ARTICLE IN PRESS

I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y 33 (2008) 3651 – 3659

Available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/he

Biohydrogen production by anaerobic co-digestion of

municipal food waste and sewage sludges

Heguang Zhua,b,, Wayne Parkerb, Robert Basnarc, Alexander Prorackic, Pat Fallettaa,
Michel Bélanda, Peter Setoa
a
Aquatic Ecosystem Management Research Division, Water Science and Technology, Environment Canada,
867 Lakeshore Road, P.O. Box 5050, Burlington, Ontario, Canada L7R 4A6
b
Civil and Environmental Engineering, University of Waterloo, 200 University Ave. W., Waterloo, Ontario, Canada N2L 3G1
c
Chemical Engineering, University of Waterloo, 200 University Ave. W., Waterloo, Ontario, Canada N2L 3G1

art i cle info ab st rac t

Article history: Food waste (FW), primary sludge (PS) and waste activated sludge (WAS) were characterized
Received 1 May 2007 and found to be complementary in the concentrations of carbohydrates, total Kjeldahl
Received in revised form nitrogen (TKN), PO4–P and some metal for biological hydrogen production. Moreover, FW
29 January 2008 was found to have low pH buffering capacity while the values for PS and WAS were
Accepted 20 April 2008 relatively higher. An anaerobic toxicity analysis (ATA) derived from a methanogenic ATA
Available online 6 June 2008 protocol showed that these waste materials had no toxicity to hydrogen production.
Adding phosphate buffer to the FW significantly improved hydrogen production while
Keywords:
initial pH was 7.0. Co-digestion of FW and sewage sludge was studied using a batch
Hydrogen production
respirometric cultivation system. All combinations of the feedstocks (FW+PS, FW+WAS and
Co-digestion
FW+PS+WAS) showed enhanced hydrogen production potential as compared with the
Anaerobic toxicity assay
individual wastes. A mixing ratio of 1:1 was found to be the best among the ratios tested for
pH buffering effect
all three co-digestion groups. A hydrogen yield of 112 mL/g volatile solid (VS) added was
obtained from a combination of FW, PS and WAS. This yield was equivalent to 250 mL/g VS
added if only FW contributed to hydrogen production. The reason for the enhancement of
hydrogen production was postulated to be multifold in which the increase in buffer
capacity in the co-digestion mixture was verified.
& 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights
reserved.

1. Introduction factors can influence the bacterial growth, fermentative

Fermentative hydrogen production has been reported from a
variety of substrates including glucose [1–3], sucrose [4,5],
starch [6,7], cellulose [8–10] and waste materials containing
these saccharides [11,12], using pure or mixed cultures. The
efficiency of hydrogen production can be affected by many
factors such as type of inoculum, substrate composition, pH,
organic loading and hydraulic retention time [13,14]. These
pathways and bacterial community (if mixed culture is used),
and finally determine the overall hydrogen production.
Food waste (FW) is an important waste material largely
produced from municipalities and the business sector. It is
also referred to as the organic fraction of municipal solid
waste (OFMSW) if combined with irrecoverable paper resi-
dues. Several studies on the feasibility of using FW or OFMSW
for hydrogen production have shown promising results

