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Bioresource Technology 99 (2008) 36513658

Impacts of sterilization, microwave and ultrasonication pretreatment

on hydrogen producing using waste sludge
Liang Guo, Xiao-Ming Li *, Xie Bo, Qi Yang, Guang-Ming Zeng, De-xiang Liao, Jing-Jin Liu
College of Environmental Science and Engineering, Hunan University, Hunan, Changsha 410082, China

Received 16 May 2007; received in revised form 15 July 2007; accepted 16 July 2007
Available online 11 September 2007

Abstract

Hydrogen production by sterilization, microwave and ultrasonication pretreated waste sludge was investigated in this study. A new
strain of Pseudomonas sp. GZ1 (EF551040) was inoculated in pretreated waste sludge to produce hydrogen. The experimental results
showed that dierent pretreated sludge had evident dierences in the yield of hydrogen production and lag time. Sterilized sludge
had the largest yield of hydrogen production, and the maximum yield was 15.02 ml/gTCOD. The lag time of using sterilized sludge
was 15 h, longer than other two pretreated sludge. Using the ultrasonicated sludge, the hydrogen production yield was smallest and
lag time was shortest in the three pretreated sludge. Protein and carbohydrate could be released from waste sludge by pretreatment. Pro-
tein was the main nutrient used for hydrogen production. The concentration of protein, carbohydrate and SCOD increased after pre-
treatment and fermentation. The impacts of dierent pretreatments on hydrogen production were also discussed in detail.
 2007 Elsevier Ltd. All rights reserved.

Keywords: Waste sludge; Hydrogen; Sterilization; Microwave; Ultrasonication

1. Introduction
Biomass (waste sludge, plants, animals, and their
organic waste products) provides approximately 14% of
the worlds energy needs. In developing countries, which
often have limited supplies of fossil fuels but abundant bio-
mass resources, as much as 35% of the energy demand is
supplied by biomass. However, in developed countries with
still abundant fossil fuel supplies, reliance on biomass as an
energy resource is very limited, despite its huge energy
potential. Waste-activated sludge from a wastewater treat-
ment plant is rich in polysaccharides and proteins and thus
it is a potential substrate for producing hydrogen (Wang
et al., 2003a). In 2001, about 4220 million t of municipal
wastewater was treated in more than 150 municipal waste-
water treatment plants in China, producing about 0.55
1.06 million t of dry sludge (Statistical Gazette, 2001).
*
Corresponding author. Tel.: +86 731 8823967; fax: +86 731 8823701.
E-mail address: xmli@hnu.cn (X.-M. Li).
Landll is one of the most common methods that are used
in the nal disposal of sludge world wide (Ozsoy et al.,
2006). The disposal of sludge generated in urban wastewa-
ter treatment plants is an important environmental prob-
lem (Solis et al., 2002). Unused, discarded biomass
residues are a potential energy resource, which at present
are not well managed and thus pose signicant environ-
mental problems (Levin et al., 2007).
Recently, some studies are focusing on using the sludge
to produce hydrogen by anaerobic fermentation. The
hydrolysis of the microbial cell limits the speed of the
anaerobic digestion of sludge and results in a long HRT
(hydraulic retention time) (Weemaes and Verstraete,
1998; Alvarez et al., 2000). To improve anaerobic digestion
of waste sludge, the most logical approach is to disrupt the
microbial cells of sludge. Several pretreatments have been
shown to promote the hydrolysis and disintegration of
sludge ocs and thus enhance the eciency of digestion
(Lee and Mueller, 2001). Due to low hydrogen yield from
the raw sewage sludge, a lot of pre-treatments had been
used in previous studies like heating (Wang et al., 2003a;

