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Anaerobic mesophilic co-digestion of sewagesludge with glycerol: Enhanced biohydrogenproduction

M. Rivero, R. Solera, M. Perez*

Department of Environmental Technologies, Faculty of Sea and Environmental Sciences, University of Cadiz,

Avda. Republica Saharaui s/n, 11510 Puerto Real, Cadiz, Spain

a r t i c l e i n f o

Article history:

Received 4 September 2013

Received in revised form

27 November 2013

Accepted 1 December 2013

Available online 10 January 2014

Keywords:

Co-digestion

Glycerol

Sewage sludge

Two-phase process

Biohydrogen

Methane

* Corresponding author. Tel.: þ34 956 016158E-mail addresses: montserrat.perez@uca

0360-3199/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.ijhydene.2013.12.0

a b s t r a c t

Anaerobic mesophilic co-digestion of mixed sewage sludge from wastewater treatment

plants, WWTP, with crude glycerol, the major byproduct of the biodiesel industry, has been

examined using a two-phase digestion process in a semi-continuous CSTR at laboratory

scale. The objective was to determine the operational conditions that enhanced bio-

hydrogen and methane production and to evaluate the effect of the organic loading rate

(OLR) applied to the process. It was concluded that the Hydraulic Retention Time HRT of

the methanogenic stage did not have an important influence on the operational process of

co-digestion of sewage sludge and glycerol in terms of efficiency of organic removal and

biogas yield. Hence, the results obtained were 73e77% organic matter removal (as CODr)

with 0.032 LH2/gCODr and 0.16 LCH4/gCODr when the system operated at OLRs in the range

of 15.33e17.90 gCODs/L d with HRTs of 3 days in the acidogenic digester and 6, 8, and 10

days in the methanogenic digester. In terms of volatile solids, the results obtained were 92

e88% organic matter removal (as VSr) with 0.20 LH2/gVSr and 1.27 LCH4/gVSr when the

system operated at OLRs in the range of 1.94e2.79 gVS/L d.

However, the OLR had an important influence on the H2 and CH4 yields. Hence, the best

operational condition was an OLR of 7.82 gCOD/L d, with removal of 93% CODr and

hydrogen and methane yields of 0.026 LH2/gCODr and 0.29 LCH4/gCODr, respectively.

In terms of volatile solids, the best operational condition was an OLR of 1.01 gVS/L d,

with removal of 89% VSr and hydrogen and methane yields of 0.50 LH2/gVSr and 1.48 LCH4/

gVSr, respectively.

Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights

reserved.

1. Introduction

Excess sludge generation is the main problem facing waste-

water treatment plants (WWTPs). Its production has been

increasing rapidly due to growth in the number ofWWTPs due

to European demands regarding sanitation, which have been

; fax: þ34 956 016411..es, montserrat.perez@gm2013, Hydrogen Energy P06

driving an increase in the level of water treatment. The sludge

treatment accounts for over 50% of the operating costs of the

WWTP, leading to agronomic or energetic recovery.

The National Sewage Sludge Plan was established in

2007 and set a target of recovering 80% of WWTP sludge and

reducing the amount of sludge sent to landfill by 20%. The

.uca.es (M. Perez).ublications, LLC. Published by Elsevier Ltd. All rights reserved.

Nomenclature

AMD acidogenic mesophilic digester

CODr Chemical Oxygen Demand

CSTR two-phase digestion process in a semi-

continuous

HRT Hydraulic Retention Time

MMD methanogenic mesophilic digester

OLR organic loading rate

PNIR The Integrated National Waste Plan

SRT solids retention time

TPAD temperature-phased anaerobic digestion

TS total solids

VFAs volatile fatty acids

VSr volatile solids

WWTP wastewater treatment plants

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 9 ( 2 0 1 4 ) 2 4 8 1e2 4 8 82482

increase in WWTP sludge production due to the application

of both Directive 91/271/EEC [7] and the National Sewage

Sludge Plan brings with it the need to manage this

sludge correctly. Approximately 0.5e2% of the treated

water in a WWTP becomes sludge that needs to be treated

before final disposal in the environment. One of the solu-

tions that can be applied to achieve environmentally

friendly sludge management is to use this sludge as agri-

cultural fertilizer. The Integrated National Waste Plan

(PNIR) of 22 December 2008 devotes its Chapter 13 to urban

sewage sludge and sets qualitative and quantitative targets

for 2015. Thus, recovery of digestate in soil for agricultural

purposes or other types of recovery, including anaerobic

digestion, should reach 18% of the total final destination of

sludge in 2015.

