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 e n e r g y 3 8 ( 2 0 1 3 ) 6 1 4 6 e6 1 5 3

Available online at www.sciencedirect.com

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

Effect of acid, heat and combined acid-heat

pretreatments of anaerobic sludge on hydrogen
production by anaerobic mixed cultures

Thitirut Assawamongkholsiri a, Alissara Reungsang a,b,*, Sakchai Pattra c

a
Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand
b
Research Group for Development of Microbial Hydrogen Production Process from Biomass, Khon Kaen University, Khon Kaen 40002,
Thailand
c
Program of Public Health, Department of Science and Technology, Faculty of Arts and Science, Chaiyaphum Rajabhat University,
Chaiyaphum 36000, Thailand

article info abstract

Article history: Activated sludge (AS) from wastewater treatment plant of brewery industry was used as
Received 10 September 2012 substrate for hydrogen production by anaerobic mixed cultures in batch fermentation
Received in revised form process. The AS (10% TS) was pretreated by acid, heat and combined acid and heat.
27 December 2012 Combined acid- heat treatment (0.5% (w/v) HCl, 110
C, 60 min) gave the highest soluble
Accepted 31 December 2012 COD (sCOD) of 1785.6  27.1 mg/L with the highest soluble protein and carbohydrate of
Available online 29 January 2013 8.1  0.1 and 38.5  0.8 mg/L, respectively. After the pretreatment, the pretreated sludge
was used to produce hydrogen by heat treated upflow anaerobic sludge blanket (UASB)
Keywords: granules. A maximum hydrogen production potential of 481 mL H2/L was achieved from
Bio-hydrogen the AS pretreated with acid (0.5% (w/v) HCl) for 6 h.
Activated sludge Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
Heat reserved.
Acid

1. Introduction wastewater treatment plant, such as, poultry slaughterhouse

Nowadays, the environmental problems have brought the
interests toward the development of alternative energy [1].
Among the alternative energy sources, hydrogen has been
receiving an increasing attention due to its environmentally
friendly characteristic and high energy content.
Hydrogen can be biologically produced from various kinds
of substrate ranges from simple sugars such as glucose [2,4];
xylose [3]; arabinose [4]; starch containing waste such as
cassava wastewater [5]; lignocellulosic materials such as
sugarcane bagasse [6]; and activated sludge (AS) from
[7,8]; and diary industry [9]. Among the aforementioned sub-
strates, AS, the major solid waste from aerobically biological
wastewater treatment processes [10], has recently received an
attention as substrate for producing renewable fuel i.e.,
methane [11] and hydrogen [11,12] due to its rich poly-
saccharide and protein content indicating its potential as
substrate for hydrogen production [13]. It is reported that in
aerobic degradation, one unit of substrate carbon is converted
to 0.5 units of CO2 and 0.5 units of cell carbon [14]. Therefore,
a utilization of AS as the substrate for renewable energy pro-
duction not only contributes to a safe and clean energy (i.e.,

* Corresponding author. Research Group for Development of Microbial Hydrogen Production Process from Biomass, Khon Kaen Uni-
versity, Khon Kaen 40002, Thailand. Tel./fax: þ66 43 362 121.
E-mail addresses: reungsang4@gmail.com, alissara@kku.ac.th (A. Reungsang).
0360-3199/$ e see front matter Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ijhydene.2012.12.138
 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 e n e r g y 3 8 ( 2 0 1 3 ) 6 1 4 6 e6 1 5 3 6147