0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2007.07.026
3652 L. Guo et al. / Bioresource Technology 99 (2008) 36513658
Zhu and Beland, 2006), alkaline (Cai et al., 2004), acidi-
cation (Zhu and Beland, 2006), sterilization (Wang et al.,
2003a), methanogenic inhibitor (Wang et al., 2003a; Zhu
and Beland, 2006), freezing and thawing (Wang et al.,
2003a), ultrasonication (Wang et al., 2003a) to enhance
the hydrogen yield. Hydrogen yield was improved to
1.4 mgH2/gDS (dissolved sludge) using the boiled sludge
and 1.52.1 mmol H2/gCOD with sludge pretreated by
sterilization or freezing and thawing (Wang et al., 2003a).
Until recently, there was little information about Pseu-
domonas to produce hydrogen with waste sludge and the
reason that the same sludge using dierent pretreatment
and releasing similar nutrients had dierent hydrogen yield
and lag time (the time phase from the beginning of inocu-
lation to the beginning of gas production) was not clear. In
this study, the waste sludge pretreated by microwave, ultra-
sonication, and sterilization was used to produce hydrogen
by a new strain of Pseudomonas sp. GZ1 (EF551040) and
the variety of organic matters released from microbe in
waste sludge such as protein, carbohydrate, SCOD (soluble
chemical oxygen demand) and VFA (volatility fatty acid)
was discussed to investigate the impact of microwave,
ultrasonication and sterilization pretreatment on waste
sludge to produce hydrogen, and the possible reason was
also analyzed in detail. The objective of this study was to
discuss the impacts of pretreatment on hydrogen produc-
tion, which would make possible to further enhance the
hydrogen production using waste sludge.
2. Methods
2.1. Substrate
Wastewater sludge was taken from the recycled stream
of the secondary treatment stage of a Municipal Wastewa-
ter Treatment Plant in Changsha which handles 140,000 m3
wastewater daily. The collected sludge settled about one
week to yield solids content of 15.42 g/L SS and 8.29 g/L
VSS. The pH value of the sludge was about 7.57. The
TCOD (total chemical oxygen demand) and SCOD (solu-
ble chemical oxygen demand) of raw waste sludge was
12,133 mg/L and 587 mg/L, respectively.
2.2. Pre-treatment
Three pre-treatments were applied to waste sludge.
These pre-treatment not only released insoluble organic
matters into water to increase the eciency of fermenta-
tion, but also inactivated methanogenic bacteria in the sub-
strate to reduce their consumption of hydrogen. The
pretreated sludge was the substrate used in the hydrogen
fermentation tests.
(1) Microwave the sludge was heated in the microwave
for 2 min at a power of 560 w (Galanz, P7023TP-K7,
China).
(2) Ultrasonication sonication tests were performed
with the help of a cell-breaker at a sonication power
density of 2 w/ml. Original sludge (200 ml) was
placed in a 1000 ml beaker with the ultrasonic probe
positioned 2 cm submerge the surface of the sludge.
The sonication time was 5 min to release the insoluble
organic matters from the solids (Branson, digita son-
ier 450, USA).
(3) Sterilization sample of sludge was sterilized at
121 C for 20 min (Hirayama Manufacturing Corpo-
ration, HV-50, Japan).
2.3. The inoculum
A Pseudomonas sp. GZ1 (EF551040) was used as seed
bacteria, which was isolated from the anaerobic granule
sludge collected from Yueyang paper mill. The applied
puried procedures include: (1) Heat pre-treating at 80 C
for 2 h to inactivate the methanogenic bacteria; (2) Tritu-
rating the granule sludge using sterilized apparatus. (3) Iso-
lating and purifying the incubated sludge on solid culture
medium rst by Hungate method (Hungate, 1969) and
about three days later transferred the colony to liquid cul-
ture medium. These procedures were repeated for three
times. (4) The preliminary hydrogen production was tested,
and the strains with the highest hydrogen yields were
selected as the inoculum in this study. The strain was ana-
lyzed at the following steps: DNA extraction; PCR ampli-
cation of 16S DNA gene (Ren et al., 2005); puried the
PCR and sequence analysis of the 16S rDNA. The results
showed that strain GZ1 was a member of the Pseudomonas
family.
2.4. Fermentation and testing
Substrate (100 ml) was mixed with 5 ml of inoculum sus-
pension and was anaerobically (blow N2 for 10 min) incu-
bated at 35 C in 250 ml serum bottles with stirring of
125 r/min and was not added other nutrients. The bottles
were capped with silica gel stoppers. Under identical condi-
tion three fermentation bottles were measured and their
average of data was reported to prevent any possible errors
introduced by sampling procedure. The COD, protein, car-
bohydrate, pH and VFA were measured during fermenta-
tion. The concentration of H2 was tested frequently
during fermentation and the H2 production was also
recorded during the whole examination. The samples were
extracted every 37 h.
Volatile fatty acids (VFA) were measured with a gas
chromatograph (Agilent Technologies 6890N, USA)
equipped with a ame-ionization detector (FID) and the
automatic liquid sampler. The gas chromatograph (GC)
was equipped with a capillary free fatty acid phase (polar-
ity) column (DB-FFAP, 30 m 0.25 mm 0.25 lm). The
temperatures of the injector and detector were 250 and
300 C, respectively. The initial temperature of oven was
70 C for 3 min followed with a ramp of 20 C/min for
 L. Guo et al. / Bioresource Technology 99 (2008) 36513658 3653