Glycerol is the major byproduct of the biodiesel industry

[30]. In general, for every 100 kg of biodiesel produced,

approximately 10 kg of crude glycerol is generated. This sup-

poses the 10% of the total manufactured biodiesel [23]. Crude

glycerol generated by homogeneous base-catalyzed trans-

esterification contains approximately 50e60% glycerol,

12e16% alkalis, especially in the form of alkali soaps and hy-

droxides, 15e18% methyl esters, 8e12% methanol, and 2e3%

water. In addition to methanol and soaps, crude glycerol also

contains a variety of elements such as Ca, Mg, P, and S as well

as other components [14,27].

The price of glycerol has decreased significantly in recent

years. The continued decline is due to the increased con-

sumption of biodiesel and, therefore, increased waste itself.

However, directives of the European Union have determined

that the use of biodiesel should reach 20% of fuels used in

2020.

Despite the wide applications of pure glycerol in the

pharmaceutical, food, and cosmetic industries, the refining of

crude glycerol to a high purity is too expensive, especially for

small and medium biodiesel producers [18]. Alternate ways of

using the crude glycerol phase have recently been studied.

Possibilities such as combustion, co-burning, composting,

animal feed, thermochemical conversion, and biological

conversion have been applied to crude glycerol processing [6]

[11], [17] [20], [21] [25], [28].

Among these different options, the biological production of

methane from crude glycerol by anaerobic digestion has

several advantages [29]. Hence, several studies have demon-

strated that the use of crude glycerol as a C source for

fermentation and biogas generation is a promising alternative

for this waste material. Besides the production of methane,

the advantages include low nutrient requirements, energy

savings, and generation of a stabilized digestate which im-

proves soil quality.

Therefore, glycerol is an attractive alternative for use and

recovery through its co-digestion with other waste, as it is a

substance that is readily biodegradable and has a pH suitable

for anaerobic processes and there are a variety of microor-

ganisms that use glycerin anaerobically as a carbonated

source [6]. Also, the high C content of glycerol increases the

C:N ratio in the mixture, avoiding inhibition of the process

through an excess of N and increasing the methane produc-

tion of digesters by 50e200%. Anaerobic co-digestion of glyc-

erol and a variety of residual biomasses may be a good

integrated solution for managing these wastes and simulta-

neously producing a source of bioenergy in an environmen-

tally friendly way.

Among the objectives of the National Plan II Sludge and

Integrated National Waste Plan is the energy evaluation in

15% of sewage sludge by anaerobic digestion. Efficient man-

agement of anaerobic digestion reduces the amount of dehy-

drated sludge by 30e40% and stabilizes the sludge for

agricultural use, and subsequent recovery generates biogas

rich in about 50e60% methane, which can be used as fuel. If

45% is digested organic matter, average yields of 800e1000 m3

biogas/t digested volatile are possible and can be used for

energy recovery.

The productivity of anaerobic digesters can be enhanced by

the addition of readily biodegradable co-substrates such as

corn maize, maize silage, swine and cattle manure, municipal

solid wastes, and mixtures of wastewater from olive mills,

slaughterhouses, and potato processing [1] [9], [19]. Some re-

searches [23] show that glycerol does not constitute a suitable

substrate for obtaining high yields of biogas when it is used

alone. However, when it is digested with proper mixtures of

co-substrates, biogas production is optimized. The feasibility

and performance of anaerobic co-digestion of glycerol with

other organic waste materials has been studied to various

extents. It requires lower investment and simpler operational

conditions compared to more sophisticated preprocessing

technologies, which makes it ideal for local applications. Less

biomass sludge is produced in comparison to aerobic treat-

ment technologies. The digestate is an improved fertilizer for

plants. A source of C-neutral energy is produced in the form of

biogas.

Thus, anaerobic co-digestion of sewage sludge with glyc-

erol may be an alternative valorization treatment with tech-

nical and economical viability for energy recovery from both

wastes.

The anaerobic digestion process is affected significantly by

the environmental and operating conditions. Two-phase

anaerobic digestion allows acidogenic and methanogenic

processes to occur in the best environmental conditions and

with the best operational parameters. Each stage of the two-

phase anaerobic degradation not only applies various

Table 1 e Main characteristics of used feeds (pretreatedsewage sludge D glycerol).

