax,ac monitoring of an unplanned full-scale

digester upset. Note: (a) shows the Digester 4 feeding rate,

volatile acids and Vmax,ac for 150 days after the first

digester upset. (b) Shows the continued response of Vmax,ac

and Vplant,ac for Digester 4 and the control digesters for 430

days after the upset in Digester 4 (Conklin et al., 2005).

Page 6: Monitoring the role of aceticlasts in anaerobic digestion: Activity and capacity

w a t e r r e s e a r c h 4 2 ( 2 0 0 8 ) 4 8 9 5 – 4 9 0 44900

At the peak in the second upset (day 54), the Vmax,ac

monitoring began (Fig. 2a). Based on the acetate concentra-

tion, by day 70 (10 days after the second acetate peak) the

digester might be assumed to be have recovered from the

upset. However, Vmax,ac values were very low, indicating that

the digester would be able to use only a small portion of the

normal feed load. The increase in Vmax,ac rates followed the

same trend as the increase in feeding rate, which was

controlled by the operators (Fig. 2a). The possibility of another

digester failure was minimized by slowly increasing the

feeding rate.

In order to compare the effects of the upset in Digester 4

with the measured Vmax,ac values, Vmax,ac of comparable

digesters was monitored at the same time (Digesters 2 and 5 in

Fig. 2b). The results of the testing show that the Vmax,ac of

Digester 4 was consistently below the Vmax,ac of the reference

digesters, even a year after the upset.

The Vplant,ac for reference digesters and Digester 4 was

similar after 120 days (Fig. 2b). However, the Vmax,ac for the

reference digesters was always higher than for Digester 4. The

ACN of Digester 4 averaged 1.3� 0.14, while the ACN of the

reference digesters (Digesters 2 and 5) averaged 1.6� 0.16.

3.3. Application of Vmax,ac test to full- and pilot-scaledigesters

The Vmax,ac test method was used for various pilot and full-

scale digesters, including the WP digesters, both stages of the

WP pilot plant temperature-phased anaerobic digesters

(TPAD, thermophilic phase followed by a mesophilic phase)

(WP TPAD1 and WP TPAD2, respectively), the SP digesters (in

Renton, Washington), both stages of the SP TPAD pilot plant

(SP TPAD1 and SP TPAD2, respectively), both stages of the SP

mesophilic staged digestion test (SP 1 and SP 2), the three

staged digesters at the Central Wastewater Treatment Plant

Table 1 – Vplant,ac, Vmax,ac and ACN values for anaerobic digest

Source Temp.(�C)

Stage N Vmax,ac

ðLCH4=Lsludge dÞVplant,ac

ðLCH4=Lsludge

SP 1 35 1st 3 1.17 (0.1) 1.0 (0.1)

CC 37 1st 1 1.09 (NA) 0.8 (NA)

WP 35 1st 46 0.93 (0.1) 0.6 (0.1)

CC 35 1st 2 0.88 (0.0) 0.4 (0.0)

SP 35 1st 4 0.68 (0.0) 0.4 (0.0)

WP TPAD1 55 1st 1 2.17 (NA) 2.8 (NA)

Tac 1 55 1st 4 1.49 (0.8) 1.1 (0.3)

SP TPAD1 55 1st 2 1.32 (0.1) 1.2 (0.2)

AI 1 55 1st 2 1.79 (0.2) 0.6 (0.1)

Tac 2 38 2nd 1 0.16 (NA) 0.1 (NA)

WP TPAD2 35 2nd 1 0.11 (NA) 0.2 (NA)

SP TPAD2 35 2nd 3 0.29 (0.1) 0.1 (0.1)

Tac 3 35 3rd 2 0.09 (0.0) 0.1 (0.0)

SP 2 35 2nd 4 0.38 (0.0) 0.0 (0.0)

AI 2 55 2nd 2 0.83 (0.5) 0.2 (0.0)

Notes: N¼number of samples analyzed, SP¼ South Treatment Plant, CC

Treatment Plant, AI¼Annacis Island Treatment Plant, TPAD¼ tempe

SRT¼ solids retention time.

Values provided in parenthesis are standard deviations of the N samples

(Tac 1 (thermophilic), Tac 2 (unheated, 38 �C) and Tac 3

(mesophilic)), the first and second thermophilic stages from AI

(AI 1 and AI 2, respectively) and CC. The operating conditions

of the digesters at the time the samples were collected are

summarized in Table 1.