5.5 min and to nal temperature of 180 C for 3 min. N2
was used as carrier gas with a ow rate of 2.6 ml/min. A
GC-TCD (Agilent Technologies 6890N, USA), equipped
with a capillary column packed with CARBONPLT
(30 m 0.53 mm 3 lm) at the temperature of 80 C
(oven), 150 C (injector), 200 C (detector), measured the
hydrogen and methane concentrations in the gas phase.
The ow rate of N2 used as carrier gas was 1 ml/min.
The TCOD (total chemical oxygen demand) of the sludge
was measured by the microwave digestion method (model
MS-3 Microwave Digestion System for COD Determina-
tion, China). The SCOD of the sludge sample was obtained
after ltering through a 0.45 lm membrane. SS and VSS
were determined by standard method (He, 1998). The pH
was measured with a digital pH-meter (pH315i, WTW
82362 Weilheim, Germany). Protein and carbohydrate
were determined by their respective standard method
(Ren et al., 2005). The concentrations of Zn, Cu, Pb and
Cd in waste sludge were determined by atomic absorption
spectrophotometry analysis (AAS-700, Perkin Elmer,
USA). The accumulative volume of hydrogen produced
(H) over the time course during the batch tests was tted
with the Gompertz equation (Lay et al., 1999)
  
Rm e
H P exp  exp k  t 1 enhanced and maintained stable from sewage sludge with
P both sterilization and microwave pretreated sludge. In
where P is the hydrogen potential (ml), Rm is the maximum
hydrogen production rate (ml/h), k is the lag phase time
(h), and e is 2.718281828.
3. Results and discussions
3.1. Hydrogen production
In this study, the inuence of dierent pretreatment on
the hydrogen production using waste sludge by anaerobic
fermentation was investigated. Sterilization, microwave
and ultrasonication were all eective for solubilizing
Table 1
The character of raw and pretreated waste sludge used in this studya
Parameter Sterilization Microwave Ultrasonication
Proteinr (mg/L) 25.40 85.88 0
Proteinp (mg/L) 410.52 597.01 72.07
Carbohydrater (mg/L) 18.72 14.26 0
Carbohydratep (mg/L) 202.65 450.51 101.96
organic matters from waste sludge (Table 1). Protein, car-
bohydrate and SCOD were all increased considerably,
but the change of TCOD, SS and VSS were inconspicuous.
It was shown that the pretreatments could destroy cellular
wall, and release dissoluble matters. So pretreatments were
advantageous for hydrogen production. In this study, the
lag time was 3 h using ultrasonicated sludge, the shortest
comparing to that using other pretreated sludge. But the
hydrogen production was the lowest of all. The maximal
yield of hydrogen was 4.68 ml/gTCOD. The largest hydro-
gen production was observed using sterilization pretreat-
ment. The maximal yield of hydrogen was 15.02 ml/
gTCOD. But the lag time was 15 h. The microwave pre-
treated sludge was then used for bio-hydrogen production,
with a maximal hydrogen yield of 11.44 ml/gTCOD, and
the lag time was 10 h (Fig. 1). However, hydrogen accumu-
lated in the headspace of bottles was then consumed by
hydrogen consumers in the following fermentation time,
which was observed in all the pretreated sludge. A common
phenomenon, higher hydrogen yield and slower hydrogen
consumption was observed in the tests. Although, the
hydrogen yield with microwave pretreated sludge was less
than sterilizations, the lag time was also shorter. These
results showed that the hydrogen production could be
addition, the estimated parameters of P, Rm and k were
quite consistent with these experimental data (Lay et al.,
1999; Chen et al., 2005) (Fig. 1). It was found that no meth-
ane was present in bio-hydrogen production during exper-
imentation and the hydrogen concentration in the gas
phase was more than 70%. Results of hydrogen production
in this study were compared with other results in the liter-
atures as shown in Table 2. It was shown that the Pseudo-
monas sp. GZ1 was an excellent hydrogen production
bacteria using waste sludge.
3.2. Hydrolyzing organic matters releasing from waste
bio-sludge
Anaerobic hydrogen fermentation needs carbohydrate-
rich organic wastes as its substrate (Lay et al., 2003).
Organic matters contained in the sewage sludge mainly
consist of protein, which is dierent from glucose or
hydrogen yield (ml/gTCOD)