Parameters Feed 1 Feed 2 Inoculum

pH 5.65 6.29 7.50

Conduct (mS/cm) 9.88 11.34

TS (g/kg) 45.02 51.28 32.00

VS (g) 34.59 39.28 18.00

COD (g O2/l) 49.41 23.45

TOC (g/l) 15.83 8.14

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reactor configurations but also uses different values of tem-

perature, pH, HRT, organic loading rate (OLR) and so on to

obtain the optimum results.

In this research, a two-phase digestion process in a semi-

continuous CSTR at laboratory scale was used to treat both

mixed sewage municipal sludge and glycerol. This process

will encourage hydrogen production in the acidogenic first

stage of the process and methane production in the meth-

anogenic second stage, in addition to producing a final prod-

uct with better digested dewaterability characteristics and

suitable for agricultural recovery. In addition, this technology

makes it possible to impose in each reactor the conditions

suitable for the microorganisms responsible for each stage,

leading to increased metabolic rates. This allows the produc-

tion of hydrogen to be maintained continuously, and the

acidogenic reactor effluent contains high concentrations of

volatile fatty acids (VFAs), which will be consumed in the

methanogenic second stage.

The objective of this study is to evaluate the potential

benefits of mesophilic co-digestion of sewage sludge mixed

with glycerol in technologically separate stages to achieve

better utilization of both wastes in terms of reduction of

organic matter for energy recovery (obtaining hydrogen and

methane).

Fig. 1 e Experimental set-up of two-phase process.

2. Material and methods

2.1. Feed and inoculum

Experimental work was carried out with sewage sludge sam-

ples (mixed primary sludge and activated sludge) from Cadiz-

San Fernando WWTP (located in Cadiz-Spain, which handles

more than 50,000 m3 of wastewater daily). All the sludge

samples were characterized on reception at the laboratory

and were kept under refrigeration at 4 �C before they were

used for the experiments so as to prevent biodegradation.

The sewage sludge used consisted of a mixture of the

sewage sludge mixed at Cadiz-San Fernando (primary sludge

and activated sludge) that had been previously chemically

pretreated by adding 10 M NaOH [3] to increase the pH to near

to 10. In these conditions the mixture remains for 2 h at room

temperature in order to solubilize the organic matter. Subse-

quently the pH was acidified by addition of HCl [31] to values

of 5.5, the optimum for the acidogenic stage of the anaerobic

digestion process. The pretreated sludge was mixed with 1%

v/v glycerol commercial household Panreac, which consti-

tuted the acidogenic reactor feed. According to Fountoulakis

et al. [10], the most appropriate concentration of glycerol in

co-digestion with sewage sludge in anaerobic processes was

1%. This study showed that the addition of glycerol can in-

crease biogas yields if it does not exceed 1% (v/v) concentra-

tion in the feed, and any further increase in glycerol causes a

large imbalance in the anaerobic digestion process [10].

Table 1 provides the average feed compositions of the

acidogenic reactor (two sludge samples obtained after chem-

ical pretreatment and mixed with glycerol). The feed to the

methanogenic digester was the acidogenic effluent, which

meant that neutralization was always necessary. Both re-

actors were initially filled with mesophilic digested sludge

from the Cadiz-San Fernando WWTP, which acts as the

inoculum in the process. The main characteristics of the

digestedmesophilic sludge used as inocula are summarized in

Table 1.

Since the amount of glycerin is constant in all feeds, vari-

ations in parameter values must be the result of changes in

the composition of the sludge, which was collected in the

WWTP periodically and could change according to the daily

operation of the plant.

2.2. Experimental set-up

The experiments were carried out using two digesters oper-

ating as completely stirred tank reactors and connected as

shown in Fig. 1. The lab-scale system consisted of a 5 L

acidogenic digester (working volume of 4.5 L) followed by a

10 L methanogenic digester (working volume of 9.0 L). Both

experimental digesters shared similar characteristics: the

cover of each reactor incorporated three separate ports for

different functions: feeding; mechanical agitation, and mea-

surement of the biogas generation (using a 20 L Tedlar bag).

The reactors were kept at the selected temperature by water

circulating in the water jacket surrounding the reactors.