hydrogen in this study) but also one of the solutions to get rid
of this abundant waste.
AS from the wastewater treatment plant has microbial
cells as the main component [13]. Most of the organics are
contained within microbial cell membranes and the semi rigid
structure of the cell envelope prevents the osmotic lysis of the
cells [15]. In addition, AS contains a significant amount of
extracellular polymeric substances of [16] which only 30e50%
can be biodegraded [17]. Thus, the hydrolysis of the microbial
cells is needed in order to disrupt the cells and release the
organics into liquid so as to enhance the anaerobic digestion
of AS [18], subsequently increase in hydrogen yield (HY) and
shorten the HRT [19]. The AS can be pretreated/hydrolyzed
using one or a combination of a number of methods. The
pretreatment methods can be divided into 5 categories
including thermal, mechanical, chemical, ultrasound and
biological hydrolysis (bacterial and enzyme hydrolysis) [20].
These pretreatments are distinctively different in their modes
of action. Thermal pretreatment depends on the high tem-
perature. Cell wall and cell membrane of the microorganisms
were disrupted by heat that was applied during thermal pre-
treatment, resulting in a solubilization of the cell components.
Mechanical pretreatment physically disintegrate the cells and
the cell contents are partially solubilized. Chemical pretreat-
ment include acid and alkaline hydrolysis. Acid hydrolysis
depends on the free Hþ while an alkaline hydrolysis depends
on free OH [21]. This method hydrolyzes the cell wall and cell
membrane. Consequently, the solubility of the organic matter
contained within cells is increased [20]. Ultrasonic pretreat-
ment depends on the shear produced in the sonication [22,23].
Lastly, biological hydrolysis breaks the cell wall compounds
by an enzyme catalyze reaction. As a result, the organic
matter inside the cells is released. In this study, acid and
thermal (heat) methods were chosen to pretreat the AS due to
its simplicity, no special equipments required, and less time-
consuming. Xiong et al. [24] reported that an increase in the
reaction temperature improved the efficiency of hydrolytic
and acidogenic waste AS in anaerobic fermentation. Thus, it is
hoped that a combined acid-heat pretreatment would
enhance the lysis of the microbial cells and more hydrogen
would be produced.
Therefore, the main objective of this study was to inves-
tigate the effects of pretreatment (acid, heat and combined
acid-heat) on solubilization of organic matter from AS as well
as on the hydrogen production. It is hoped that the pretreat-
ment methods would improve the rate of digestion and
facilitate the hydrogen production process.
2. Materials and methods
2.1. Seed and inoculum preparation
The anaerobic seed sludge was upflow anaerobic sludge blan-
ket (UASB) granules from Khon Kaen Brewery CO., Ltd., Khon
Kaen, Thailand. The UASB reactor is used to treat wastewater
from beer production process. Anaerobic sludge was heated at
105
C for 3 h in order to inhibit methanogenic bacteria. For
inoculum preparation, 5% (w/v) heat treated UASB granules
were enriched with 20 g/L glucose, 0.5 g/L yeast extract, 1 mL/L
nutrient stock solution [25], and 1.29 mL/L stock solution of
155 g/L NH4HCO3. The initial pH of the culture was adjusted to
5.5 by 4 mol/L HCl. The culture was incubated at room tem-
perature and shaken at 150 rpm. After 24 h of incubation, 50%
(v/v) of the culture was transferred into fresh medium. After
ten cycles of subculture, the culture was used as an inoculum
in batch experiment. The volatile suspended solids (VSS) con-
centration of the inoculum was 14.26  0.21 g-VSS/L.
2.2. Feedstock
The feedstock is AS obtained from the wastewater treatment
of Khon Kaen Brewery CO., Ltd., Khon Kaen, Thailand. The AS
was stored at 4
C prior usage. Characteristics of raw AS were
shown in Table 1.
2.3. Pretreatment of AS
AS (10% total solid (TS)) was pretreated by acid, heat and
combined acid-heat. For the acid pretreatment, the AS was
treated by 0.5% (w/v) HCl and incubated at room temperature
for 0, 6, 12, 18 and 24 h. For heat pretreatment, the AS was
heated in a hot air oven at 110
C for 15, 30, 45 and 60 min. For
combined acid-heat pretreatment, the AS was added with
0.5% (w/v) HCl and then heated in a hot air oven at 110
C for
15, 30, 45 and 60 min. AS (10% TS) without pretreatment (raw
AS) was used as a control. The treatment codes are showed in
Table 2.
2.4. Biohydrogen production
Biohydrogen production was conducted in 120 mL serum
bottles with a working volume of 70 mL. The serum bottle
contained 65 mL of pretreated AS (10% TS), 4.5 mL of seed
inoculums (14.26  0.21 g-VSS/L), 1 mL/L of nutrients stock
solution [25] and 1.29 mL/L of NH4HCO3 stock solution. An
initial pH was adjusted to 5.5 by 2 mol/L HCl or 2 mol/L NaOH.
The serum bottle was closed with a rubber stopper and capped
with aluminum cap. After replacement of gas phase with ni-
trogen to create anaerobic condition, the serum bottles were
incubated at room temperature (30  2
C) and horizontally
shaken at 150 rpm. The fermentation process was continued
until no biogas generated. At each time interval, the total gas
Table 1 e Characteristics of raw activated sludge.
pH (5% TS AS) 7.05
Total solid (TS) (g/kg sludge) 867.95  0.16
Volatile solid (VS) (g/kg sludge) 76.23  0.06
Total chemical oxygen demand 113.91  14.78
(tCOD) (g/kg TS)
Soluble chemical oxygen demand 407  47
(sCOD) (mg/kg TS)
Carbohydrate concentration 10326  654
(mg/kg TS)
Protein concentration (mg/kg TS) 4000  866
Fat concentration (mg/kg TS) 1949  534
Total Nitrogen (mg/kg TS) 1644
NHþ3 (mg/kg TS) 572
Total Phosphorus (mg/kg TS) 677
6148 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 e n e r g y 3 8 ( 2 0 1 3 ) 6 1 4 6 e6 1 5 3

Table 2 e Estimated values of parameters by the modified Gompertz equation and hydrogen yield.
Treatment Time Code Hmax Rm l (h) R2 H2 yield
(mL H2/L) (mL H2/L h) (mL H2/g VSadded)

Raw sludge 0 min raw sludge 66AB 8.38B 7.40A 0.99 9AB
Acid 0h A0 167BC,a 13.63CDE,a 44.80D,a 0.99 12ABCD,a
6h A6 481G,bc 15.17DE,a 84.07GH,bc 0.99 34EF,bc
12 h A12 212CD,a 18.74F,b 81.90FG,bc 0.99 14BCD,ab
18 h A18 340DE,ab 34.56H,c 78.20F,b 0.99 23D,ab
24 h A24 600G,c 12.76CD,a 87.30HI,c 0.99 41F,c
Heat 0 min raw sludge 66AB,b 8.38B,bc 7.40A,a 0.99 9AB,b
15 min H15 79AB,b 16.49EF,d 7.63A,a 0.99 11ABC,b
30 min H30 65AB,b 11.06BC,c 8.27A,a 0.99 9ABC,b
45 min H45 37AB,a 4.86A,ab 16.35B,c 0.99 3A,a
60 min H60 20A,a 3.23A,a 26.77C,d 0.99 2A,a
Acid combined heat 0 min AH0 167BC,a 13.63CDE,b 44.83D,a 0.99 12D,b
15 min AH15 284CDE,b 29.79G,d 79.14F,c 0.99 20BCD,a
30 min AH30 367EF,d 10.74BC,a 138.30J,e 0.99 20BCD,a
45 min AH45 324DE,c 13.49CDE,b 66.53E,b 0.99 23D,b
60 min AH60 328DE,c 27.26G,c 89.37I,d 0.99 21CD,ab