Corresponding author at: Aquatic Ecosystem Management Research Division, Water Science and Technology, Environment Canada,
867 Lakeshore Road, P.O. Box 5050, Burlington, Ontario, Canada L7R 4A6. Tel.: +1 905 319 7229; fax: +1 905 336 4858.
E-mail address: heguang.zhu@ec.gc.ca (H. Zhu).
0360-3199/$ - see front matter & 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2008.04.040
 ARTICLE IN PRESS
3652 I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y
[15–19]. To obtain effective hydrogen production, supplemen-
tation of an adequate amount of pH buffer and minerals is
typically required to optimize the pH condition and nutrient
balance. However, this will inevitably increase the cost of
operation. A study on co-digestion of FW and sewage sludge
to produce hydrogen has been reported and concluded that
the addition of sewage sludge to FW supplied a more
balanced carbohydrate/protein ratio [20]. This study indicated
that FW may be lacking in some proteinaceous nutrients
which are essential for hydrogen production. The concept of
co-digestion of FW and sewage sludge for hydrogen produc-
tion is especially significant as it could integrate the manage-
ment of municipal solid waste and sewage sludges. If co-
digestion were successful, it may be possible to use existing
wastewater treatment facilities to treat FW and produce
hydrogen. However, the practicable parameters of the co-
digestion process for hydrogen production remain largely
unknown. The impact of sludge source (primary sludge, PS or
waste activated sludge, WAS) on co-digestion has not been
evaluated. In addition, the optimal mix of these sludges with
FW for hydrogen production is unknown. A previous study
[20] investigated the mix of FW and sewage sludge with
considerable addition of buffer and mineral salts which may
not be necessary because these substances are largely
contained in the sewage sludge. More importantly, by adding
exogenous buffer and mineral salts, an advantage of co-
digestion (i.e. pH buffering capacity) was neglected. The
objective of this study, therefore, was to investigate whether
hydrogen production from co-digestion is affected by the type
of sewage sludge streams employed and to assess the impact
of the ratio of FW to sludges in the co-digestion mixture on
hydrogen production. Moreover it was also intended to
further investigate the factors effecting the biohydrogen
production and the mechanisms involved in the co-digestion
process.
A series of bench scale experiments have been conducted
in this research. An FW and two municipal sewage sludges (PS
and WAS) were characterized with respect to chemical
composition. An anaerobic toxicity analysis (ATA) for hydro-
gen production, derived from the ATA for methane produc-
tion, was performed to examine possible toxicity of the
feedstocks toward hydrogen production. Parallel tests were
performed to determine how the feedstock composition
impacted upon co-digestion to generate hydrogen. The
influence of pH buffering on hydrogen production was
investigated to obtain an improved understanding of the
mechanisms that affect hydrogen production during co-
digestion of these feedstocks.
2. Materials and methods
2.1. Preparation of feedstocks
Food residues were collected from a cafeteria for use in these
experiments and consisted of a variety of grains, vegetables
and meats. The food residues were homogenized using a
Waring blender and mixed in a container. The resulting FW
slurry was stored at 70 1C for subsequent use. The stored FW
slurry was thawed before use, and then diluted with water
33 (2008) 3651 – 3659
and sieved through No. 15 and No. 40 meshes. A small
amount of water was used to rinse the debris on the sieve. For
each gram of the slurry, a total of 10 mL of water was added.
The filtrate obtained from sieving was used as the feedstock
for the remaining experiments.
PS and WAS that were collected from a local wastewater
treatment plant were used in the experiments. The waste-
water treatment plant employed primary settling and a
typical activated sludge process with the addition of ferric
iron for phosphate removal.
2.2. Preparation of hydrogen producing inocula
The hydrogen producing inocula were cultivated in a respiro-
metric cultivation system (CES, AR, USA) using digested
sludge as the original source of organisms and a sucrose
medium as the substrate [21]. The sucrose medium contained
10 g/L sucrose, phosphate buffer, inorganic salts and protein
extracts. A volume of 80 mL of the sucrose medium and 20 mL
of digested sludge were added to each serum bottle (250 mL).
The serum bottles were capped with a silicon septum and
placed in a 35 1C water bath for cultivation. Before the bottles
were capped, they were flushed with argon gas for 10 min to
make them completely anaerobic. The contents of the bottles
were agitated with magnetic stirrers at 250 rpm. The bottles
were connected to a bubble counter to record the gas
production. The cultivation proceeded overnight until sig-
nificant hydrogen was produced.
2.3. ATA for hydrogen production
The ATA test method for hydrogen production was adapted
from the ATA test for methane production [22]. The test was
conducted using the respirometric cultivation system with a
similar procedure to that previously described for preparation
of the hydrogen producing inocula. The sucrose medium and
the hydrogen producing seeds obtained from the previous
step were used as a standard substrate and inoculum for
hydrogen production. Different amounts of test feedstock
(10, 20 or 30 mL) were added to the sucrose medium (with the
final volume maintained at 80 mL) to examine the possible
toxicity of the waste to hydrogen production from sucrose. To
obtain a consistent sucrose composition without increasing
the volume of the mixed liquor, the ingredients of the sucrose
medium were added according to the final volume (80 mL) in
each bottle. All bottles were inoculated with 20 mL of
hydrogen-producing seeds. Tests of sucrose medium alone
and also of the individual feedstocks alone were used as
controls. The initial pH in each bottle was adjusted to 7.0 with
1 M NaOH or 1 M HCl solutions. All the bottles were flushed
with argon gas before cultivation. Toxicity of the wastes was
indicated by reduction in either the total volume of hydrogen
produced or the maximum hydrogen production rates as
compared with the sucrose control.
2.4. Hydrogen production from co-digestion of FW with
sewage sludges
Hydrogen production from mixtures of FW and sewage
