Natural Science, 2009, 1, 10-16 NS
http://dx.doi.org/10.4236/ns.2009.11003
Studies on the effects of pretreatment on production
hydrogen from municipal sludge anaerobic fermentation
Feng Wu1,2, Shao-Qi Zhou1,*, Yang-Lan Lai1, Wen-Jiao Zhong2
1College of Environmental Science and Engineering, South China University of Technology, Guangzhou, 510006, China;
2Guangdong Provincial People’s Armed School, Guangzhou, 510006, China.
Corresponding author: *
Received 31 March 2009; revised 15 April 2009; accepted 21 April 2009.
ABSTRACT
Municipal sludge was rich in organic matter,
period of natural degradation was long and low
efficiency, leachate would pollute underground
water. In this paper, a comparative study of the
ways of pretreatment with acid alkali treatment,
heat digestion and ultrasonic treatment were
done. The results showed that the dehydro-
genase activity was increased, the SCOD (solu-
able chemical oxygen demand, SCOD) in-
creased more than 2.47~2.83, 1.70~1.76, 2.6~
2.77 times respectively. The hydrogen yield in-
creased more than 11.5~12.2, 24.1~24.7, 34.2~
34.9 mL.g-1 (VS) respectively. The period of pro-
hydrogen was shorten to 7.5, 8.0, 6.5 d respec-
tively. The degradation rate was up to 72.04%,
81.4%, 80% respectively, the methane concen-
tration in the gas was close to “zero” and ul-
trasonic treatment was better than others.
Gompertz model curve fitting on hydrogen
production was carried out. All the values of
correlation factor R2 were more than 0.97.
Keywords: Municipal Sludge; Pretreatment; An-
aerobic Digestion; Biological Pro-Hydrogen
1. INTRODUCTION
The wastesolid treatment mainly includes filleading,
compost and incineration. Filleading and heap in brief
results in resource waste and pollute the body of water,
even endanger the human health. Which don’t attach to
coincidence method of “circulation economy”. The
wastesolid anaerobic fermentation producted hydrogen
develope a new road of wastesolid resource. And many
research workers are interested in it [1,2,3,4,5,6,7]. Go-
mez [8] has study on the two stages of bio-hydrogen
production, hydrogen production and methanogenic,
using organic solid waste and slaughterhouse waste as
substrate, high temperature activated sludge as inoculum.
Levin’s study [9] showed the wooden fiber of delignifi-
cation is a good hydrogen production substrate. Liliana
[6] use anaerobic sludge to degraded organic solid waste
and synthetic wastewater in UASB whose capacity is
3.85 L, and produce hydrogen successfully. The volume
content and hydrogen production rate of H2 is 47%, 99 N
mL.g-1(VS); 51%, 127 N mL.g-1(VS) respectively. Zuo
Yi [10] used river sediments as seed sludge, at the opti-
mal condition of the pH of 5.0-5.2, temperature of 35, ℃
and HRT of 6-8 h, a steady anaerobic bio-hydrogen
production was obtained in a lab scale reactor success-
fully with gluose as substrate. The highest hydrogen
production was 6.7 L.d-1. Tai Mulin [11] showed that the
optimal initial pH for bio-hydrogen production from
sewage sludge was around 11.0, Under this optimal con-
dition, the bio-hydrogen yield of raw sludge was 8.1
mL.g-1, and it would reach to 16.9 mL.g-1 when the
sludge was pretreated by alkali. Steven W. Van Ginkel
[12] used food wastewater as substrate indicated Biogas
produced from all four food processing wastewaters
consistently contained 60% hydrogen, with the balance as
carbon dioxide. Heguang Zhua [13] showed enhanced
hydrogen production potential as compared with All
combinations of the feedstocks (FW+PS, FW+WAS and
FW+PS+WAS). A mixing ratio of 1:1 was found to be
the best among the ratios tested and hydrogen yield of
112 mL.g-1 volatile solid (VS). M. Krupp and R. Wid-
mann [14] studied Biohydrogen production by dark fer-
mentation, the result showed The gas amount varied with
the different OLRs, but could be stabilised on a high level
as well as the hydrogen concentration in the gas with
44~52%. Ela Eroglua [15] introduced Biological hydrogen
production from olive millwastewater with two-stage proc-
esses. In some cases of dark-fermentation, activated sludge
was initially acclimatized to the OMW to provide the ad-
aptation of microorganisms to the extreme conditions of
OMW. The highest hydrogen production potential obtained
was 29 L H2/LOMW. Dongmin Li [16] used corn straw as
substrate, Hydrogen was produced by simultaneous sac-
charification and fermentation from steam-exploded corn
straw (SECS) using Clostridium butyricum AS1.209.