Of the digestion systems tested, the majority of the Vmax,ac

tests were performed on the WP digester system. A total of 46

Vmax,ac tests were performed on these five digesters over

a period of 3 years, 2003–2005. The average Vmax,ac was

0:93 LCH4=Lsludge d, and the average standard error of each

Vmax,ac test was 0:03 LCH4=Lsludge d. This low standard error

indicates that the test is reproducible. Additionally over the 3

years of testing the standard deviation of all the tests was

0:13 LCH4=Lsludge d, indicating that the digestion system was

stable over this time period. A similar conclusion can be based

on ACN values, which averaged 1.7� 0.3 for all WP digesters

over the 3 year period, indicating stability of the digestion

system. The ACN values indicate the aceticlastic metha-

nogens were normally functioning at about 59% of their

maximum capacity, a value closer to capacity than measured

by Kaspar and Wuhrmann (1978).

The results from the Vmax,ac tests (Table 1) suggest that the

digester Vmax,ac was affected by three parameters: digester

temperature, digester SRT and staging in addition to degrad-

able COD of the feed sludge (S0). For 1st stage or single stage

digesters, the Vmax,ac for thermophilic digesters ð1:6� 0:6

LCH4=Lsludge dÞ was greater than the Vmax,ac for mesophilic

digesters ð0:9� 0:2 LCH4=Lsludge dÞ. The Vmax,ac for second stage

mesophilic digesters ð0:3� 0:3 LCH4=Lsludge dÞ was much lower

than for first stage digesters ð1:0� 0:3 LCH4=Lsludge dÞ. All

differences were significant with 95% confidence. There also

was an apparent decrease in Vmax,ac with SRT for both meso-

philic and thermophilic digesters that will be discussed later.

These observations are qualitative since Vmax,ac depends on S0,

which varied between plants and was not directly measured.

er measured in this study

dÞACN SRT

(days)VS Load(kg/m3 d)

VSR (%) Max VS Load(kg/m3 d)

1.2 (0.1) 17 (1) 3.5 (0.3) 64 (1) 4.2 (0.2)

1.3 (NA) 18 (NA) 2.7 (NA) 68 (NA) 3.6 (NA)

1.7 (0.3) 26 (3) 2.0 (0.3) 64 (3) 3.3 (0.5)

2.0 (0.1) 31 (2) 1.4 (0.1) 66 (2) 2.9 (0.1)

1.6 (0.2) 39 (2) 1.5 (0.2) 65 (3) 2.3 (0.1)

0.8 (NA) 4 (NA) 10.9 (NA) 57 (NA) 8.4 (NA)

1.3 (0.3) 8 (1) 4.1 (1.0) 58 (2) 5.7 (2.8)

1.1 (0.1) 13 (3) 3.9 (0.8) 62 (3) 4.4 (0.3)

3.0 (0.0) 18 (1) 2.1 (0.1) 58 (0) 6.4 (0.2)

1.5 (NA) 8 (NA) 1.5 (NA) 13 (NA) NA

0.5 (NA) 8 (NA) 2.3 (NA) 30 (NA) NA

2.6 (1.9) 16 (0) 1.2 (0.2) 21 (14) NA

1.1 (0.5) 7 (2) 1.5 (0.5) 12 (1) NA

8.3 (2.1) 18 (1) 1.2 (0.1) 6 (3) NA

4.4 (1.5) 6 (1) 2.9 (0.0) 10 (3) NA

¼Chambers Creek, WP¼West Point Treatment Plant, Tac¼ Tacoma

rature-phased anaerobic digestion, VSR¼ volatile solids reduction,

.

Page 7: Monitoring the role of aceticlasts in anaerobic digestion: Activity and capacity

w a t e r r e s e a r c h 4 2 ( 2 0 0 8 ) 4 8 9 5 – 4 9 0 4 4901

ACN values do not depend on S0, but are less precise than

Vmax,ac, because of dependence on plant data and seldom-

measured conversion factors for VS to COD.

The digester ACN values are also displayed in Table 1.

For first stage or single stage digesters, the ACN for the 18-

day thermophilic digester (AI 1) was 3.0, which is signifi-

cantly higher than values of 1.2 and 1.3 (SP1 and CC) for 17

and 18 day SRT mesophilic digesters. ACNs for the ther-

mophilic digesters with SRTs less than 20 days were much

lower than for the lower SRT mesophilic digesters (WP

TPAD1 at 0.8, Tac 1 at 1.3, SP TPAD1 at 1.1). Values for low

SRT thermophilic digesters (less than 13 days) were 0.8, 1.1

and 1.3 (WP TPAD1, SP TAPD 1 and Tac 1, respectively).

Thus, mesophilic digesters with SRTs less than 20 days and

thermophilic digesters with SRTs less than 13 days were

found to have very little excess capacity.

ACN values for second stage digesters were often high,

indicating considerable excess capacity, but two values were

low (less than 1), likely because of inaccurate estimates of

Vplant,ac.

The ACN value can be used to estimate the maximum VS

load that first stage anaerobic digesters can process. The max

VS load is simply the ACN multiplied by the average VS load

(Eq. (13)). The VSmax for the sampled digesters are shown in

Table 1.