sludges was examined using a modification of the ATA test
 ARTICLE IN PRESS
I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y
method, where the composition of the media was adjusted.
Serum bottles were prepared by adding 80 mL of the tested
feedstock mixture and seeded with 20 mL of hydrogen-
producing inocula. Three different co-digestion mixtures
(FW+PS, FW+WAS and FW+PS+WAS) were tested at three
different mix ratios (1:3, 1:1 and 3:1 by volume) of sludge to
FW. Equal volumes of PS and WAS were added in mixtures
that contained both sludge sources. Samples containing the
individual feedstocks were examined as controls (0:1 and 1:0).
All the mixture samples were tested in duplicate while the
test of FW alone was repeated three times. Testing of sludge
streams alone was only conducted once.
2.5. Effect of pH buffer on hydrogen production
The effect of exogenous pH buffer addition on hydrogen
production from FW was tested using the same method
described in ATA test, except that a pH buffer (K2HPO4 and
KH2PO4) was added to the culture media before incubation. A
volume of 100 mL of the FW and 25 mL of hydrogen producing
inocula were added to each serum bottle. The initial pH was
adjusted to 7.0. Varying amounts of the phosphate buffer
were added to the sample bottles to achieve final buffer
concentrations of 0.05, 0.1, 0.15 and 0.2 M after the pH was
adjusted.
2.6. Chemical analysis
Gaseous concentrations (hydrogen, methane and carbon
dioxide) were measured using a gas chromatograph (Agilent
3000, Wilmington, DE, USA), equipped with two columns
(MolSieve 5A and PLOT U). The columns were installed in
separate modules, allowing temperature and carrier gas flow
rate to be controlled separately. Module A equipped with a
MolSieve 5A column was used for the measurement of
hydrogen and methane and operated with an injector
temperature of 95 1C, a column temperature of 100 1C and
argon as the carrier gas at a pressure of 30 psi in the column.
Module B equipped with a PLOT U column was used to
measure carbon dioxide and hydrogen sulfide at an injector
temperature of 70 1C, a column temperature of 70 1C and
helium as the carrier gas at a pressure of 15 psi in the column.
Each module was equipped with a separated TCD as detector.
Soluble carbohydrates in the culture liquid were examined
using the anthrone method [23]. A spectrophotometer (DR/
2000; HACH) was used to determine the absorbance at
620 nm. COD (total and soluble); total Kjeldahl nitrogen
(TKN); Soluble PO4–P; total solids (TS) and volatile solids (VS)
were measured using standard methods [24]. Alcohols and
volatile fatty acids (VFAs) in liquid samples were analyzed by
gas chromatography (GC) (5890 Series II Plus, Hewlett Packard,
Wilmington, DE). A column (Restek Stabilwax-DA) with a
dimension of 30 m  0.25 mm (I.D. 0.25 mm) was employed in
the GC; the carrier gas was hydrogen with a flow rate of
1.2 mL/min at 0.77 kg/cm2; and the detector was FID. The pH
was measured with a pH meter (Barnant 30, USA). Liquid
samples for metal analysis were digested with HNO3 and
analyzed with either an atomic absorption spectroscopy
(Perkin Elmer 5100, for potassium, sodium, calcium and iron)
or an inductively coupled plasma-mass spectrometer (Agilent
33 (2008) 3651 – 3659 3653
ICP-MS 7500a, for barium, copper, magnesium, manganese,
molybdenum and zinc).
Total acidity and total alkalinity of the feedstocks were
measured to assess their pH buffering capacity. Each of the
wastes (50 mL) was placed in a 100 mL beaker, stirred with a
magnetic stirrer and titrated with 1 M HCl and 1 M NaOH for
alkalinity and acidity, respectively. The pH of the sample was
monitored using a pH meter (Barnant 30). The pH buffering
capacity of the solution was quantified based on the total
acidity and alkalinity computed from the titration curves. The
pH end points used for acidity and alkalinity calculations
were 9.0 and 4.5, respectively.
3. Results and discussions
3.1. Characteristics of the FW, PS and WAS
The results of the feedstock characterization are summarized
in Table 1. The FW contained soluble carbohydrate concen-
trations as high as 4480 mg/L, while the soluble carbohydrates
contained in PS and WAS were negligible (124 and 31 mg/L,
respectively). These data suggested that FW was favorable for
hydrogen production as carbohydrates are recognized as
suitable substrates for hydrogen production. The PS and
WAS feedstocks alone were considered to be unlikely
substrates for hydrogen on the basis of the low carbohydrate
content. The TS and VS concentrations of the FW were 11.4
and 10.5 g/L, respectively, indicating a high organic content,
pertaining to be readily digestible. The volatile fraction of the
PS and WAS solids was somewhat less (75%) indicating a
larger undigestible fraction in these two feedstocks. It has
been reported that the majority of the volatile fraction of the
sewage sludges is proteinaceous materials and a proteinac-
eous COD is more than 50% of the total COD in the sewage
sludges [20]. VFAs were detected in the FW and PS suggesting
that acidogenesis of these two wastes had started although
the distribution of the concentrations of the three acids
(acetic, propionic and butyric acid) were different in the two
feedstocks. There was no significant VFAs detected in WAS.
All the feedstocks contained significant concentrations of
TKN that ranged from 505 mg/L in the FW to 1233 mg/L in the
PS. The high concentration of TKN in PS and WAS also
suggests that the protein concentration in the sludge samples
was high. Soluble PO4–P was only detected in significant
concentrations in the FW and PS, indicating that phosphorous
might limit the digestion of the WAS. The metals that are
considered important for bacterial growth were present in all
three feedstocks. The PS and WAS contained relatively high
concentrations of most metals except potassium and sodium
which were higher in the FW. Iron concentrations in the PS
and WAS were as high as 735 and 558 mg/L, respectively. The
impact of such high concentrations of iron on hydrogen
production was not specifically investigated in this study.
However, as shown later the overall toxicity to hydrogen
production of these two sludges was not observed according
to the ATA tests.
The FW had a low total acidity and alkalinity (340 and
40 mg/L as CaCO3, respectively) which indicated that it had a
low pH buffering capacity. On the other hand, PS and WAS
 ARTICLE IN PRESS
3654 I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y 33 (2008) 3651 – 3659