Difference capital letters indicated the differences among treatments in column by Duncan test (P < 0.05); Difference lower case letters indi-
cated the difference results obtained by the same pretreatment method affected by difference pretreatment time.
Hmax: maximum cumulative hydrogen production; Rm: maximum hydrogen production rate; l: lag phase time; R2: the determination coefficient.

volume was measured by releasing the pressure in the bottles hydrogen produced at each time interval, using the mass
using wetted glass syringe [26]. All treatments were conducted balance equation [31]. A modified Gompertz equation [32] was
in four replicates. used to fit the cumulative hydrogen production curve for
biohydrogen production.
2.5. Analytical methods

Biogas composition was analyzed using a gas chromatograph
(GC) (GC-2014, Shimadzu, Japan) equipped with a thermal
conductivity detector (TCD) and a 0.2 m  3 mm diameter
stainless column packed with Shin carbon (50/80 mesh). The
operational protocols followed the method of Saraphirom and
Reungsang [27].
Volatile fatty acids (VFAs) and ethanol concentrations in
the liquid samples were analyzed by high performance liquid
chromatography (HPLC) (LC-10AD, Shimadzu, Japan) with ul-
traviolet detector using a VertiSep polymer-based HPLC
column (7.8 mm  300 mm, VertiSep OA 8 mm HPLC column,
Vertical). The temperature of column oven was 40
C. 4 mM
H2SO4 was used as a mobile phase at a flow rate of 0.5 mL/min
for 22 min followed by 0.4 mL/min for 8 min. Preparation of the
liquid samples prior the analysis by GC and HPLC followed the
method of Saraphirom and Reungsang [27].
Closed reflux method was used to determine total COD
(tCOD) and soluble COD (sCOD) concentrations [28]. Phenol
sulfuric method [29] with glucose as a standard was used to
determine total and soluble carbohydrate concentrations.
Lowry method [30] with a bovine serum albumin as standard
was used to measure total and soluble protein concentrations.
pH was measured by a digital pH meter (Sartorius, Germany).
Concentrations of TS, volatile solid (VS), total suspended
solid (TSS) and VSS were measured according to standard
method [28].
2.6. Kinetic analysis
Hydrogen gas production was calculated from the headspace
measurement of gas composition and the total volume of
3. Results and discussion
3.1. Effect of pretreatment on AS solubilization
Every pretreatment method could partially break down and
solubilize the solid components of AS to the liquid phase as
indicated by an increase in concentrations of sCOD (Fig. 1a),
soluble carbohydrate (Fig. 1b) and soluble protein (Fig. 1c).
Carbohydrate was more solubilized than protein for all pre-
treatment methods. Effect of acid (A6eA24) and heat
(H15eH60) pretreatments on carbohydrate solubilization was
not markedly different. Significant higher amount of increased
sCOD was observed in heat pretreatment than acid pretreat-
ment. Combined acid-heat pretreatment (AH15eAH60) is the
most effective method for solubilization of organic matters
from AS as indicated by highest percentage increased in sCOD,
soluble carbohydrate and soluble protein concentrations.
Combination of acid and heat pretreatment provided a stron-
ger reaction than heat or acid pretreatment alone hence larger
amount of organic matters could be degraded and solubilized
to the liquid phase.
Treatment time significantly affected the increase in sCOD
concentration for all pretreatments. In the acid pretreatment,
increased sCOD observed in A24 (111.49%) is significantly
higher than A6eA18 (39.27e76.90%). For heat pretreatment, an
increase in the treatment time from 15 (H15) to 45 (H45) min
significantly enhanced the percentage increase in sCOD con-
centration from 30.91% to 218.32%; further prolong the heat
treatment time to 60 min did not affect COD solubilization.
Percentage increase in soluble carbohydrate concentration
significantly rose from 100% to 162.16% when acid treatment
 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 e n e r g y 3 8 ( 2 0 1 3 ) 6 1 4 6 e6 1 5 3 6149

that obtained in treatment AH45 (408.57%) as well as treat-

ments AH30 (374.60%) and AH15 (212.30%), respectively.
A distinct result from combined acid-heat pretreatment of
sludge obtained from this study was on the %increase in sol-
ubilization of sCOD, soluble carbohydrate, and soluble protein
that was shown to be greater when compared to other studies
(Fig. 2). This high % increase in solubilization indicated that
more organic matters in the sludge was solubilized as the
result of combined acid-heat hydrolysis, which could be the
result of more acidic in our study (approximately pH of 1) than
the other studies used for a comparison (pH of 2 [33] and 3
[34]). It should be noted that our study used HCl while the
other studies used H2SO4 to pretreat the sludge. Smith and
Goransson [35] reported that thermal acidic hydrolysis by
using HCl gave slightly higher solubilization rate than H2SO4.
A hydrolysis yield obtained was 30e50%. Unfortunately, there
is limited information in the literature with regard to the use
of combined acid-heat pretreatment of the sludge. Most of the
previous studies were on the utilization of combined alkaline-
heat to pretreat the sludge. It is important to note that our
findings showed a similar trend to the results of a combined
alkaline-heat pretreatment in which the degree of solubiliza-
tion of sCOD increased with increasing pH (more basic con-
dition) [36,37] (or a solubilization rate increase with decreasing
pH (more acidic condition)). The increase in acid or base
concentration in the acidic or basic-hydrolyzing process,
respectively, could provide a strong or complete reaction for
breaking down the cell wall or cell membrane of the micro-
organisms present in the sludge yielding a better solubiliza-
tion of sCOD, soluble carbohydrate and soluble protein. Thus,
a strong acidic or basic condition is recommended for pre-
treatment of the sludge.