2.3. Experimental procedures

Two studies were developed: a) the optimization of HRTs of

acidogenic and methanogenic reactors for the mesophilic

anaerobic co-digestion process of sewage sludge and glycerol

in a two-phase process to maximize the production of

hydrogen and methane; and b) the study of the influence of

the OLR applied to the process on hydrogen and methane

yields.

a) Optimization of HRTs of acidogenic and methanogenic

reactors for the mesophilic anaerobic co-digestion process

of sewage sludge and glycerol in a two-phase process to

maximize the production of hydrogen and methane.

Table 2 e Operational conditions in experimental TESTs.

TEST OLR gDQO/L d HRT AMD (d) Feed volume AMD (L/d) HRT MMD (d) Feed volume MMD (L/d) Testing period (d)

1 15.33 3 1.66 10 1.00 37

2 16.68 3 1.66 8 1.25 35

3 17.90 3 1.66 6 1.66 28

4 7.82 3 1.66 6 1.66 31

Control 0.53 20 0.25 55

AMD: acidogenic mesophılic digester; MMD: metanogenic mesophilic digester.

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 9 ( 2 0 1 4 ) 2 4 8 1e2 4 8 82484

For this study, three two-phase experiments were carried

out. Table 2 shows the initial operational data of the TEST

used in this research: TESTs 1, 2, and 3. The information in-

cludes the initial OLR (gCOD/L d) applied to each TEST the HRT

in each phase (days), the feed volume (L), and the period of

operation (days).

Initially, the OLR applied to the acidogenic mesophilic

digester (AMD) in TEST 1 was 15.33 gCOD/L d (1.94 gVS/L d)

with an HRT of 3 days, and these conditions were kept

constant until steady-state conditions were reached. The

methanogenic mesophilic digester (MMD) was later fed with

the effluent generated in the previous acidogenic digester

with an HRT of 10 days. The attainment of the steady state

for all stages of the study was verified after a period

equivalent to three times the HRT by checking whether

effluent characteristic values of total (TS) and volatile solids

(VS), gas production and composition (H2 and CH4), and

volatile fatty acid (VFA) levels were constant. The sampling

during each steady-state period was performed for five

consecutive days.

The other two TESTs were operated for methanogenic

HRTs of 8 and 6 days, respectively. A single-stage mesophilic

digester (5 L), whichwas operatedwith a 15-day SRT, was used

as the control system.

b) Influence of the OLR applied to the process on biodegra-

dation process efficiency and hydrogen and methane

yields.

Two TESTs were developed which operated with two

different initial OLRswhilemaintaining the sameHRTs: 3 days

for AMD and 6 days forMMD (TESTs 3 and 4). The OLRs studied

were 17.90 and 7.82 gCOD/L d, as summarized in Table 2.

Table 3 e OLR expressed as gCODs/L d, for food andeffluents of acidogenic and methanogenic digesters.

TEST 1 TEST 2 TEST 3 TEST 4 Control

Feed 15.33 16.68 17.90 7.82 0.53

AMD 13.05 14.07 14.89 4.32

MMD 4.03 3.76 3.87 0.55 0.38

Global organic

removal

COD%

73 74 77 93 29

AMD: acidogenic mesophılic digester; MMD: metanogenic meso-

philic digester.

2.4. Analytical methods

The digesters were monitored by analysing daily samples of

the influent and effluent streams. The main parameters were

determined in order to study the process stability. The pa-

rameters controlled were soluble Chemical Oxygen Demand

(COD), TS, and VS, which were determined according to the

StandardMethods [2]. The pHwasmeasured using a pH-metre

(Crison 20 Basic model), and total alkalinity was measured by

pH titration to 4.3. Volatile fatty acids (VFAs) and biogas

composition (H2, CH4, and CO2) were determined by gas

chromatography as described in Riau et al. [22].

The volume of gas produced in the reactor was measured

directly with a high precision flow gas metre e a Ritter Wet

Drum TG 0.1 (mbar) e and a Tedlar bag.

3. Results and discussion

A) Optimization of HRTS of acidogenic and methanogenic

reactors for the mesophilic anaerobic co-digestion pro-

cess of sewage sludge and glycerol in a two-phase

process.