F. Wu et al. / Natural Science 1 (2009) 10-16 11
Maximum specific hydrogen production rate and maximal
hydrogen yield were 126 mL.g-1 (VSS) d and 68 mL.g-1
SECS, respectively. The yield of soluble metabolites was
197.7 mg.g-1 SECS. Acetic acid accounted for 46% of the
total was the most abundant product and this shows that
hydrogen production from SECS was essentially ace-
tate-type fermentation.
Consequently, fermentative bio-hydrogen production
technique is at the stage of laboratory research, many
hydrogen production bottlenecks binding factors are
urgently needed to be solved. This study focused on the
factors of fermentative bio-hydrogen production of mu-
nicipal sludge. In this paper, a comparative study on the
effect of pretreatment-acid alkali treatment, heat digestion
and ultrasonic treatment on hydrogen production were
done, and the optimum pretreatment approach was ascer-
tained, which break new a way for sludge treatment.
2. MATERIALS AND METHODS
2.1. Source and Characteristic of Sludge
Concentrated sludge came from a sewage treatment
plant in Guangzhou, China. Table 1 showed The char-
acteristics of the municipal sludge. In the experiment,
the proper complement of N, P and inorganic micro-
nutrients should be added in the sludge. The nutrient
solution contained: NH4HCO3 2.0 g·L-1, MgSO4·7H2O
50 mg·L-1, NaCl 10 mg·L-1, Na2MoO4·2H2O 10 mg·L-1,
CaCl2·2H2O 10 mg·L-1, MnSO4·7H2O 15mg·L-1, FeCl2
70 mg·L-1, KH2PO4 10 mg·L-1.
2.2. Experimental Equipment
Cylindrical anaerobic reactor (patent number: ZL 20052
0053384.X) with the dimensions: ødiameter = 22 cm, øexternal
diameter = 24 cm, h = 30 cm, effective volume = 11 liters;
JY99-IID ultrasonic cell disruptor (Ningbo Xinzhi);
XLJ-IIB low-speed tabletop centrifuge (Shanghai an ting
Scientific Instruments and Apparatus Co.); SC-15 ther-
mostatic water-circulator bath box (Ningbo Xinzhi); JJ-4
digital display motor stirrer (Jintan City Zhengji Instru-
ments Co. Ltd); BSD0.5 wet-gas flow meter (Shanghai
Blue Jewelry); GC-7900 gas chromatograph, thermal
conductivity detector, and FID detector (Shanghai Tian-
mei); ZXZ-1 sliding vane rotary vacuum pump (Zheji-
ang Huangyanqiujing, modified as shown in Fig. 1).
2.3. Experimental Methods
1kg dried sludge was dissolved in 10 L water，stirred
uniformly, divided into A, B, C group. This sample
would carry out acid alkali treatment, heat digestion and
ultrasonic treatment, using 2#, 3#, 4# to mark the sample
performed cid alkali treatment, 5#, 6#, 7# to mark the
sample performed heat digestion and 8#, 9#, 10# to mark
the sample performed ultrasonic treatment. Put 200 mL
the liquor in a cone type bottle as reference object which
was marked 1#. They were respectively performed an-
aerobic digestion in shaking table whose rate was 1,050
rpm at 36℃.
2.4. Analysis Methods
Gas components were detected using a gas chromato-
graph (model: GC-7,900). A flame ionization detector
(FID) and a 2-m stainless steel column packed with 5A
Table 1. Characteristics of condensed sludge from municipal
wastewater treatment plan.
pH SS
(g/Kg)
Water content
(%) TN COD
6.7~7.913~2778.7~90 1750~2000 3900~5000
1. SC-15 water bath; 2. pH adjusting port; 3. organic reactor; 4. agitating blade; 5. reaction substrate
outlet; 6. material inlet; 7. vent; 8. digital stirrer; 9. thermometer; 10. CO2 removal; 11. desiccant; 12.
wet gas flowmeter; 13. GC-7900 gas chromatography; 14. nitrogen purging port.