Table 1 – Characteristics of the FW, PS and WAS

FW PS WAS

TS (g/L) 11.470.75 30.675.6 8.871.5

VS (g/L) 10.570.78 19.873.9 6.571.3
Carbohydrates (mg/L) 44807106 124744 31710
Soluble COD (mg/L) 92307300 448072160 2407110
Total COD (mg/L) 1925071360 35900712600 1060072890
Acetic acid (mg/L) 6787205 11407516 n.d.
Propionic acid (mg/L) 3027156 5817284 n.d.
Butyric acid (mg/L) 65720 2897183 n.d.
TKN (mg/L) 505745 12337350 7097190
PO4–P (mg/L) 327730 2167130 n.d.
Ba (mg/L) 0.03 2.41 0.45
Ca (mg/L) 38 418 108
Cu (mg/L) 0.17 3.22 2.22
Fe (mg/L) 1.35 735 558
K (mg/L) 160 70.9 60.3
Mg (mg/L) 12.5 77.9 29.3
Mn (mg/L) 0.12 2.05 1.66
Mo (mg/L) 0.01 0.06 0.14
Na (mg/L) 143 148 109
Zn (mg/L) 0.36 3.43 1.28
Total acidity as CaCO3 340 1972 2200
Total alkalinity as CaCO3 40 1960 840
pH 4.770.1 5.970.1 6.870.1