3.2. Effect of pretreatments on hydrogen fermentation

Pretreatment methods significantly affected hydrogen pro-

duction (Table 2). Significant higher maximum cumulative
hydrogen production (Hmax) and hydrogen yield (HY) were
obtained from acid and AH pretreated sludge when compared
to heat pretreatment (Table 2). Treating the sludge by acid for

Fig. 1 e Percentages increase in soluble COD (a), soluble

protein (b) and soluble carbohydrate (c) for difference
pretreatment methods.

time was extended from 6 (A6) to 12 (A12) h; further extend the

treatment time to 18 (A18) and 24 (A24) h did not affect car-
bohydrate solubilization. The increased soluble carbohydrate
was statistically increased as the treatment time was exten-
ded from 15 to 30, 45 and 60 min for both heat and combined
acid-heat (AH) pretreatments. Protein solubilization in acid
pretreatment was not affected by the treatment time. For heat
pretreatment, higher percentage increase in soluble protein
was observed at treatment time of 30 min (H30) (168.89%) than
15 min (H15) (23.57%); prolong the treatment time to 45 (H45)
and 60 (H60) min did not increase protein solubilization.
Treatment AH60 gave the highest percentage increase in sol- Fig. 2 e Comparison of percentages increase in
uble protein of 586.90% which was significantly higher than solubilization.
6150 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 e n e r g y 3 8 ( 2 0 1 3 ) 6 1 4 6 e6 1 5 3
6 h (A6) gave the Hmax and HY of 480 mL H2/L and 34 mL H2/g
VSadded. Under these conditions, the maximum hydrogen
production rate (Rm) of 15.17 mLH2/L h was achieved with the
lag time (l) of 84.07 h. The highest Hmax and HY of 600 mLH2/L
and 41 mL H2/g VSadded were obtained from the treatment A24
(treating AS by acid for 24 h) with the Rm and l of 12.76 mLH2/
L h and 87.30 h, respectively. However, these results were not
statistically different from the treatment A6 (Table 2). Con-
sidering that a shorter treatment time is needed for econom-
ical efficient and practical application, treatment A6 was the
most efficient pretreatment of AS prior the hydrogen fer-
mentation. Our results on hydrogen production from sludge
pretreated with acid for 6 h showed superior results over the
other literature search (Table 3). This might be due to the
treatment temperature used in our study (30
C) was higher
than Xiao and Liu (15e24
C) [38] and Wang et al. (4
C) [13]
resulted in more release of soluble organic matter which
was subsequently used as a substrate to produce hydrogen. It
is worth noting that it is difficult to compare our results with
other researchers’ results because the sludge and the seed
inoculum used in each study are different in characteristics
which can lead to the differences in hydrogen production
efficiency.
Fig.3 depicted the ratios of sCOD/tCOD at initial and at the
end of hydrogen fermentation. The initial sCOD/tCOD for acid
pretreatment was approximately 0.16 and was not sig-
nificantly affected by treatment time. For heat treatment,
sCOD/tCOD significantly increased with the prolong treat-
ment time within the range of 0.20e0.26. Though initial sCOD/
tCOD for AH pretreatments were not statistically different, the
trend of increased initial sCOD/tCOD with the extended
treatment time was observed. After fermentation, significant
increase in sCOD/tCOD was observed in all treatments, except
for treatment A12. The results indicated that microorganisms
in the fermentation system could further break the microbial
cells in AS. As a result, organic matters were released or sol-
ubilzed to liquid phase causing an increase in sCOD concen-
tration. The results also indicated that solubilization of
organic matter was more effective than utilization. Non-
markedly difference between initial and final sCOD/tCOD
observed in treatment A12 indicated the balance of organic
matter solubilization and utilization. Treatments H45-H60
and AH45-AH60 had a significant higher final sCOD/tCOD than
other treatments. This is because a long heat treatment time
of AS (45e60 min) could change the structures of solid portion
in AS and made the protein to be easier to digest by micro-
organisms in the fermentation system. However, there is no
correlation between hydrogen production efficiency and ini-
tial or final sCOD/tCOD concentration. Our results were dif-
ferent from other published report which mostly found that
Table 3 e Comparison of other literature search on hydrogen production using acid pretreated sludge as the substrate.
Substrate Seed inoculums Pretreatment
methods
Wastewater sludge Clostridium bifermentans HClO4, pH 3.0
Sewage sludge without extra-seeds 6 mol/L HCl, pH 2.0
Waste activated sludge Anaerobic mixed 0.5% (w/v) HCl,
from brewery industry culture pH z 1.0
Fig. 3 e Changes of sCOD/tCOD in the hydrogen
fermentation system.
hydrogen production efficiency increased when soluble
organic matter was more solubilized [39,40].
Different pretreatment methods affected the concentra-
tion of soluble organic compounds and consequently affected
hydrogen production. Increased soluble carbohydrate con-
centrations (Table 4), caused by different pretreatment
method, resulted in an increase of Hmax and HY (Table 2).
Carbohydrate was found as the main substrate for hydrogen
production from AS in the previous reports [39]. Increase in
solubilization of carbohydrate made it readily to be consumed
by hydrogen producing bacteria hence increasing hydrogen
production efficiency. Significant higher soluble protein con-
centration (10.72e14.64 mg/L) obtained from treatments H45
and H60 apparently caused a reduction in Rm (Table 4). Initial
concentration of lactic acid (HLa), propionic acid (HPr) and
acetic acid (HAc) were not obviously different. Higher initial
butyric acid (HBu) concentrations in heat treatment than in
acid and AH pretreatments were observed which might be
responsible for lower Hmax and HY values in heat treatment.
The inhibitory effect of high initial HBu concentration on
hydrogen fermentation was reported elsewhere [31,41,42].
Change of soluble organic matter concentrations in the
fermentation system reflected the substrate utilization and
metabolite production. After fermentation, concentration of
carbohydrate and HLa decreased in all treatments implying
that they were consumed for microbial growth and/or
hydrogen production by the presence microorganisms in the
fermentation system. Concentration of HPr in all treatments
and HAc and HBu in some treatments were increased which
suggested that they were the metabolic products during
Digestion time Hydrogen yield Reference
(mLH2/g COD)
6 h (at 4
C) 8.9 [13]
12 h (at room temperature) 2.53 [38]
6 h (at room temperature) 42.72 This study
 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 e n e r g y 3 8 ( 2 0 1 3 ) 6 1 4 6 e6 1 5 3 6151