VSmax

�kg VSm3 � d

�¼ VS Load

�kg VSm3 � d

��ACN (13)

Table 2 – Monod and hydrolysis parameters used topredict Vmax,ac and ACN values

Mesophilic Thermophilic

k (mgCOD/mgVSS d) 6a 18a

kd (d�1) 0.082b

Y (gVSS/gCOD) 0.032c

KI (d�1) 0.33d 0.34d

Degradable fraction 0.75e

a Batstone et al. (2002).

b Average value measured for digester sludge.

c Measured for a mesophilic culture of Methanosaeta sp. (Conklin,

2004).

d Moen et al. (2003).

e Newton (1999).

4. Discussion

There has long been some controversy about the fate of

acetate in methanogenic systems. It is widely recognized that

60–70% of the energy flow from complex substrates to

methane is via acetate as an intermediate. There are only two

genera of methanogens that transform acetate to methane,

Methanosaeta and Methanosarcina, and often one or two species

will predominate in an anaerobic system. With the high

maximum specific substrate utilization rate (k), half satura-

tion coefficient (KS) and decay coefficient (kd), Methanosarcina

will dominate when acetate concentrations are high (above

approximately 100 mg/L). However, Methanosaeta with a low k,

KS and kd values is expected to dominate when acetate

concentrations are at levels typically found in mesophilic

digesters. Acetate can also be oxidized by syntrophic bacteria,

associated with hydrogenotrophic methane production. This

pathway has been shown to be more important in thermo-

philic than in mesophilic digestion. Petersen and Ahring (1991)

and Griffin et al. (1998) have shown that the aceticlastic genera

were relatively less common in a thermophilic digester than

in a mesophilic digester. However, Zinder et al. (1984) found in

a thermophilic digester that about 2/3 of methane produced

from a municipal solid waste feed was from aceticlasts, and

Karakashev et al. (2006) found that when Methanosaetaceae

were present in thermophilic and mesophilic anaerobic

reactors, aceticlastic methanogenesis predominated. In work

related to this study most digester samples were analyzed by

quantitative polymerase chain reaction methods for the

number of 16S rRNA gene copies for Methanosaeta and Meth-

anosarcina. Abundant aceticlasts were found in all reactors

with gene copy numbers ranging from 4� 107 to

8� 108 copies/mL, indicating likely aceticlastic activity in both

mesophilic and thermophilic digesters. Methanosaeta was

present at 25 to 1000 times the concentration of Meth-

anosarcina in the mesophilic systems, while the ratios ranged

from 16 to 1.6 in the thermophilic digesters. The overall

predominance of methane production through acetate seems

clear, and in our system very likely is via aceticlastic

methanogens.

This research found that the Vmax,ac of a digester was

affected by three parameters: SRT, temperature and staging,

and the ACN was affected by SRT and staging. The model for

aceticlastic activity (more properly, acetotrophic activity) was

used to explore these relations, using kinetic coefficients lis-

ted in Table 2.

Using Eqs. (7) and (9) and the kinetic coefficients listed in

Table 2, a relationship between Vmax,ac, ACN and SRT can be

developed and is shown in Fig. 3 for mesophilic digestion with

a typical S0 of 78,000 mg COD/L. The measured and calculated

Vmax,ac values for the mesophilic digesters are similar (Fig. 3a),

and both calculated and measured values decrease with SRT.

The washout SRT for methanogens is around 7 days, and thus

the low SRT portions of the curves are not shown. For SRTs

above approximately 7 days the extent of COD hydrolysis

begins to plateau, the decay terms tends to reduce Xa, and

Vmax,ac decreases with increasing SRT. Calculated and

measured ACN values, on the other hand, increase with

increasing SRT (Fig. 3b), since Vplant,ac decreases more rapidly

than Vmax,ac with SRT.

It was seen that second stage digesters had very low Vmax,ac

rates and high ACN values for mesophilic and thermophilic

digestion systems. Fig. 4a displays the Vmax,ac predictions for

mesophilic series configuration with equal SRT in both stages,

along with measured Vmax,ac values from four sets of samples

from series operation at South Plant. Both 1st and 2nd stage

results fit model predictions when kd values are between 0.08

and 0.1 d�1. These high kd values are consistent with our

measurements (Conklin, 2004), using a modified Vmax,ac test

method, but they are higher than values commonly used in

digestion models, which may range from 0.02 to 0.05 d�1.