Figure 1. Equipment and sequence of steps in the anaerobic digestion of sludge.

12 F. Wu et al. / Natural Science 1 (2009) 10-16
Molecular Sieve were used to analyze the methane con-
centration. The temperatures of the injector, detector,
and packed column were, respectively, 150, 180, and
100°C. H2 was used as the carrier gas at a flow rate of 30
mL· mi n -1. The N2 flow rate was 30 mL·min-1 and the air
velocity 260 mL·min-1. The hydrogen concentration was
analyzed using a thermal conductivity detector (TCD)
and a 2-m stainless steel column packed with 5A Mo-
lecular Sieve. The temperatures of the injector, detector,
and packed column were, respectively, 180, 200, and
100°C. N2 was used as the carrier gas at a flow rate of 29
mL· mi n -1. The injection volume was 10 μL. Quantitative
analysis was carried out using external standards.
Otherwise, The quantity of chemical oxygen de-
mand in sludge supernatant fluid was used to estimate
the performance of sludge disintegration. The value of
TCOD was equal to that of waste activated sludge su-
pernatant fluid. The value of SCOD was equal to that
of COD of sludge supernatant fluid which has been
treated by centrifugal separation and filtration [17].
Determination of COD followed the standard methods
[18]. The centrifuge worked for 20 min at 1,050 rpm,
COD was determined according to International Stan-
dard [3]. Dehydrogenase activity of sludge was deter-
mined according to the method reported in the litera-
ture [19,20].
2.5. Cumulative Hydrogen Yield
Cumulative hydrogen yield was estimated using the fol-
lowing equation [21,22]:
V=V0γi+∑Viγi (1)
where V is the cumulative hydrogen yield (mL), V0 the
volume above the liquid level in the reactor (mL), Vi the
volume of gas extracted in phase i (i=1,2,3...) (mL), and
γi the concentration of hydrogen in the gas extracted in
phase i (i = 1, 2, 3...) (i = 1, 2, 3...)(%).
2.6. Kinetic Model of Hydrogen Production
The Gompertz equation was used in the regression
analysis of the anaerobic hydrogen production data in
order to determine the lag time of hydrogen production,
the hydrogen production potential, and the hydrogen
production rate [22,23]:

exp exp1
s
s
s
Re
HP t
P



 




(2)
where H is the cumulative hydrogen yield (mL), Ps the
maximum hydrogen yield (mL), Rs the maximum hy-
drogen yield rate (mL.h-1), and λ the lag time of hydro-
gen production (h).
3. RESULTS AND DISCUSSIONS
3.1. Effect of Acid and Alkali Treatment on
Hydrogen Production
Under normal temperature, the SCOD value of different
sludge changed. 2#, 3#, 4# were used to mark the sludge
pH ＝ 10, 11, 12, respectively. 1# is control group, it is
neutral. Fig. 2 showed the changes of SCOD value of
sludge treated by acid alkali treatment. Fig. 3 showed
the state of sludge anaerobic digestion bio-hydrogen
production. Fig. 2 showed that the SCOD value of 2#,
3#, 4# changed with time in the same regular but in dif-
ferent level: 9,266, 9,477, 10,624.5 mg.L-1. Compared
with 1#, The SCOD value of 2#, 3#, 4# were separately
increased 2.47, 2.53, 2.83 times, The data indicated the
dissolution of organic increased because of acid alkali
treatment. The value of SCOD reached maximum at
24th hour, beginning to decrease at about 24-28th hour.
The degradation rate was up to 72.04% from 60.4%,
increased by 12%. The reason was that after the sludge
was treated by acid alkali treatment, most of the organic
has been dissolved, some of the difficult dissolved or-
Figure 2. Change of SCOD about Sludge for anaerobic digestion.