3.1. Evolution of pH

pH values remained in the course of the biodegradation pro-

cess and fairly close to the optimal value in each reactor:

around 5e6 in the acidogenic phase, the optimal range for

acidogenic bacteria [24], and between 7 and 8 in the meth-

anogenic digester, the optimal range for methanogenic bac-

teria [25]. All TESTs showed similar behaviours.

3.2. Efficiency of organic matter removal

Table 3 shows the values of OLR expressed as gCOD/L d for

food and effluents of the AMD and MMD in the three HRT

TESTs.

As can be seen, for TEST 1 (3:10), the OLR fed was 15.33

gCODs/L d, reaching an average output with an OLR of 14.35

gCODs/L d. This represents a degradation rate of 9%. In this

acidogenic stage, most of the organic matter present in the

medium is hydrolyzed and converted into short-chain VFA,

a substrate which is readily biodegradable in the methano-

genic phase. In the methanogenic second phase, a higher

percentage of soluble COD is consumed. In this case, the

average OLR input to this stage is the acidogenic output,

that is, 14.35 gCODs/L d, while the average OLR effluent was

4.03 gCODs/L d. This represents a degradation percentage of

71% CODr. Taken together, the overall effectiveness of sol-

uble COD removal found in the overall process was 73%

CODr.

Table 4 e Main values of VFA as mg/L.

Acetic Propionic n-Butyric n-Valeric Total ac. (mg acetic ac.)

Feed 4.19Eþ02 2.43Eþ02 8.40Eþ01 8.09Eþ01 9.80Eþ02

TEST 1 AMD 3.65Eþ02 3.16Eþ03 3.69Eþ02 2.97Eþ03 9.85Eþ03

MMD 4.11Eþ02 3.23Eþ02 3.01Eþ02 1.70Eþ03 4.14Eþ03

TEST 2 AMD 3.68Eþ02 2.87Eþ03 3.03Eþ02 9.78Eþ02 6.01Eþ03

MMD 4.16Eþ02 3.12Eþ02 2.67Eþ02 8.22Eþ02 2.59Eþ03

TEST 3 AMD 4.59Eþ02 3.09Eþ03 3.50Eþ02 1.00Eþ03 6.48Eþ03

MMD 1.15Eþ02 1.73Eþ03 2.80Eþ02 6.30Eþ02 3.73Eþ03

TEST 4 AMD 2.94Eþ02 1.29Eþ03 5.29Eþ02 1.01Eþ03 4.38Eþ03

MMD 7.54Eþ01 2.38Eþ02 8.96Eþ01 7.83Eþ01 6.33Eþ02

Control 264.37 640.885 219.360 381.106 2039.438

AMD: acidogenic mesophılic digester; MMD: metanogenic mesophilic digester.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 9 ( 2 0 1 4 ) 2 4 8 1e2 4 8 8 2485

For the second TEST (3:8), the initial OLR fed to the AMD

was 16.68 gCOD/L d, reaching an average output with an OLR

of 14.07 gCOD/L d, with a clearance rate of around 17%. In the

methanogenic phase the average output had an OLR of 3.76

gCODs/L d, showing a removal rate of 68%. Taken together, the

overall effectiveness of soluble COD removal found in the

overall process was 74% CODr.

Finally, for TEST 3 (3:6), the initial OLR was 17.90 gCOD/L d

and an average effluent OLR of 14.39 gCOD/L d was reached.

The percentage removal was 19%. In themethanogenic phase,

the average effluent OLR was 3.87 gCOD/L d. This represents a

percentage removal of 71%. The overall effectiveness of sol-

uble COD removal found in the overall process was 77%.

It was clearly observed that the percentages of purification

of sludge-glycerol mixtures were very similar in the three

TESTs, increasing slightly with decreases in the HRT of the

methanogenic phase and thus reducing the overall HRT of the

process. This allows to food to be treated quickly with an OLR

in the range of 15.33e17.90 gCOD/L d, maintaining high

removal rates of the order of 73e77% for an HRT of 3:6.

In terms of VS removals, the overall organic removals were

in the range of 92e88% VSr. These values are very high in

relation to the data obtained for single-stage anaerobic mes-

ophilic digesters treating sewage sludge (only 40% VSr), but

were similar to those published by Riau et al. [22] for a TPAD 3/

15 system (78%VSr). Hence, these authors showed that the co-

digestion of sewage sludge and glycerol in the two-phase

process was found to be capable of higher VS removal at

retention times in the range of 13e9 days compared to single-

stage mesophilic or thermophilic digestion for 15 days [22].