n.d.—not detectable.
The detect limits for acetic, propionic, butyric acids and PO4–P were 0.28, 0.64, 0.55 and 0.08 mg/L, respectively.

Table 2 – Anaerobic toxicity analysis of FW, PS and WAS for hydrogen production

Media FW PS WAS

Hmaxa Rate Final Hmaxa Rate Final Hmaxa Rate Final

(mL) (mL /h) pH (mL) (mL /h) pH (mL) (mL/h) pH

80 mL suc 85 9.0 4.3 82 8.3 4.3 109 8.5 4.4

80 mL waste 23 3.5 3.9 0.8 0.5 6.0 0 0 6.5
80 mL suc+10 mL waste 128 10.4 4.3 95 9.0 4.4 112 10.5 4.4
80 mL suc+20 mL waste 132 10.6 4.3 93 9.0 5.3 134 10.6 5.0
80 mL suc+30 mL waste 147 10.9 4.3 88 8.9 5.6 127 14.7 5.2

a
Hmax—total volume of hydrogen produced.

had a much higher acidity and alkalinity, and therefore had
much higher pH buffering capacity. In general, the FW has a
complementary nutrient composition and pH buffering
capacity to the two sewage sludges, which suggested the
potential of co-digestion for the purpose of hydrogen
production.
3.2. ATA tests
ATA tests were conducted to investigate if the individual
feedstocks exerted any toxicity to hydrogen fermentation and
the results are presented in Table 2. Toxicity was identified
when hydrogen production from the bottles containing both
the feedstock and the sucrose medium was reduced com-
pared with that from the sucrose-alone bottles. The hydrogen
production from bottles with the sucrose medium alone was
stable, with approximately 2 mol hydrogen from 1 mol
sucrose and a final pH of 4.3. Although this pH was lower
than the typical optimal pH for hydrogen production (5–6), it
seemed not to affect hydrogen production significantly. In all
cases hydrogen production from the test feedstocks alone
was less than that of the sucrose medium. The hydrogen
produced from the bottle containing FW alone was 23 mL
which was only 27% of the hydrogen volume obtained from
the control bottle with sucrose medium and hydrogen
production from either PS or WAS alone was insignificant.
These results indicate that FW, if not appropriately condi-
tioned, was less amenable for hydrogen production as
compared with the sucrose medium, although substantial
quantities of carbohydrates were present in the feedstock.
 ARTICLE IN PRESS
I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y 33 (2008) 3651 – 3659 3655

As presented in Table 2, the final pH was only 3.9, far lower
than the typical optimal pH range for hydrogen production.
The limited hydrogen production from PS and WAS could be
attributed to the relatively low carbohydrate content of these
feedstocks.
The addition of the test feedstocks to the sucrose medium
improved both the total hydrogen production and the
maximum hydrogen production rate in all cases; although
the extent of the improvement was different among the three
wastes. The greatest increase in total hydrogen produced
were observed when FW was added to the sucrose medium;
the hydrogen production increased as the quantity of FW
increased. The final pH was not affected by the addition of the
FW showing that the pH buffer in medium was enough to
maintain the pH unchanged compared with the pH in the
bottle with sucrose medium. PS had the least effect on
the improvement of hydrogen production, and the increase in
the PS fraction did not further improve the hydrogen
production. The final pH values in the bottles with municipal
sludges present were between those observed in the bottle
with sucrose medium and the individual sludge, indicating
that the high alkalinity in the sludges altered the pH
conditions in the mixture. These observations showed that
the three wastes were not toxic to the hydrogen production
under the conditions tested and in contrast were beneficial
for hydrogen production. FW which had hydrogen production
potential by itself probably improved the hydrogen produc-
tion mainly through the addition of more substrates (carbo-
hydrates) into the medium. PS and WAS which had negligible
hydrogen production potential by themselves likely improved
The total hydrogen production observed in the batch tests
is summarized in Fig. 2. Insignificant quantities of hydrogen
were produced in the bottles that contained only sludge
feedstocks which was consistent with that observed in the
ATA tests. Hydrogen production from the FW alone demon-
strated considerable variability amongst the replicate tests
and this was attributed to differing storage times after
thawing of the stock. The hydrogen production from FW
alone was relatively low as was observed in the ATA tests and
this was attributed to the low pH (3.8) of the fermentation
process. As will be subsequently demonstrated carbohydrate
conversion was limited under the pH conditions that were
established in these tests.
The results presented in Fig. 2 indicate that, with the
appropriate mixture of FW and sludges, co-digestion im-
proved hydrogen production as compared with the digestion
of the single component wastes. The impact of co-digestion
was however dependent upon the type of sludge used and the
mixing ratio. Mixtures that contained 1:1 v/v ratios of FW to
sludge demonstrated enhanced hydrogen yields as compared
with the FW control for all sludge combinations. Mixtures
7.5
7.0 FW+PS
FW+WAS
6.5 FW+PS+WAS
6.0
Final pH