20 40 60 80100120140160
0
50
100
150
200
250
300
Hydrogen yield (mL)
Time (h)
1# 2#
3# 4#
Figure 3. Change of hydrogen production about Sludge for
anaerobic digestion ed and modified , and then solidified.

F. Wu et al. / Natural Science 1 (2009) 10-16 13
ganic lignose, cellulose and hemicellulose etc had struc-
tural changes, hydrolyzed by cellulase, ligninase, etc.
24h after start-up, almost all the organic absorbed by
sludge has dissolved. The rate of organic dissolution was
faster than that of bio-degradation result in the accumu-
lation of organic in liquor, therefore, SCOD value rose
obviously. 28~32 h after start-up, the number of biomass
and live bacteria is max in the system. Plenty of carbon
source was needed to maintain biological metabolism, so
the anaerobic digestion speeded up, the organic was con-
sumed by anaerobic bacteria as nutrient, and the COD
started to decline. At the moment a large number of
small bubbles attached to the conical flask because of
biohydrogen bacterium starting to produce enormous
hydrogen. In order to facilitate gas emissions, turn down
the rate of shaking table to 1,050 rpm to reduce gas-liq-
uid interface pressure. The peak hydrogen productionin
was observed at 32-48th hour, then the sludge hydrogen
production ability weakened gradually, and the hydrogen
content began to decline. 7.5 d after start-up, litter hy-
drogen was produced. The hydrogen production yield of
2#, 3#, 4# were 11.5, 12.2, 11.7 mL.g-1 (VS) respectively.
A little gas was produced at the first day in 1#, and the
hydrogen content was low, Less than 30%. The amount
of hydrogen started to increase linearly in the second day
and reach its peak at the sixth day, but the maximum rate
of hydrogen production lasted no more than 2 h. Until
the 13th day the gas was too little to collected, therefore
the hydrogen production period is 13d. hydrogen pro-
duction yield is 9.2 mL.g-1 (VS). Consequently, after the
sludge was treated by acid alkali treatment, the period of
hydrogen production was shortened obviously, acid al-
kali treatment is an effective solution to the problem that
the hydrogen production period is too long in sludge
anaerobic digestion system. After acid pretreatment, the
amount of dissolved organic in sludge increased, which
is the same as the effect of alkali pretreatment. The dif-
ference between acid pretreatment and alkali pretreat-
ment was that acid pretreatment provided methanogen a
good growth condition because of the acidification of
substrate and the formation of menthanogenic phase, in
order to maintain the systems ability of hydrogen pro-
duction, it is needed to control the pH value or to use
methanogen inhibitor such as acetylene, BES to make
the system pro-hydrogen instead of methane.
3.2. Effect of Heat Digestion Pretreatment
on Bio-Hydrogen Production
Fig. 4 showed the amount of SCOD in 5#, 6#, 7# rose
linearly with the heat digestion time extended and the
temperature rose. When the sludge temperature is
80~100℃, the amount of dissolved organic increased
obviously. The dissolution rate reached the peak at 93℃,
however the rate decreased when the temperature rose
20 40 60 80100120140
3500
4000
4500
5000
5500
6000
6500
7000
SCOD (mg.L-1)
Heat digestion time (h)
5#
6#
7#
Figure 4. Change of SCOD about Sludge for heat digestion.
to 120℃. There was refractory fiber as well as easily
degradable organic in the sludge. Fig. 5 showed the
structure of the fiber. When the temperature was higher
than 120℃, the fiber structure was destroyed, decom-
posing into cellobiose and penta-disaccharide, and then
transforming into glucose, degraded by bacteria finally.
Therefore, the SCOD value increased slowly. The
amount of SCOD in 5#, 6#, 7# were separately increased
1.73, 1.76, 1.70 times. The changes of fermentative
bio-hydrogen production were depicted in Fig. 6 hydro-
gen production of 5#, 6#, 7# increased considerablely 27
h after start-up, and reached the peak in 40~48h, then
declined. Little gas could be collected at the 8th day,
which was considered as the end of cycle period. The
hydrogen production yield of 5#, 6#, 7# is 24.7, 24.1,
24.1 mL.g-1 (VS). Heat digestion dissolved the de-lipid
of cell, weakened the tolerance ability of cell wall
against heat, promoting the hydrolysis of sludge. It was
observed that the color of sludge mixed liquor turned
into reddish-brown, and the liquor was covered by a
layer of film because of the effect of heat. the reason
maybe a part of microbiology protein dissolve.