However, the addition of glycerol on the one hand and the

two-phase process on the other hand could enhance the VS

biodegradation rate.

3.3. Evolution of volatile fatty acids (VFAs)

The concentrations of individual and total VFAs in feed and

the three TESTs are shown in Table 4 (expressed as mg acetic

acid/L). The average VFA concentration in the feed throughout

all the experiments was 908 mg of acetic acid/L; mainly acetic

acid was present in the feed.

As can be observed, all TESTs showed similar behaviours in

relation to the evolution of volatile acids. In all TESTs, the

experimental results indicated that the %VFA removals were

in the range 88e92%.

In general, VFA concentrations and the total acidity

increased during the acidogenic stage, with acetic, propionic

and valeric acids showing higher concentrations. Propionic

acid was the predominant acid in all TEST with concentra-

tions in the range of 2800e3100 mg/L.

In the methanogenic phase these values fall significantly

due to their consumption by the methanogenic microorgan-

isms responsible for this last phase of the process.

In fact, the methanogenic stage reduced VFA concentra-

tions in the effluent by 42e58%. Thus, the VFA concentration

in the effluent of AMD was found to be two orders higher

(9585e6000 mg of acetic acid/L) than that in the effluent of

AMD (4000e3000 mg of acetic acid/L). This indicated that a

significant amount of the glycerol fed to the acidogenic

digester should be partially degraded to VFAs in the first stage,

resulting in better performance in the second-stage [8]. How-

ever, the final values of total acidity were very high, indicating

the bad quality of the effluent.

However, the accumulation of VFA in MMD not influenced

the pH inMMD, and pH valueswere greater than 7 in all TESTs.

In relation with the experimental data obtained for the

control digester treating sewage sludge, the total %VFA

removal is slightly lower than the value obtained in all TESTs,

reaching 77% VFA removal in a single-stage.

3.4. Biogas generation and yields of hydrogen andmethane

Table 5 summarizes the biogas generation in both AMD and

MMD in L/L d and the percentage of H2, CH4 and CO2 in biogas

in digesters.

Biogas generation was very constant in all TESTs, showing

values in the range of 0.28e0.35 L/L d and 3.01e3.25 L/L d for

AMD and MMD respectively. This supposes global quantities

of 3.52e3.35 L/L d. Hence, the daily hydrogen generation oc-

curs exclusively in the AMD, showing values in the range of

0.07e0.09 LH2/L d.

The daily hydrogen generation occurs exclusively in the

AMD, showing values in the range of 0.07e0.09 LH2/L d. The

daily methane generation was in the range of 1.55e1.78 LCH4/

L d for TESTs 1, 2, and 3 respectively. These values are in

accordance with those published by Fountoulakis et al. [9] for

the co-digestion of sludge with 1% glycerol.

As shown in Table 5, the generation of H2 takes place

exclusively in the AMD. In this phase the registered

Table 5 e Biogas generation in acidogenic andmethanogenic phase (L/L d) and percentual biogascomposition (%).

TEST 1 TEST 2 TEST 3 TEST 4 Control

AMD. L/L d 0.28 0.34 0.35 0.37

H2 (%) 24.84 27.42 26.46 24.18

CH4 (%) 1.26 1.36 1.21 1.39

CO2 (%) 73.90 71.22 72.34 74.53

MMD. L/L d 3.25 3.01 3.16 1.71 0.38

H2 (%) 0 0 0 0 0

CH4 (%) 49.93 51.59 56.32 62.39 71.32

CO2 (%) 50.07 48.41 43.68 37.61 29.68

Total biogas L/L d 3.52 3.35 3.41 2.08 0.38

AMD: acidogenic mesophılic digester; MMD: metanogenic meso-

philic digester.

Table 6 e Hydrogen and methane yield.

LH2/gCODr LCH4/gCODr LH2/gVSr LCH4/gVSr

TEST 1 0.032 0.16 0.20 1.27

TEST 2 0.036 0.15 0.23 0.91

TEST 3 0.031 0.16 0.17 0.99

TEST 4 0.026 0.29 0.50 1.48

Control e 0.33 e 0.63

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 9 ( 2 0 1 4 ) 2 4 8 1e2 4 8 82486

components comprised mainly CO2 (71e73%), followed by H2

(25e27%), with the remainder corresponding to CH4 (approx.