5.5
hydrogen production by supplying a better nutrient balance.
5.0
Hydrogen production in the bottles with these sludges
present was not proportional to the amount of sludge added 4.5
suggesting that the amount of useful nutrients added with 4.0
the sludge was more than required. 3.5
3.0
80 mL FW 80 mL 60 mL FW 40 mL FW 20 mL FW
3.3. Co-digestion of FW, PS and WAS (1:0) Sludge + 20 ml + 40 ml + 60 ml
(0:1) Sludge Sludge Sludge
(3:1) (1:1) (1:3)
The pH of anaerobic fermentations has been demonstrated to
substantially influence the metabolic pathways for carbohy- Fig. 1 – Final pH in different co-digestion combinations
drate conversion and hence hydrogen production. Fig. 1 under different mixed ratios.
presents the pH values that were observed for each of the
co-digestion mixtures and control samples. Although the
initial pH in each bottle was adjusted to 7, the final pH varied 120
from 3.8 to 6.9. The largest pH decline was observed in the FW+PS
H2 production based on

100 FW+WAS
VS added (mL/ g VS)

bottles with only FW while the bottles containing only sludge FW+PS+WAS
feedstock had the least decline. In the bottles with mixtures 80
the declines increased as the FW fraction of the feedstock
60
increased and were between those of the FW sample and the
sludge-only samples. The composition of the sludge feed- 40
stocks with respect to PS and WAS did not appear to have a
20
significant impact on the final pH of the fermentations.
Previous studies have indicated that the optimum pH for 0
hydrogen production from FW was in the range of 5.5–6.0. 80 mL FW 80 mL 60 mL FW 40 mL FW 20 mL FW
Based upon pH alone it would be expected that the 1:1 and 1:3 (1:0) Sludge + 20 ml + 40 ml + 60 ml
(0:1) Sludge Sludge Sludge
mixtures of FW to sludge feedstocks would have the max-
(3:1) (1:1) (1:3)
imum hydrogen production. The relationship between pH
and generation of fermentation products will be discussed Fig. 2 – Hydrogen production from different co-digestion
subsequently. combinations under different mixed ratios.

3656 I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y
containing 1:3 v/v ratios of FW to sludge consistently pro-
duced lower hydrogen yields as compared with FW alone.
Mixtures that contained 3:1 v/v ratios of FW to sludge
demonstrated mixed results with the binary mixtures of FW
and sludge producing less hydrogen than FW alone while the
ternary mixture of FW, PS and WAS produced substantially
more hydrogen than the control. The largest hydrogen yields
(104 and 112 mL/g VS) were produced from ternary mixtures
of FW, PS and WAS at volumetric ratios of 3:1 and 1:1 v/v of
FW to sludge.
With some exceptions, the hydrogen yields observed in the
co-digestion tests appeared to be a function of the availability
of suitable substrates (i.e. carbohydrates from FW) and pH.
The 3:1 mixtures of FW to sludges had lower hydrogen
production and this was attributed to the relatively low pH of
these fermentations (approximately 4.2) despite the avail-
ability of carbohydrates that were present in the FW. The 1:1
mixture of FW to sludges had the most hydrogen production
and this was attributed to the more suitable pH values
(approximately 5.3) and the availability of carbohydrates from
the FW. The 1:3 mixture of FW and sludges had considerably
less hydrogen production than the other mixtures. In these
mixtures the availability of carbohydrates from the FW was
reduced while the pH appeared to be in a satisfactory range
for hydrogen generation. Hence it appeared that in this
mixture the availability of suitable substrates was limited.
It should be noted that pH and FW availability could not
have been the only factors impacting hydrogen generation
since there were considerable differences in hydrogen yields
for co-digestions that had different combinations of PS and
WAS. For the 3:1 and 1:1 v/v mixtures of FW to sludge there
were minimal differences between the pH values of the
fermentations however the ternary mixtures produced sub-
stantially more hydrogen than the binary mixtures.
Fig. 3 presents the concentrations of soluble carbohydrates
that were measured upon completion of the co-digestion
fermentations. From Fig. 3 it can be seen that the significant
concentrations of residual carbohydrates were only observed
in the bottles that contained FW alone. The residual
concentrations of soluble carbohydrates in the sludge-alone
and co-digestion bottles were consistently less than 500 mg/L
and did not differ substantially between bottles. Based upon
the feedstock characterization, the FW-alone bottles were
3500
3000 FW+PS
Carbohydrates (mg/L)
FW+WAS
2500
FW+PS+WAS
2000
1500
1000
500
0
80 mL FW 80 mL 60 mL FW 40 mL FW 20 mL FW
(1:0) Sludge + 20 ml + 40 ml + 60 ml
(0:1) Sludge Sludge
(3:1) (1:1)
Fig. 3 – Carbohydrates remaining at end of the co-digestion.
ARTICLE IN PRESS
33 (2008) 3651 – 3659
expected to have the highest concentrations of carbohy-
drates. However, the differences in soluble carbohydrate
concentrations between the FW-alone and the co-digestion
bottles could not be explained by dilution of the FW alone. It is
believed that the low pH conditions that were established in
the FW bottles inhibited carbohydrate conversion. The
reduced carbohydrate conversion was believed to have been
at least partially responsible for the reduced hydrogen
production.
Fig. 4 presents the net production of VFAs and ethanol that
were observed in the co-digestion tests. From Fig. 4 it can be
seen that the fermentations with FW alone produced
relatively low concentrations of VFAs (43, 30, 515 mg/L of
acetic, propionic and butyric acids, respectively) as compared
with the VFA produced in the other co-digestion conditions.
Moderate concentrations of ethanol (approximately 440 mg/L)
were generated from FW alone. The low VFA production from
FW when considered along with the elevated residual
carbohydrate concentrations and the low hydrogen produc-
tion tend to confirm the low overall conversion of carbohy-
drates in these tests. The moderate concentrations of ethanol
suggest that metabolic pathways that lead to alcohol
production were somewhat active under these conditions
and are consistent with the previous studies that have
indicated that pathway for ethanol production is favored
under low pH conditions [25].
The fermentation of sludges alone resulted in relatively
high productions of acetic acid (1155–1439 mg/L), elevated
generations of propionic and butyric acids (396–843 mg/L) and
negligible alcohols. The high VFA production without sig-
nificant hydrogen or alcohol production that was observed in
the sludge-alone tests likely resulted from the fermentation
of proteinaceous materials. Fermentation of proteins was not
expected to produce significant quantities of hydrogen or
alcohols. These results suggest that negligible quantities of
hydrogen would have been generated from the organic
components of the sludges during co-digestion with FW.
Hence, the observed differences in the yields of hydrogen
could be reasonably attributed to the differing extent of
utilization of the FW components.
Co-digestion of FW and sludges resulted in patterns of VFA
and alcohol production that were dependent on the types of
feedstocks and their relative contribution to the feed mixture.
The net production of acetic and propionic acid generally
increased as the fraction of the sludge in the mixture
increased. Butyric acid production peaked for the 1:1 mixture
of FW and sludges and the ethanol decreased as the sludge
fraction increased. Only the production of propionic acid
differed substantially with the composition of the sludge
component of the mixture. The production of this component
appeared to increase with the fraction of PS that was present
in the mixture.
3.4. Effect of pH buffering on fermentation
As previously described the PS and WAS had very differing pH
buffering capacity as compared with the FW. Hence, a
Sludge
separate experiment was conducted to examine the effect
(1:3)
of pH buffering on hydrogen production from FW. The volume
of hydrogen produced and the final pH of the fermentations
 ARTICLE IN PRESS
I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y 33 (2008) 3651 – 3659 3657