Table 4 e Final pH and changes in soluble compounds in the liquid phase in the fermentation system.
Treatment Final sCarbohydrate sProtein HLa HPr HAc HBu tVFAs (mg/L)
pH (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L)

Initial Final Initial Final Initial Final Initial Final Initial Final Initial Final Initial Final

Raw 5.93 5.83 3.67 1.66 2.09 1843.48 10.98 38.08 379.24 342.14 285.12 167.71 484.62 2391.41 1159.96
sludge
A0 5.77 15.28 5.78 2.34 3.33 1969.10 3.44 14.39 155.12 354.37 390.38 94.34 1141.74 2432.20 1690.68
A6 5.86 19.05 6.13 2.53 3.20 1992.90 22.09 18.28 96.64 330.92 179.92 59.89 1282.11 2401.98 1580.75
A12 5.73 16.59 6.08 2.53 3.09 1810.98 5.95 12.25 178.55 341.58 195.00 44.64 1162.95 2209.45 1542.44
A18 5.87 20.36 9.21 2.81 4.27 1942.10 5.55 13.12 66.86 366.35 307.67 54.92 1087.01 2376.49 1467.10
A24 5.86 20.32 7.57 2.86 3.48 1445.77 120.22 8.81 88.04 258.82 353.38 64.88 1074.24 1778.28 1635.87
H15 6.08 6.63 4.73 2.26 2.56 1818.01 56.28 11.29 437.66 288.24 532.16 466.89 440.02 2584.42 1466.12
H30 6.02 7.66 4.30 5.38 3.27 1845.00 78.45 5.68 295.84 328.12 672.55 578.24 420.92 2757.04 1467.76
H45 6.08 8.93 4.35 10.72 4.38 1758.70 3.48 16.88 532.22 332.62 908.08 533.76 855.32 2641.96 2299.10
H60 6.02 12.30 6.14 14.64 5.48 1629.11 1.89 32.72 992.32 322.45 924.38 522.46 421.00 2506.74 2339.59
AH15 5.92 21.31 10.63 2.60 3.52 1928.40 3.28 7.86 106.94 361.36 325.65 40.45 1171.76 2338.06 1607.64
AH30 5.75 27.94 15.30 3.43 4.66 1703.20 89.98 11.23 73.19 305.82 426.96 15.68 935.90 2035.94 1526.03
AH45 5.97 40.56 28.22 4.59 3.78 1828.95 17.62 5.99 127.29 342.49 241.67 49.20 1232.33 2226.63 1618.91
AH60 5.90 52.54 29.27 5.89 5.45 1846.22 6.01 6.21 105.83 348.80 360.33 59.27 1079.56 2260.50 1551.74

sCarbohydrate: soluble Carbohydrate; sProtein: soluble Protein; HLa: lactic acid; HPr: propionic acid; HAc: acetic acid; HBu: normal butyric acid.
tVFAs (total volatile fatty acids) ¼ HLa þ HPr þ HAc þ HBu; initial: prior hydrogen fermentation; final: after hydrogen fermentation.