TCODr (mg/L) 14,800 11,600 10,000 18

TCODp (mg/L) 16,000 12,800 9200 16
SCODr (mg/L) 1520 160 80 14
12
SCODp (mg/L) 2840 1120 1200 10
SSr (g/L) 16.25 14.81 15.21 8
SSp (g/L) 14.55 14.88 15.14 6
4
VSSr (g/L) 8.42 7.81 8.63 2
VSSp (g/L) 7.91 8.01 7.98 0
pHr 7.53 7.99 7.20 0 5 10 15 20 25 30 35 40 45
pHp 7.38 8.32 6.94 Time (h)

Xr: raw waste sludge. Xp: pretreated waste sludge. Fig. 1. The hydrogen yield using sterilization (), microwave (h) and
a
All data is shown in their average. ultrasonication (D) pretreated sludge, using unit of ml/gTCOD.
3654 L. Guo et al. / Bioresource Technology 99 (2008) 36513658

Table 2
Comparison of the literature data on biohydrogen production using waste sludges
Ref. Seed inocula Substrate Reactor Maximal hydrogen yield
mlH2/gDS mgH2/gDS mlH2/gCOD
This study Pseudomonas sp. GZ1 Sterilization pretreated sludge Batch reactor 30.38 27.12 15.02
This study Pseudomonas sp. GZ1 Microwave sludge Batch reactor 18.28 16.32 11.44
This study Pseudomonas sp. GZ1 Ultrasonication sludge Batch reactor 5.40 4.82 4.68
Cai et al. (2004) Raw sludge Raw sludge Serum bottles 9.13 0.81 18.48
Cai et al. (2004) Alkaline treated sludge Alkaline treated sludge Serum bottles 16.59 1.48 10.08
Wang et al. (2003a) Digester sludge Waste-activated sludge Batch reactor na 0.16 na
Wang et al. (2003b) C. bifermentans Waste-activated sludge Serum bottles na 1.80 13.40
Wang et al. (2003a) C. bifermentans Freezing and thawing and Serum bottles na na 40.32
sterilization pretreated sludge
Wang et al. (2003a) Boiling treated sludge Concentrate of boiling treated Batch reactor na na 15.90
Wang et al. (2003a) Boiling treated sludge Boiling treated sludge Batch reactor na 1.03 4.48
Cheng et al. (2000) Boiling treated sludge Boiling treated sludge Serum bottle na na 15.68
Huang et al. (2000) Boiling treated sludge Boiling treated sludge Serum bottle 1.79 na na
na: not analyzed.