3.3. Effects of Ultrasound Treatment on
Anaerobic Sludge Digestion Hydrogen
Production
Fig. 7 illustrated changes of anaerobic sludge digestion
hydrogen production. On the conditions of P = 1,800 W
and f = 35 kHz, the sludge samples 8#, 9#, 10# are
treated with ultrasound for 20 min respectively. An
increase of dissolved chemical oxygen demand of sludge
was observed. The rate of organic matter dissolution can
be calculated by Equation (2).
0
0
100%
tt pHSCOD SCODSCOD
DDCOD TCOD SCOD


 (3)
In the equation:
DDCOD───rate of organic matter dissolution, %;

14 F. Wu et al. / Natural Science 1 (2009) 10-16
Figure 5. The schematic diagram about the structure of the cellulose.
02468
-20
0
20
40
60
80
100
120
140
160
180
Hydrogen yield (mL)
Time (d)
5#
6#
7#
Figure 6. production hydrogen of anaerobic digestion from
sludge by heat digestion.
-20020 40 60 80100120140160180
-100
0
100
200
300
400
500
600
700
8# hydrogen yield
9# hydrogen yield
10# hydrogen yield
8# hydrogen production rate
9# hydrogen production rate
10# hydrogen production rate
Time (h)
Hydrogen yield (mL)
0
10
20
30
40
50
60
70
Hydrogen concentration (%)
Figure 7. production hydrogen of anaerobic digestion from
sludge by ultrasonic wave.
TCOD───the COD of supernatant obtained from the
sludge solution, mg/L;
SCODpH───the COD of filtering supernatant of
sludge solution under pH, mg·L-1;
SCODt0───the COD of supernatant obtained from
the sludge solution been centrifuged, mg·L-1;
SCODt───the COD of filtering supernatant of
sludge solution under different radiation time, mg·L-1.
The SCOD value was 2.70, 2.77, 2.64 times than that
of 1# respectively, organic solution rate was 48~65%.
Adaptation time of hydrogen-producing bacteria is 17.4
h, and logarithmic phase time is 9 h. The growth and
reproduction of microorganisms went into stationary
phase after 26 h, which has the largest biomass and the
most hydrogen production. Simultaneously, hydrogen
production of 8#, 9#, 10# went into the peak phase, and
the production of 10# reached 210 mL.d-1, then the pro-
duction began to decline. 7 days after start-up, hydrogen
production was less than 10 mL.d-1 reaching almost zero
at the end of the cycle period. Degradation rate of COD
is more than 80%, and the hydrogen production yield is
34.2, 34.9, 34.5 mL.g-1 (VS) respectively, and the meth-
ane concentration close to “zero”. The analysis showed
that sludge solution was affected by ultrasonic energy
experience dynamic processes of vibration, growth, col-
lapse and closure. At the moment of the collapse of the
bubble, high-temperature and high pressure will be cre-
ated in a very small space around the bubble, which will
destruct the floceulent structure of sludge and crush the
cell of microorganisms. Intermolecular hydrogen bond
of Cellulose which is refractory broke by ultrasonic irra-
diation, producing organic matter easily biodegradable
such as sugar. Consequently the dissolved organic,
which provided enough carbon source for the growth
and reproduction of anaerobic microbe, multiplied in
sludge solution. It was also found that after ultrasonic
irradiation, the permeability of cell membrane and cell
wall have changed, and the looseness of extracellular
polymers increased, which promoted biological mass-
transfer and improved the enzyme activity, so the TF
(The activity of dehydrogenase was evaluated by the
amount of TF which generated by the reaction between
per unit mixture liquid sludge and TTC in unit time, the
unit is mg·L-1·h-1) value rose. Fig. 7 showed the change
trend of dehydrogenase activity. because of the ultra-
sound pretreatment, the catalysis of dehydrogenase and
nitrogenase etc. was improved, as well as the decompo-
sition and absorption ability of anaerobic bacteria and
facultative anaerobe [24]. Therefore the degradation of
organic speed up. In the stage of peak hydrogen produc-
tion lots of bio-hydrogen heterotrophic bacteria were
observed, such as clostridium, enterobacter, Escherichia
coli, Citrobacter, Bacillus, Thiobacillus, etc. by micro-
scopic examination, the most bacteria were enterobacter
aerogenes, candida maltose. Synergistic effect between
strain is good, which inhibited the accumulation of me-
tabolites and then provided a good environment for hy-
drogenogens, therefore it was given full play to hydro-

F. Wu et al. / Natural Science 1 (2009) 10-16 15
genogens, the hydrogen production yield rose.