hydrogen fermentation. The decrease in concentrations of were considered as substrate for growth and activity of mi-
HAc and HBu might be the results of anaerobic oxidation of croorganisms in the fermentation system while HPr, HAc and
these two metabolites to carbon dioxide [43,44]. In addition, HBu were considered as the SMPs.
the reduction of HLa concentration can also be caused by tCODconsumed showed a strong positive correlation with
anaerobic oxidation process. Hmax. This result indicated that organic matter was consumed
In order to give more explanation about the phenomena on to produce hydrogen in the fermentation system. Level of
substrate consumption and metabolites production, data on tCODconsumed correlated positively with the increased level of
organic matter concentration (Table 4) and hydrogen pro- HBu. This could be explained by the fact that, the production
duction (Table 2) were tested for the correlations between of HBu from organic matters in the AS, for example carbohy-
hydrogen production and substrate consumption, substrate drate, released carbon dioxide and hydrogen to the biogas
consumption and SMPs as well as the correlation between (this also called butyrate-type fermentation) (Eq. (1)) [45]
hydrogen production and SMPs (Table 5). tCODconsumed rep- resulting in a decrease of tCOD as reflected by increased
resented overall utilization of organic matter in the fermen- tCODconsumed along with the decrease in HBu production.
tation system. Soluble carbohydrate, soluble protein and HLa tCODconsumed showed the significant negative correlation with
HPr and HAc. The possible pathway to explain this phenom-
ena are HPr production (Eq. (2)) [46,47] and homoacetogenesis
pathways (Eq. (3)) [48] which can be proceeded by propiono-
Table 5 e Correlations between hydrogen production, genic and acetogenic bacteria, respectively. As shown in Eqs.
substrate utilization and soluble metabolite products (2) and (3), hydrogen and carbon dioxide in gas phase can be
(SMPs). converted to HPr and HAc and accumulated in the liquid
HPr HAc HBu Hmax phase, as a result, tCOD concentration in the liquid phase
increased increased increased increased. These phenomena were reflected by decreased
tCOD consumed 0.766* 0.06* 0.794** 0.991**
tCODconsumed along with HPr and HAc production.
sCarbohydrate 0.655* 0.564* 0.894** 0.857**
consumed
sProtein 0.889** 0.852* 0.622* 0.567* C6H12O6 þ 2H2O / 2H2 þ CH3CH2CH2COOH þ 2CO2 (1)
consumed
HLa consumed 0.223 0.407 0.243 0.222
HPr increased e 0.812** 0.796** 0.732** C6H12O6 þ 2H2 / 2CH3CH2COOH þ 2H2O (2)
HAc increased e e 0.788** 0.591*
HBu increased e e e 0.774**