sucrose (Ozsoy et al., 2006). Cai et al. (2004) reported that 1600
protein was the major substrate for bio-hydrogen fermen- 1400 sterilization

tation from sewage sludge, protein concentration sharply Carbohydrate (mg/L) microwave
1200
ultrasonication
decreased in the rst 24 h of fermentation then remained 1000
stable, but little change in carbohydrate concentration 800
occurred throughout the period, meanwhile, almost no 600
changes in lipid were found. The main components in the 400
microbe were carbohydrate, protein and lipid. It was 200
known that carbohydrate and protein were the main nutri- 0
tion released from the bio-sludge used for hydrogen pro- 0 5 10 15 20 25 30 35 40

duction. In this study, it was shown that pretreatment Time (h)

could enhance the releasing of protein and carbohydrate
into the liquid phase, and during fermentation, protein
and carbohydrate could be released further. Before fermen-
tation, the protein in the liquid phase of waste sludge was
37.09 mg/L (average), while after sterilization, microwave
and ultrasonication pretreatment, the protein rose to
410.52, 597.01 and 72.07 mg/L, respectively. At the begin-
ning of hydrogen production fermentation, the concentra-
tion of protein increased again. Along with the process of
fermentation, the protein was used for hydrogen produc-
tion, and the concentration of protein decreased (Fig. 2).
The variety of carbohydrate during fermentation was sim-
ilar to protein, but the amount of carbohydrate released
and consumed after pretreatment and fermentation was a
1800
1600
1400
Protein (mg/L)
Fig. 3. Changes of carbohydrate concentrations versus time plot using
pretreated waste sludge.
little lower than protein (Fig. 3). It indicated that the
organic matter in waste sludge used for hydrogen produc-
tion was protein, and then was carbohydrate.
Cai et al. (2004) reported that for the alkaline pretreated
sludge, both the initial SCOD concentration and initial
ratio of SCOD/TCOD were much higher than those in
the raw sludge. The experimental results of this study indi-
cated that the initial SCOD/TCOD was 0.01, after pre-
treatment, the value rose to 0.18 (sterilization), 0.09
(microwave) and 0.13 (ultrasonication), respectively. Dur-
ing fermentation, the ratio of SCOD/TCOD increased fur-
ther (Fig. 4). It was also found that the value of TCOD and
sterilization
SCOD were not reduced (data no shown), probably due to
microwave the balance of consuming and releasing in the waste sludge.

1200 ultrasonication This result revealed that pretreatment and fermentation

1000 were all eective for solubilizing organic matters from
800 waste sludge, and the sludge was advantage for further
600
treatment.
400
200 The soluble metabolites include acetic acid (HAc), pro-
0 pionic acid (HPr), butyric acid (HBu), and valeric acid
0 5 10 15 20 25 30 35 40
(HVa). In this study, the main VFA was HAc and HBu,
Time (h) and there was nearly no HPr detected except in ultrasoni-
Fig. 2. Changes of protein during biohydrogen production from pre- cation pretreated sludge. The HVa could be detected in
treated waste sludge. microwave and ultrasonication pretreated sludge. The con-
 L. Guo et al. / Bioresource Technology 99 (2008) 36513658 3655

0.8 0.6
sterilization sterilization microwave ultrasonication
0.7 0.58
microwave
0.6
SCOD/TCOD

ultrasonication 0.56

VSS/SS
0.5
0.54
0.4
0.52
0.3
0.5
0.2
0.48
0.1
0 0.46
0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40
Time (h) Time (h)

Fig. 4. The temporal variation of SCOD/TCOD for waste sludge. Fig. 6. Changes of VSS/SS for waste sludge during fermentation.

Table 3 to reduce waste sludge, and produce more fuel, the combi-
The VFA concentration during hydrogen fermentationa nation of hydrogen and methane fermentation using con-
VFA Sterilization Microwave Ultrasonication tinuous ow would be a better method. The detailed
Acetate 9569.77 9065.81 24881.17
microbiologic information of Pseudomonas sp. GZ1 was
Propionate 0 0 568.85 discussed in another paper.
Butyrate 4912.59 549.65 4239.99
Valerate 0 672.12 3747.26
a
All units are in mg/L.
3.3. Impact of pretreatment