1.3%), which is assumed to come from H2-utilizing microbial

populations (methanogenic archaea, acetogenic and sul-

phate-reducing bacteria).

The CH4 production occurs mainly in the methanogenic

phase (1.55e1.78 LCH4/L d). The CO2 presents percentages at or

below those for CH4 (50e56%) during all TESTs. These values

are very similar to those published by Luostarinen et al. [16]

for the anaerobic co-digestion of sewage sludge with grease

trap sludge from a meat processing plant. However, single-

stage digester control showed higher percentages of CH4,

approx. 71%CH4. It is noteworthy that in a stable conventional

single-phase reactor the CH4:CO2 ratio was approximately

70:30 while in these TESTs the ratio was approximately 50:50.

The hydrogen production was very stable and constant

during the whole process in the acidogenic digester, showing

a representative value of 0.07e0.09 LH2/L d. The stability of

hydrogen along the entire processmust be due to the presence

of glycerol, as explained by Seifert et al. [24] in their studies.

In practice, one of the best terms for judging the perfor-

mance of a digester is the specific hydrogen andmethane yield

(the volume of hydrogen or methane produced per unit of COD

consumed orVS reduced). Specifichydrogen andmethane yield

productions from each stage are shown in Table 6. The mean

overall hydrogen and methane yields were expressed as litres

of H2 or CH4 per grams of COD and VS removal.

Hydrogen yield was in the range of 0.031e0.036 or

0.17e0.23, expressed as LH2/gCODr or LH2/gVSr, respectively.

Data published by Kotay [15] on the feasibility of biohydrogen

production from sewage sludge using a defined microbial

consortium (Bacillus coagulans) indicated values of 0.037 LH2

per gram of COD removal, similar to the data found in this

study.

Methane yield was in the range of 0.15e0.16 or 0.91e1.27,

expressed as LCH4/gCODr or LCH4/gVSr, respectively. In rela-

tion with the experimental data obtained for the control

digester treating sewage sludge, the methane yield related to

COD removal is higher, reaching 0.33 LCH4/gCODr (or 0.63

LCH4/gVSr).

However, the data of methane yield published by Chen [4]

are in the range of 0.23e0.10 LCH4/gCODr. This range includes

the values obtained in our work.

In terms of VS removal, these data are higher than those

reported by Cheunbarn and Pagilla [5], who obtained 0.66 L of

CH4 per gram of VSr in mesophilic conditions. The data pub-

lished by Riau et al. [22] were also lower (between 0.52 and 0.62

LCH4/gVSr) in TPAD 3/15 studies.

In conclusion, sewage sludge and glycerol can be efficiently

co-digested in a two-phase process. Under the experimental

condition tested (OLR of 15.33e17.90 gCOD/L d), the results

obtained were 73e77% CODr, with 0.07e0.09 LH2/L d in AMD

and 1.55e1.78 LCH4/L d in MMD, showing depurative yields of

0.03 LH2/gCODr and 0.16 LCH4/gCODr (0.2 LH2/gVSr and 1.1

LCH4/gVSr).

b) Influence of the organic loading rate (OLR) applied to the

process on biodegradation process efficiency and H2 and

CH4 yields.

3.5. Efficiency of organic matter removal

Table 3 shows the values of OLR expressed as gCOD/L d for

food and effluents of the AMD and MMD in TESTs 3 and 4.

In TEST 4, the OLR fed to the acidogenic stage was 7.82

gCODs/L d, and an average effluent OLR of 3.32 gCODs/L d was

reached. This means that the organic matter was hydrolyzed

and converted into short-chain VFAs in the order of 45%CODr.

For an OLR of 17.90 gCOD/L d, the organic removal in the

acidogenic phase was only 17% CODr. In the second meth-

anogenic stage, a higher percentage of soluble COD was

consumed. In this case, the average OLR fed was the acido-

genic effluent, that is, 3.32 gCOD/L d, while the average OLR

output was 0.95 gCOD/L d. This represents 87% CODr. Hence,

the overall effectiveness of soluble COD removal in the global

process was 93%, while for an OLR of 17.90 gCOD/L d the

overall CODr reached 77%.

Clearly an important increase in the organicmatter removal

was detected in TEST 4, which showed a higher amount of

organic matter removal (93%) compared to TEST 3 (77%).