*Pearson correlation is significant at the 0.05 level (2-tailed),
**Pearson correlation is significant at the 0.01 level (2-tailed).
tCarbohydrate: total Carbohydrate; sCarbohydrate: soluble Carbo-
hydrate; sProtein: soluble Protein; HLa: lactic acid; HPr: propionic
acid; HAc: acetic acid; HBu: normal butyric acid; Hmax: maximum
cumulative hydrogen production.
4H2 þ 2CO2 / CH3COOH þ 2H2O (3)
Soluble carbohydrate consumption has a strong positive
correlation with Hmax and HBu production. The result indi-
cated that soluble carbohydrate was mainly consumed for
hydrogen production by HBu production pathway. The
6152 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 e n e r g y 3 8 ( 2 0 1 3 ) 6 1 4 6 e6 1 5 3
negative correlations between soluble carbohydrate con-
sumed and HPr and HAc production was observed. The
described possible reason for negative correlation between
tCODconsumed and HPr and HAc could also be used to explain
these results.
Soluble protein consumption negatively correlated with
Hmax and HBu production, whereas for HPr and HAc, the cor-
relations were positive. These results implied that protein
might be used for HPr and HAc production and degradation of
soluble protein might cause negative impact on butyrate-type
hydrogen production. However, it should be noted that in
some treatments, the rate of protein solubilization was
greater than the rate of protein utilization and the soluble
protein concentration was increased after fermentation. This
might cause the errors in correlation analysis.
Hmax had a strong positive correlation with HBu production
which confirmed that in this study, hydrogen was produced
from butyrate-type fermentation. Significant negative corre-
lation between Hmax and HAc indicated that the HAc was not
produced from acetate type fermentation but from aceto-
genesis pathway which consume hydrogen for HAc produc-
tion. Hmax had strong negative correlation with HPr confirming
that hydrogen was consumed for the production of HPr.
Previous research reported that HLa can be used as sub-
strate for hydrogen production by some bacteria species such
as Desulfovibrio desulfuricans strain New Jersey (NCIMB 8313)
[49], Clostridium acetobutylicum, Butyribacterium methylo-
trophicum, Clostridium tyrobutyricum [50]. However, it could be
seen from our result that HLa consumption has no significant
correlation with both Hmax and SMPs. These implied that mi-
croorganisms in the fermentation system might utilize HLa
not mainly for hydrogen production but for their growth.
However, the biomass production was not considered in this
study.
In this study, initial pH was adjusted to 5.5. Dramatically
change of pH was not observed in this fermentation system.
After fermentation the pH rose to be in the range of 5.77e6.08
(Table 4). The variation of pH in this hydrogen fermentation
system could be explained by the combination of VFAs con-
sumption, solubilized protein from AS, and accumulation of
ammonia from protein degradation. Consumption of VFAs
during the fermentation (Table 4) could cause an increase in
pH in the hydrogen fermentation system. Protein is an
amphoteric substance and has large buffering capacity;
therefore, the solubilized protein can prevent the dramatic
changes of pH in the fermentation system. Formation of
ammonia ion led to the increase of pH and alkalinity in the
fermentation system and prevented the drop of pH caused by
VFAs production [7,19].
4. Conclusions
Different pretreatment methods i.e., acid, heat and AH affect
the solubilization of organic matter from AS to liquid phase in
which AH pretreatment gave the highest percentage increase
in sCOD, soluble carbohydrate and soluble protein concentra-
tion. Prolonged pretreatment time tended to increase the sol-
ubilization of organic matter for all investigated pretreatment
methods. However, the solubilization efficiency did not
correlate with hydrogen production efficiency. Application of
different AS pretreatment method yielded different concen-
trations of soluble organic matter concentrations which con-
sequently affected hydrogen fermentation. After pretreatment
higher HBu concentration was found in heat pretreatment
than acid and AH pretreatment; HBu inhibit hydrogen fer-
mentation resulted in lower Hmax and HY for heat pretreat-
ment, accordingly. Correlation analysis revealed that hydrogen
was produced by butyrate-type fermentation. HPr and HAc
were negatively correlated with Hmax values. Carbohydrate
was the substrate which showed the strong positive correlation
between its consumption and hydrogen production.
Acknowledgments
The authors acknowledge the Royal Golden Jubilee Ph.D.
Program (Grant No.PHD/0194/2552) for a Ph.D. scholarship to
TA. Additional acknowledge goes to research funds from
Fermentation Research Center for Value Added of Agricultural
Products and the National Research University Project
through Biofuels Research Cluster-Khon Kaen University, Of-
fice of the Higher Education Commission.
references
[1] Jeong TY, Cha GC, Yoo IK, Kim DJ. Hydrogen production from
waste activated sludge by using separation membrane acid
fermentation reactor and photosynthetic reactor. Int J
Hydrogen Energy 2007;32:525e30.
[2] Amorim ELCd, Sader LT, Silva EL. Effect of substrate
concentration on dark fermentation hydrogen production
using an anaerobic fluidized bed reactor. Appl Biochem
Biotechnol 2012;166:1248e63.
[3] Khamtib S, Reungsang A. Biohydrogen production from
xylose by Thermoanaerobacterium thermosaccharolyticum
KKU19 isolated from hot spring sediment. Int J Hydrogen
Energy 2012;37:12219e28.
[4] Abreu AA, Karakashev D, Angelidaki I, Sousa DZ, Alves MM.
Biohydrogen production from arabinose and glucose using
extreme thermophilic anaerobic mixed cultures. Biotechnol
Biofuels 2012;5:6. http://dx.doi.org/10.1186/1754-6834-5-6.
[5] Cappelletti BM, Reginatto V, Amante ER, Antônio RV.
Fermentative production of hydrogen from cassava
processing wastewater by Clostridium acetobutylicum. Renew
Energy 2011;36:3367e72.
[6] Arunsri F, Reungsang A. Biohydrogen production from
sugarcane bagasse hydrolysate by elephant dung: effects of
initial pH and substrate concentration. Int J Hydrogen Energy
2011;36:8687e96.
[7] Sittijunda S, Reungsang A, O-thong S. Biohydrogen
production from dual digestion pretreatment of poultry
slaughterhouse sludge by anaerobic self-fermentation. Int J
Hydrogen Energy 2010;35:13427e34.
[8] Thungkin P, Sittijunda S, Reungsang A. Hydrogen production
from sludge of poultry slaughterhouse wastewater treatment
plant pretreated with microwave. Int J Hydrogen Energy
2011;36:8751e7.
[9] Venkata Mohan S, Lalit Babu V, Sarma PN. Effect of various
pretreatment methods on anaerobic mixed microflora to
enhance biohydrogen production utilizing dairy wastewater
as substrate. Bioresour Technol 2008;99:59e67.
 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 e n e r g y 3 8 ( 2 0 1 3 ) 6 1 4 6 e6 1 5 3
[10] Bougrier C, Delgenes JP, Carrere H. Effects of thermal