2500 2500
a FW+PS b FW+PS
FW+WAS FW+WAS
2000 2000
FW+PS+WAS FW+PS+WAS
HAc (mg/L)

HPr (mg/L)
1500 1500

1000 1000

500 500

0 0
2500 2500
c FW+PS
d FW+PS
FW+WAS
2000 FW+WAS 2000 FW+PS+WAS
FW+PS+WAS

Ethanol (mg/L)
HBu (mg/L)

1500 1500

1000 1000

500 500

0 0
80 mL FW 80 mL 60 mL FW + 40 mL FW + 20 mL FW + 80 mL FW 80 mL 60 mL FW + 40 mL FW + 20 mL FW +
(1:0) Sludge (0:1) 20 ml 40 ml 60 ml (1:0) Sludge (0:1) 20 ml Sludge 40 ml Sludge 60 ml Sludge
Sludge (3:1) Sludge (1:1) Sludge (1:3) (3:1) (1:1) (1:3)

Fig. 4 – VFAs and alcohols produced from the co-digestion.

Total H2 produced (mL/g VS)

120 The maximum production of hydrogen was observed when

a
100 the pH was in the range of 5.5–6. When the pH was below this
range negligible hydrogen was produced and above this range
80
it was slightly reduced as compared with the peak value. The
60 results of this experiment were consistent with those
40 observed in the co-digestion tests in that the pH range for
20 optimal hydrogen production was the same in both tests.
However, the hydrogen yields from the co-digestion bottles
0
6.5 under optimal pH conditions were substantially greater than
b those observed in the pH-buffered FW-alone digestions.
6