In order to further increase the yield of hydrogen pro-

centration of VFA was highest in ultrasonication pre-
treated sludge after fermentation (Table 3). Fermentative
bacteria hydrolyze and ferment carbohydrates, proteins,
and lipids to volatile fatty acids, which are further con-
verted to acetate, and CO2/H2 by acetogenic bacteria;
The products of acetogenesis, acetate and CO2/H2, are
nally converted to methane by methanogenic bacteria
(Bitton, 1994). Mizuno et al. (2002) and Ginkel et al.
(2001) suggested that H2 production might be lower at high
substrate concentrations as a result of the product inhibi-
tion of hydrogen and organic acids. It implied that if it
could weaken the inhibition of VFA, the hydrogen produc-
tion would increase largely. It was found that pH value
decreased in the hydrogen production tests. The initial
pH of raw waste sludge was 78, and after pretreatment
pH changed slightly. During the fermentation tests using
pretreated sludge, the pH drop sharply to 5.56.5 at rst
and then rose a little (Fig. 5). VSS/SS was presented in
Fig. 6. It was shown that there was a little change of
VSS/SS, which suggested that the releasing of carbon to
the air was too small to reduce the rate of VSS/SS. In order
8.5
8
7.5
7
pH
duction, the impacts of dierent pretreatments on hydro-
gen production using waste sludge should be examined.
The reason that the same sludge, using dierent pretreat-
ment had dierent hydrogen yield and lag time was demon-
strated below.
3.3.1. Impact of ultrasonication pretreatment on hydrogen
production
Ultrasonic could produce cavitation in waste sludge,
and cell lysis and particle size reduction would be caused
which the digestion process was enhanced (Clark and Nuj-
joo, 2000). It appeared from the study that the mechanical
shear forces caused by ultrasonic capitations could be a key
factor for sludge disintegration and collapse of cavitations
bubbles could signicantly alter the sludge characteristics
(Mao et al., 2004).
Although, the lag time of using ultrasonicated sludge
was only 3 h, the yield of hydrogen was also very little,
about 4.3 ml (4.68 ml/gTCOD). It was reported that ultra-
sonication could increase the SCOD by 501200% (Chu
and Lee, 2004), and the same phenomenon was also found
in this experiment. Ultrasonication caused disruption of
the sludge ocs, releasing a high concentration of deoccu-
sterilization lated sludge particles (DSP) and relatively low concentra-
microwave tions of extracellular polymers (ECPs) such as protein,
ultrasonication RNA, DNA and polysaccharides (Kakii et al., 1994).
Ultrasonic-assisted methods could extract some metals

6.5 e.g., Cu, Ni and Zn from sludge (Perez-Cid et al., 1999).