treatments on five different waste activated sludge samples
solubilisation, physical properties and anaerobic digestion.
Chem Eng J 2008;139:236e44.
[11] Lin Y, Liang J, Wu S, Wang B. Was pretreatment beneficial for
more biogas in any process? Chemical pretreatment effect
on hydrogenemethane co-production in a two-stage
process. J Ind Eng Chem 2013;19:316e21.
[12] Kan E. Effects of pretreatments of anaerobic sludge and
culture conditions on hydrogen productivity in dark
anaerobic fermentation. Renew Energy 2013;49:227e31.
[13] Wang CC, Chang CW, Chu CP, Lee DJ, Chang BV, Liao CS.
Producing hydrogen from wastewater sludge by Clostridium
bifermentans. J Biotechnol 2003;102:83e92.
[14] Gallert C, Winter J. In: Jordening HJ, Winter J, editors.
Bacterial metabolism in wastewater treatment systems.
Environmental Biotechnology. Weinheim, Germany: WILEY-
VCH Verlag GmbH&Co.KGaA; 2005. p. p.4e8.
[15] Muller J, Lehne G, Schwedes J, Battenberg S, Naveke R, Kopp J,
et al. Disintegration of sewage sludges and influence on
anaerobic digestion. Water Sci Technol 1998;38:425e33.
[16] Frolund B, Palmgren R, Keiding K, Nielsen PH. Extraction of
extracellular polymers from activated sludge using a cation
exchange resin. Water Res 1996;30:1749e58.
[17] Li Y, Noike T. Upgrading of anaerobic digestion of waste
activated sludge by thermal pretreatment. Water Sci Technol
1992;26:857e66.
[18] Kim J, Park C, Kim T, Lee M, Kim S, Kim SW, et al. Effects of
various pretreatment for enhanced anaerobic digestion with
waste activated sludge. J Biosci Bioeng 2003;95:271e5.
[19] Cai ML, Liu JX, Wei YS. Enhanced bio-hydrogen production
from sewage sludge with alkaline pretreatment. Environ Sci
Technol 2004;38:3195e202.
[20] Appels L, Baeyens J, Degreve J, Dewil R. Principles and
potential of the anaerobic digestion of waste-activated
sludge. Prog Energ Combust 2008;34:755e81.
[21] Elbeshbishy E, Hafez H, Dhar BR, Nakhl G. Single and
combined effect of various pretreatment methods for
biohydrogen production from food waste. Int J Hydrogen
Energy 2011;29:1e9.
[22] Weemaes MPJ, Verstraete WH. Evaluation of current wet
sludge disintegration techniques. J Chem Technol Biotechnol
1998;73:83e92.
[23] Muller JA. Prospects and problems of sludge pre-treatment
processes. Water Sci Technol 2001;44:121e8.
[24] Xiong H, Chen J, Wang H, Shi H. Influence of volatile solid
concentration, temperature and solid retention time for the
hydrolysis of waste activated sludge to recover volatile fatty
acids. Bioresour Technol 2012;119:285e92.
[25] Lay JJ, Lee YJ, Noike T. Feasibility of biological hydrogen
production from organic fraction of municipal solid waste.
Water Res 1999;53:435e40.
[26] Owen WF, Stuckey DC, Healy Jr JB, Young LY, McCarty PL.
Bioassay for monitoring biochemical methane potential and
anaerobic toxicity. Water Res 1979;13:485e93.
[27] Saraphirom P, Reungsang A. Biological hydrogen production
from sweet sorghum syrup by mixed cultures using an
anaerobic sequencing batch reactor (ASBR). Int J Hydrogen
Energy 2011;36:8765e73.
[28] APHA, AWWA, WEF. In: Chairman AEG, Clescerri LS,
Eaton AD, editors. Standard methods for the examination of
water and wastewater. 19th ed. Washington DC: American
Public Health Association; 1995.
[29] Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Phenol
sulfuric total sugar. Anal Chem 1956;28:350e6.
[30] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein
measurement with the folinephenol reagent. J Biol Chem
1951;193:265e75.
6153
[31] Zheng XJ, Yu HQ. Inhibitory effects of butyrate on biological
hydrogen production with mixed anaerobic cultures. J
Environ Manage 2005;74:65e70.
[32] Zwietering MH, Jongenburger L, Rombouts FM, Vant RK.
Modeling the bacterial growth curve. Appl Environ Microbiol
1990;56:1875e81.
[33] Barlindhaug J, Ødegaard H. Thermal hydrolysate as a carbon
source for denitrification. Water Sci Technol 1996;33:99e108.
[34] Liu X, Liu H, Chen J, Du G, Chen J. Enhancement of
solubilization and acidification of waste activated sludge by
pretreatment. Waste Manage 2008;28:2614e22.
[35] Smith G, Goransson J. Generation of an effective internal
carbon source for denitrification through thermal hydrolysis
of pre-precipitated sludge. Water Sci Technol 1992;25:211e8.
[36] Chen Y, Jiang S, Yuan H, Zhou Q, Gu G. Hydrolysis and
acidification of waste activated sludge at different pHs.
Water Res 2007;41:683e9.
[37] Rajesh Banu J, Khac UD, Adish Kumar S, Ick-Tae Y,
Kaliappan S. A novel method of sludge pretreatment using
the combination of alkalis. J Environ Biol 2012;33:249e53.
[38] Xiao BY, Liu JX. Effects of various pretreatments on
biohydrogen production from sewage sludge. Chin Sci Bull
2009;54:2038e44.
[39] Devlin DC, Esteves SRR, Dinsdale RM, Guwy AJ. The effect of
acid pretreatment on the anaerobic digestion and
dewatering of waste activated sludge. Bioresour Technol
2011;102:4076e82.
[40] Guo L, Li XM, Bo X, Yang O, Zeng GM, Liao DX, et al. Impacts
of sterilization, microwave and ultrasonication pretreatment
on hydrogen producing using waste sludge. Bioresour
Technol 2008;99:3651e8.
[41] Chin HL, Chen ZS, Chou CP. Fedbatch operation using Clostridium
acetobutylicum suspension culture as biocatalyst for enhancing
hydrogen production. Biotechnol Prog 2003;19:383e8.
[42] Wang JL, Wan W. Comparison of different pretreatment
methods for enriching hydrogen-producing cultures from
digested sludge. Int J Hydrogen Energy 2008;33:2934e41.
[43] Amani T, Nosrati M, Mousavi SM. Using enriched cultures for
elevation of anaerobic syntrophic interactions between
acetogens and methanogens in a high-load continuous
digester. Bioresour Technol 2011;102:3716e23.
[44] Hattori S, Galushko AS, Kamagata Y, Schink B. Operation of
the CO dehydrogenase/acetyl coenzyme a pathway in both
acetate oxidation and acetate formation by the
syntrophically acetate-oxidizing bacterium
Thermacetogenium phaeum. J Bacteriol 2005;187:3471e6.
[45] Levin DB, Pitt L, Love M. Biohydrogen production: prospects
and limitations to practical application. Int J Hydrogen
Energy 2004;29:173e85.
[46] Li J, Zheng G, He J, Chang S, Qin Z. Hydrogen-producing
capability of anaerobic activated sludge in three types of
fermentations in a continuous stirred-tank reactor.
Biotechnol Adv 2009;27:573e7.
[47] Guo LA, Li XM, Zeng GM, Zhou Y. Effective hydrogen
production using waste sludge and its filtrate. Energy 2010;
35:3557e62.
[48] Drake HL, Küsel K, Matthies C. Ecological consequences of
the phylogenetic and physiological diversities of acetogens.
Antonie Van Leeuwenhoek 2002;81:203e13.
[49] Carepo M, Baptista JF, Pamplona G, Fauque G, Moura JJG,
Reis MAM. Hydrogen metabolism in Desulfovibrio desulfuricans
strain New Jersey (NCIMB 8313)-comparative study with D.
vulgaris and D. gigas species. Anaerobe 2002;8:325e32.
[50] Hashsham SA, Fernandez AS, Dollhopf SL, Dazzo FB,
Hickey RF, Tiedje JM, et al. Parallel processing of substrate
correlates with greater functional stability in methanogenic
bioreactor communities perturbed by glucose. Appl Environ
Microbiol 2000;66:4050e7.