Because aceticlasts grown in the first stage enter the

second stage digester, the Xa values for the second stage

Page 8: Monitoring the role of aceticlasts in anaerobic digestion: Activity and capacity

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 5 10 15 20 25 30 35 40 45 50

SRT, days

Vm

ax,ac,L

CH

4/L

slu

dg

e-d

ay

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 5 10 15 20 25 30 35 40 45 50

SRT,days

AC

N

a

b

Fig. 3 – Full- and pilot-scale Vmax,ac and ACN

Measurements. Note: data points represent measured fist

stage Vmax,ac (a) and ACN (b) values from nine full- and

pilot-scale digesters from around the North West. Solid

line shows the model results, using the kinetic coefficients

in Table 2 and a typical initial substrate concentration of

78,000 mg COD/L.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 5 10 15 20 25 30 35 40

SRT (days)

Vm

ax

,a

c (L

CH

4/L

slu

dg

e/d

ay

)

1st Stage2nd StageSP 1SP 2

0

2

4

6

8

10

12

0 5 10 15 20 25 30 35 40

SRT (days)

AC

N

1st Stage2nd StageSP 1SP 2

a

b

Fig. 4 – Measured and predicted Vmax,ac and ACN for 1st and

2nd stage mesophilic digesters. Note: data points represent

measured first and second stage Vmax,ac (a) and ACN (b)

values from the SP series operation pilot-scale digesters.

Solid line shows the model results, using the kinetic

coefficients in Table 2 and an initial substrate

concentration of 78,000 mg COD/L.

w a t e r r e s e a r c h 4 2 ( 2 0 0 8 ) 4 8 9 5 – 4 9 0 44902

digester (and Vmax,ac values) might be expected to be higher

than the first stage. However, in the second stage, both the

Vmax,ac and Vplant,ac values are depressed. The Vplant,ac rates

are considerably more affected due to the low VS destruction

rates in the second stage, so ACN values are high. This trend is

shown with the measured values listed in Table 1 and the

measured and modeled data presented in Fig. 4b.

The Vmax,ac values for mesophilic second stages of TPAD

systems are influenced by the reduced temperature, as well as

factors incorporated in Eq. (11). Effects of temperature on rates

of microbial processes are often modeled with the Arrhenius

equation. We found that the Vmax,ac for a thermophilic sludge

decreased by 73% when incubated at 35 �C (Conklin, 2004).

This decrease corresponds to a temperature coefficient (q) of

1.067, which is similar to values presented in the literature

(Wu et al., 1995). Using q equal to 1.067, and accounting for the

effects of staging (Eq. (11)) for the three temperature-phased

systems, the calculated ratios of Vmax,ac values for second

stage digester to the first stage digesters were 0.28, 0.17 and

0.25 for the WP TPAD, SP TPAD and the Tacoma digesters,

respectively. These ratios are significantly higher than the

measured ratios of 0.05, 0.12, 0.03 and 0.1 for the WP TPAD, SP

TPAD and Tacoma digesters (first and second measurements),

respectively. This difference could be due to a lack of

understanding of the hydrolysis rates (KI) or decay rates for

thermophilic sludge. As KI and kd values increase, the ratio of

the Vmax,ac values for the second stage to the first stage

decrease.

5. Conclusions

This paper found that the Vmax,ac gives a good indication of the

maximum acetate utilization rate for the acetotrophic

activity, which is likely dominated by aceticlastic metha-

nogens. For the best-characterized West Point digesters, the

average method error was approximately 3% of the average

Vmax,ac. Additionally over 3 years of testing (after the 2002

upset) the standard deviation of all the tests was

0:13 LCH4=Lsludge d, indicating that the digestion system was

stable over this time period.

Vmax,ac provides an additional tool to assess digester

conditions and changes in operating conditions. It does not

Page 9: Monitoring the role of aceticlasts in anaerobic digestion: Activity and capacity

w a t e r r e s e a r c h 4 2 ( 2 0 0 8 ) 4 8 9 5 – 4 9 0 4 4903

displace other monitoring tools, but because it allows a means

to understand the state of the aceticlastic methanogenic

population, it provides additional advantages that are useful

for controlling operation and performance. These are:

1. It can indicate the degree of damage to a digester due to

a toxic shock or feed upset.

2. It can assess the recovery condition of a digester after an

upset.

3. It can be used to determine safe loading changes when

higher feeding conditions are desired.

4. It provides a measure of the digester reserve capacity and

risk for upset.

Acknowledgments

This research was funded by the National Science Foundation

grant number BES-0332118 and the King County Department

of Natural Resources and Parks, Advanced Wastewater

Technology Program.

Anne Conklin, Jeffrey Zahller and Tom Chapman were

students at the University of Washington when the research

was conducted. Anne Conklin is now with Carollo Engineers

in Seattle, WA, Jeffrey Zahller is with HDR Inc. in Bellevue, WA

and Tom Chapman is with Brown and Caldwell in Seattle, WA.

H. David Stensel and John Ferguson are professors in the

Department of Civil and Environmental Engineering at the

University of Washington in Seattle.

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