5.5
Final pH

5
4. Discussion
4.5

4 The results of this study show that hydrogen was not

3.5
0.0
Fig. 5 – Effect of buffer addition on hydrogen production
from FW.
are presented in Fig. 5. It can be observed that for buffer
concentrations less than or equal to 0.05 M, only small
amounts of hydrogen (0.6 and 1.3 mL H2/g VS, respectively)
were produced. When the buffer concentration was increased
to 0.1 M, a total hydrogen production of approximately
90 mL H2/g VS was observed. Further increase in buffer con-
centrations to 0.15 and 0.2 M resulted in slightly less hydrogen
production (approximately 70 mL H2/g VS).
produced in significant quantities from any of the single
0.5 1.0 1.5 0.2 waste streams when the initial pH was adjusted at 7.0.
However, co-digestion of FW with either PS, WAS or a mixture
Buffer Concentration (M)
of PS and WAS significantly improved hydrogen production at
this initial pH condition. Overall, co-digestion of a ternary
mixture of FW, PS and WAS produced the most hydrogen and
a 1:1 v/v mix FW with a 1:1 v/v blend of PS and WAS was found
to be optimal. The maximum hydrogen yield obtained from
the co-digestion was 112 mL/g VS added when the ternary
mixture of FW, PS and WAS was used. The results suggested
that hydrogen was mostly produced from FW and hence, the
hydrogen yield based on the FW added was 250 mL H2/g VS.
This yield was higher than the previously reported values
for FW. Values of 180 mL H2/g VS added [15], 310 mL/g VS
degraded (no more than 100 mL/g VS added) [17], 129 mL H2/g
carbohydrate-COD [20] and 92 mL/g VS added in [26] have
been reported in the literature.
 ARTICLE IN PRESS
3658 I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y
The results of this study indicate that the mixture of FW
and the sewage sludges provided a unique condition for
hydrogen production. The addition of sewage sludges to FW
provided pH buffering capacity that allowed the fermentation
to establish a pH (5.5–6.0) that was conductive to hydrogen
production. The response in hydrogen production to pH that
was observed in the co-digestion tests was confirmed with
separate tests where the pH was buffered using inorganic
buffer solutions. However, even under the optimal buffer
condition (0.1 M as shown in Fig. 5), the hydrogen yield from
FW was 90 mL/g VS and this was substantially less than the
yield of 250 mL/g VS observed from the mixture of FW, PS and
WAS. This suggests that factors other than pH buffering play
an important role in hydrogen production during co-diges-
tion. This conclusion was also indicated by the observation
that the different sludges had different effect on hydrogen
production although they had similar pH buffer capacity.
The enhanced hydrogen yields from co-digestion may have
been due to the provision of nutrients that stimulated
hydrogen producing organisms or may have resulted from
modifications of the physiological state of the organisms
which resulted from changes in the availability of FW. The
provision of protein from the sludge streams may have
provided an improved C/N ratio that has been reported as
an important parameter for hydrogen production [20].
Proteinaceous substances, including peptone, meat extract
and yeast extract, have been found to be necessary for growth
of hydrogen producing bacteria [27,28]. Micronutrients such
as biotin and vitamins have also been found to be important
for the growth of hydrogen producing bacteria [27,28] and
could possibly have been present in the sewage sludge. The
reduced concentration of substrates present in the co-
digestion tests may also have affected hydrogen production
as the concentration of FW was diluted to differing extents by
the sludge streams. Van Ginkel et al. [29] have reported that
lower organic loading can increase hydrogen production.
Further studies are required to specifically identify the impact
of these factors.
The production of the liquid products (VFAs and alcohols)
which reflects the metabolic pathways were affected by both
the substrate composition and reaction conditions in the
co-digestion hydrogen production. The concentrations of
acetic and propionic acids and ethanol that were produced
in the co-digestion bottles were generally consistent with the
concentrations that were observed in the tests that were
conducted with the individual feedstocks (Fig. 5). Hence, for
these components it would appear that their production in
the mixture was at least partially determined by the
composition of the feedstock. The increase in protein
availability with the increased sludge fraction likely led to
the increased production of the acids and decreased produc-
tion of ethanol as fermentation products. The decreased
ethanol production at higher sludge contents was also likely
due to the increased pH of the fermentations which tends to
reduce the activity of the ethanol producing pathway [25]. The
butyric acid produced in the mixtures was generally higher
than those that would be predicted based upon the individual
tests (Fig. 5). These results suggest that the environment
which was created by blending these feedstocks enhanced
the production of these substances. The metabolic pathway
33 (2008) 3651 – 3659
that leads to butyric acid production also results in hydrogen
production. It can be noted that the conditions which led to
maximum butyric acid production were the same conditions
that had high production of hydrogen. Hence, it would appear
that co-digestion of a 1:1 mixture of FW and sludge
established an environment in the bottles that favored the
butyric acid pathways. This result supports the observation
by Kim et al. [20].
The observation that co-digestion can supply pH control and
other supplements is important for practical operation of a
hydrogen reactor using FW as feedstock because it supplies a
way to reduce the cost for pH control or nutritional supple-
ments. The costs of base addition for enhanced hydrogen
production have been recognized in other studies [30].
Acknowledgments
Samuel (Sheng-Fong) Hsieh from the Biochemistry Depart-
ment of the University of Waterloo and Kara Edmonson from
the Chemical Engineering Technology Department of Mo-
hawk College, Hamilton participated in the experiments. John
Salvatore, Keri Kwong, Mohamad Al-Jamal and Stephen
Lee undertook the chemical analysis and laboratory support
work.
R E F E R E N C E S
[1] Nakamura M, Kanbe H, Matsumoto J. Fundamental studies
on hydrogen production in the acid-forming phase and its
bacteria in anaerobic treatment processes—the effects of
solids retention time. Water Sci Technol 1993;28(7):81–8.
[2] Mizuno O, Dinsdale R, Hawkes FR, Hawkes DL, Noike T.
Enhancement of hydrogen production from glucose by
nitrogen gas sparging. Biores Technol 2000;73:59–65.
[3] Logan BE, Oh SE, Kim IS, Ginkel SV. Biological hydrogen
production measured in batch anaerobic respirometers.
Environ Sci Technol 2002;36:2530–5.
[4] Liu H, Fang HHP. Hydrogen production from wastewater by
acidogenic granular sludge. Water Sci Technol 2002;47(1):153–8.
[5] Lin CY, Jo CH. Hydrogen production from sucrose using an
anaerobic sequencing batch reactor process. J Chem Technol
Biotech 2003;78:648–78.
[6] Zhang T, Liu H, Fang HHP. Biohydrogen production from
starch in wastewater under thermophilic condition. J Environ
Manage 2003;69:149–56.
[7] Hussy I, Hawkes FR, Dinsdale R, Hawkes DL. Continuous
fermentative hydrogen production from a wheat starch co-
product by mixed microflora. Biotechnol Bioeng 2003;84(6):
219–26.
[8] Taguchi F, Mizukami N, Yamada K, Hasegawa K, Taki TS. Direct
conversion of cellulosic material to hydrogen by Clostridium
sp. strain no. 2. Enzyme Microbial Technol 1995;17:147–50.
[9] Ueno Y, Kawai T, Sato S, Otsuka S, Morimoto M. Biological
production of hydrogen from cellulose by natural anaerobic
microflora. J Ferment Bioeng 1995;79(4):395–7.
[10] Liu H, Zhang T, Fang HHP. Thermophilic H2 production from a
cellulose-containing wastewater. Biotech Lett 2003;25:365–9.
[11] Roychowdhury S, Cox D, Levandowsky M. Production of
hydrogen by microbial fermentation. Int J Hydrogen Energy
1988;13(7):407–10.
[12] Yu HQ, Yu ZH, Hu WR, Zhang HS. Hydrogen production from
rice winery wastewater in an up-flow anaerobic reactor by
 ARTICLE IN PRESS
I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y
using mixed anaerobic cultures. Int J Hydrogen Energy 2002;
27:1359–65.
[13] Hawkes FR, Dinsdale R, Hawkes DL, Hussy I. Sustainable
fermentative hydrogen production: challenges for process
optimization. Int J Hydrogen Energy 2002;27:1339–47.
[14] Li CL, Fang HHP. Fermentative hydrogen production from
wastewater and solid wastes by mixed cultures. Crit Rev
Environ Sci Technol 2007;37:1–39.
[15] Lay JJ, Lee YJ, Noike T. Feasibility of biological hydrogen
production from organic fraction of municipal solid waste.
Water Res 1999;33(11):2579–86.
[16] Nielsen AT, Amandusson H, Bjorklund R, Dannetun H,
Ejlertsson J, Ekedahl LG, et al. Hydrogen production from
organic waste. Int J Hydrogen Energy 2001;26:547–50.
[17] Han HK, Shin HS. Performance of an innovative two-stage
process converting food waste to hydrogen and methane. J
Air Waste Manage Assoc 2004;54:242–9.
[18] Jo JH, Jeon CO, Lee DS, Park JM. Process stability and microbial
community structure in anaerobic hydrogen-producing mi-
croflora from food waste containing Kimchi. J Biotechnol
2007;131:300–6.
[19] Ueno Y, Fukui H, Goto M. Operation of a two-stage
fermentation process producing hydrogen and methane
from organic waste. Environ Sci Technol 2007;41:1413–9.
[20] Kim SH, Han SK, Shin HS. Feasibility of biohydrogen
production by anaerobic co-digestion of food waste and
sewage sludge. Int J Hydrogen Energy 2004;29:1607–16.
[21] Zhu HG, Béland M. Evaluation of alternative methods of
preparing hydrogen producing seeds from digested waste-
water sludge. Int J Hydrogen Energy 2006;31:1980–8.
33 (2008) 3651 – 3659 3659
[22] Owen WF, Stuckey DC, Healy JJB, Young LY, McCarty PL.
Bioassay for monitoring biochemical methane potential and
anaerobic toxicity. Water Res 1979;13:485–92.
[23] Gaudy AF. Colorimetric determination of protein and carbo-
hydrate. Ind Water Wastes 1962;January–February:17–22.
[24] APHA. Standard methods for the examination of water and
wastewater, 20th ed. Washington DC, USA: American Public
Health Association/American Water Works Association/
Water Environment Federation, 2004.
[25] Batstone DJ, Keller J, Angelidaki I, Kalyuzhnyi SV, Pavlo-
stathis SG, Rozzi A, et al. Anaerobic digestion model no. 1.
Scientific and Technical Report No. 13. IWA Publishing;
2002.
[26] Shin HS, Youn JH, Kim SH. Hydrogen production from food
waste in anaerobic mesophilic and thermophilic acidogen-
esis. Int J Hydrogen Energy 2004;29:1355–63.
[27] Cummins CS, Johnson JL. Taxonomy of the clostridia: wall
composition and DNA homologies in Clostridium butyricum
and other butyric acid-producing clostridia. J Gen Microbiol
1971;67:33–46.
[28] Tanner RS, Stackebrandt E, Fox GE, Woese CR. A phylogenetic
analysis of Acetobacterium woodii, Clostridium butyricum, and
Clostridium lituseburense, Eubacterium limosum and Eubacterium
tenue. Curr Microbiol 1981;5:35–8.
[29] Van Ginkel SW, Logan B. Increased biological hydrogen
production with reduced organic loading. Water Res
2005;39:3819–26.
[30] Kraemer JT, Bagley DM. Continuous fermentative hydrogen
production using a two-phase reactor with recycles. Environ
Sci Technol 2005;39:3819–25.