3.4. Effect of Dehydrogenase Activity by
Pretreatment Sludge
Dehydrogenase activity is defined as the TF volume per
unit time, with the unit mg·L-1·h-1 [25]. From Fig. 8, the
initial TF was 60.6 mg·L-1·h-1, after pretreatment TF
were 71.2, 69.9, 78.8 mg·L-1·h-1 respectively.12h later,
TF declined to 32, 31.2, 27.7 mg·L-1·h-1 respectively.
The result showed Dehydrogenase activity was increased
with pretreatment. the permeability of cell membrane
and cell wall changed because of pretreatment, which
promoted the mass transfer, the production and activity
of cell enzyme, so the metabolism speeded up. Moreover,
NAD+ or NADP+ regenerated by cell can absorb and
transport substrate or TTC effectively, therefore the
amount of TF increased [26].
3.5. Analyzing Kinetic Model of Hydrogen
Production Bacteriaons
Fig. 3, Fig. 6, and Fig. 7 showed hydrogen production
closely related to the microbial growth regularity. The
change of hydrogen yield contain four phases: lag phase,
beginning of hydrogen production, continuous hydrogen
production and attenuation of hydrogen production. The
lag phase was short (0~11 h). There was no hydrogen
production until a stable hydrogen production flora
formed after acclimation, cultivation and propagation.
Subsequently, hydrogen yield increased gradually with
the exponential increase of bacteria. Hydrogen content
rose when the growth rate of bacteria was maximum.
About 27 h later, the organic content of substrate de-
clined because of the rapid bacteria propagation con-
suming considerable organic material. Meanwhile, the
accumulation of metabolites poisoned bacteria, and bac-
teria death rate rose. When the growth rate balanced the
death rate, the amount of bacteria in the system was
maximum. After 80h nutrition was exhausted, the bacte-
0 102030405060
20
30
40
50
60
70
80
TF/mg.L-1.h-1
tim e (m in )
sample1 sample2 sample3
Figure 8. Variation of dehydrogenase activity with pretreat-
ment.
ria performed endogenous respiration and even formed
spore, hydrogen yield declined until the end. The reac-
tion period was about 160 h. No methane was observed
during the reaction. Gompertz model curve fitting on
hydrogen production was carried out. All the values of
correlation factor R2 were more than 0.97. Therefore the
fitting effect of Gompertz model on describing the bio-
hydrogen production process was good.
4. CONCLUSIONS
The sludge been treated by acid and alkali, heat diges-
tion, ultrasonic treatment, most of the refractory organic
transformed into easily degradable carbohydrate. Com-
pared with control group, the dehydrogenase activity
was increased, the SCOD was increased 2.47~2.83,
1.70~1.76, 2.6~2.77 times respectively, the hydrogen
production yield were 11.5~12.2, 24.1~24.7, 34.2~34.9
mL.g-1(VS) respectively, the period of hydrogen produc-
tion was shorten to 7.5, 8.0, 6.5 d respectively. Remove
of the COD was up to 72.04%, 81.4%, 80% respectively.
the methane concentration in the gas was close to “zero”.
The hydrogen concentration can reach 99.3% after the
bio-gas was purified by Ca(OH)2 saturated solution.
Gompertz model curve fitting on hydrogen production
was carried out. All the values of correlation factor R2
were more than 0.97.
ACKNOWLEDGEMENTS
The authors wish to thank the New Century Outstanding Young
Teacher Grant of the Ministry of Education of China for financial
support (NCET-04-0819).
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