In terms of VS removal, the overall organic removal was

89% VSr, which was similar to the data obtained for TESTs

with high organic loading rates (TESTs 1, 2, and 3) and very

high in comparison with single-stage digestion of sewage

sludge [2]. Also, glycerol enhanced the VS biodegradation rate

of sewage sludge.

3.6. Evolution of volatile fatty acids (VFAs)

Table 4 shows the concentration of individual and total

VFAs in the feed and effluents of the AMD andMMD for TESTs

3 and 4.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 9 ( 2 0 1 4 ) 2 4 8 1e2 4 8 8 2487

In TEST 4, the behaviour of individual acids was similar to

that observed in the above TESTs, with propionic acid being

predominant at 1290 mg/L in AMD effluent, according to data

published by Khanal et al. [13]. This is explained by the rapid

conversion of glycerol in propionic acid [1]. In theMMD, a high

removal of all acids was detected. The individual acids were

detected at low concentrations and total volatile acid was

633 mg acetic-acid/L. This supposes a depurative efficiency of

86% in contrast with the data of TEST 3, which showed 42%

VFA removal. Hence, the fed OLR has an important influence

on propionic acid generation, which increases as the OLR

applied increases. The stability of the process could be

complicated, leading to inhibition of the process when

inhibitory concentrations are exceeded.

3.7. Biogas generation and yields of hydrogen andmethane

Table 5 shows the biogas generation in the AMD and MMD (L/

L d) and the percentage biogas composition (H2, CH4, and CO2)

for TESTs 3 and 4.

Biogas generation was 2.08 L/L d in TEST 4, with 0.37 L

biogas/L d in the AMD, slightly higher than the values ob-

tained in the above TESTs (in the range of 0.28e0.35 L biogas/

L d). However, generation in the MMD dropped to values of

1.71 L biogas/L d in relation to TEST 3 (3.16 L biogas/L d). Hence,

themain data for hydrogen andmethane generationwere 0.09

LH2/L d and 1.07 LCH4/L d respectively. The latter value is

lower compared to themethane generation obtained from the

above TESTs.

Table 6 shows the H2 and CH4 yields generated in litres per

gram of COD consumed and VS reduced. Compared to the

average values of TEST 3 at an OLR of 17.90 gCOD/L d, the

hydrogen yield in TEST 4 was very similar in terms of LH2/

gCODr, reaching 0.026 LH2/gCODr. However, although the CH4

generation was lower in these conditions (1.07 LCH4/L d), the

methane yield increased to values of 0.29 LCH4/gCODr and

1.48 LCH4/gVSr, which were very high in relation to the data

for TEST 3.

These values are considerably better than those published

by Song [26] (0.42e0.47 LCH4 per gram of VS removed) for

single-stage mesophilic anaerobic digestion of sewage sludge.

In conclusion, the co-digestion of sewage sludge and

glycerol at a low OLR (7.82 gCOD/L d) enhanced the methane

yield with respect to the above TESTs with a higher OLR (17.90

gCOD/L), providing amethane yield of 0.29 LCH4/gCODr or 1.48

LCH4/gVSr.

Also, the addition of glycerol enhanced the methane yield

in relation to single-phase mesophilic digestion of sewage

sludge in similar operational conditions [22].

4. Conclusions

Sewage sludge and glycerol can be efficiently co-digested in a

two-phase process. Under the experimental conditions tested

(OLR: 15.33e17.90 gCOD/Ld), the results obtainedwere 73e77%

CODr, with depurative yields of 0.03 LH2/gCODr and 0.16 LCH4/

gCODr (0.2 LH2/gVSr and 1.1 LCH4/gVSr). Hence, the meth-

anogenic stage was not influenced by HRT in the operational

process of co-digestion of sewage sludge and glycerol in terms

of efficiency of organic removal and biogas yield.

However, the OLR had an important influence on the H2

and CH4 yields. Hence, at an OLR of 7.82 gCOD/L d, the organic

removal was 93% CODr (89% VSr), with hydrogen and

methane yields of 0.026 LH2/gCODr or 0.50 LH2/gVSr and 0.29

LCH4/gCODr or 1.48 LCH4/gVSr, respectively.

Acknowledgements

The authors wish to express their gratitude to the Spanish

Ministry for the Environment, Rural Affairs and Marine Policy

(Project 148/PC08/3e04.3) for providing financial support.

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