6 The relative toxicity of heavy metals to H2 production
5.5 was found in the following order: Cu (most toxic)>>Ni
5 Zn > Cr > Cd > Pb (least toxic) (Li and Fang, 2007). In
0 5 10 15 20 25 30 35 40
this study, it was found that the heavy metal extracted from
Time (h)
the sludge by ultrasonication was much higher than other
Fig. 5. Changes of pH during hydrogen production. two methods, especially Cu and Zn (Table 4). This might
3656 L. Guo et al. / Bioresource Technology 99 (2008) 36513658
Table 4
The concentration of Cu, Cd, Pb and Zn in raw sludge and dierent
pretreated waste sludgea
Sample Cu Cd Pb Zn
Raw sludge 9 28 0 40
Sterilization 21 30 0 61
Microwave 34 37 0 89
Ultrasonication 105 37 0 482
a
All units are in lg/L.
be the reason that using ultrasonicated sludge had such low
yield of hydrogen.
The reason of short lag time would be discussed as fol-
lowing. A sludge oc is a bioreactor and a substrate sup-
plier. The interior structure inuences the penetration of
enzymatic substances and the release of metabolic products
from sludge ocs; the structure of a oc governs the resis-
tance of its interior to mass transfer (Chu and Lee, 2004).
Chu and Lee (2004) recommended that the power of
1.8 W/ml was too low to disrupt the wastewater sludge oc,
but it did support changing of the oc structure before
digestion. The change of the oc structure revealed that
the mass transfer could be accelerated, and it was advan-
tage for fermentation. A mass of VFA would be produced
soon. As discussed above, the VFA could restrain hydro-
gen production. So hydrogen was produced quickly and
then stopped.
3.3.2. Impact of sterilization pretreatment on hydrogen
production
It was known that sterilization could kill all the microor-
ganisms, at the same time all the pathogen was also killed.
Otabbong and Barbolina (1998) reported that sterilization
markedly increased soil phosphorus levels compared with
the non-sterilized soils. It was found that the sludge after
sterilization could also release other nutrient like protein
and carbohydrate, and the SCOD was also enhanced.
In this study, the highest yield of hydrogen production
was observed using sterilization pretreated sludge. The
maximum yield of hydrogen production was 24.03 ml
(15.02 ml/gTCOD). The lag time was 15 h. Haytoglu
et al. (2001) indicated that the waste sludge after steriliza-
tion was eective in adsorbing heavy metals, namely Pb(II),
Cr(II), Cu(II), Ni(II) and Zn(II); it was observed that
Pb(II) was adsorbed with the highest capacity. Toxic met-
als, such as Ni, Zn and Cr, can be removed from dilute
aqueous solutions by sorption onto nonliving sewage
sludge applied as nely dispersed biosorbent particles after
sterilization and drying (Solari et al., 1996). The toxic metal
was removed by sludge, and it was favorable for hydrogen
production bacteria (Table 4). There was no information
that sterilization could change the structure of oc, and
the waste sludge adsorbed toxic metals might inuence
the mass transfer, so the lag time was long consequentially.
It implied that less and slower VFA would produce. Since
the inhibitory eect of VFA and toxic metals were reduced,
the hydrogen could be produced more.
3.3.3. Impact of microwave pretreatment on hydrogen
production
The main advantages of microwave heating are the uni-
formity of heating and the precise control of the process
temperature. (Datta et al., 1994). In this study, the maximal
hydrogen production yield obtained using microwave pre-
treated sludge was 14.65 ml (11.44 ml/gTCOD), and lag
time was 10 h. It was reported that microwave could
release phosphate (Liao et al., 2005; Chan et al., 2007)
and ammonia from sludge, and signicant concentrations
of soluble COD were also obtained in this treatment (Chan
et al., 2007). Microwave digested sludge showed a reduc-
tion of capillary suction time by 11.1% and 10.7%, as com-
pared to control and conventionally heated digested sludge
(Pino-Jelcic et al., 2006), and it was found out that micro-
waves improve sludge dewaterability (Nakazato et al.,
2006). The eect listed above was ecient for hydrolyzing
sludge and producing hydrogen. The releasing of nutrient
was also found in this study. Otherwise, the anaerobic
digester fed with microwave irradiated sludge was more
ecient in inactivation of fecal coliforms than the other
two digesters fed with raw sludge and externally heated
sludge, respectively (Hong et al., 2006). So microwave pre-
treated sludge could reduce the pathogen.
But why the hydrogen yield was less than the steriliza-
tion pretreated sludge were found below. In recent years,
microwave-assisted extraction (MAE) has been developed
for the extraction of a variety of toxic organic contami-
nants, such as PAHs, PCBs, phenols and pesticides from
soils, sediments and other solid matrices (Xiong et al.,
1999). The analysis of heavy metal in sludge and wastewa-
ter were conducted using a microwave-assisted digestion
procedure (Weemaes and Verstraete, 1998). Nakazato
et al. (2006) also indicated that heavy metals in soil like
Cd, Cr, Cu, Ni, Pb, and Zn could be extracted by micro-
wave. These toxic matters were deleterious for hydrogen
production bacteria. So, when the organic nutrients
released, the toxic matters could be released simultaneously
(Table 4), and they would restrain the hydrogen production
bacteria in a way.
4. Conclusion
This work is the rst systematic study of using Pseudo-
monas to produce hydrogen from waste sludge. The eects
of three dierent pretreatments, microwave, sterilization
and ultrasonication on the hydrogen production were also
investigated. Sterilization and microwave markedly
increased the specic hydrogen yield to 15.02 ml/gTCOD
and 11.04 ml/gTCOD, respectively. However, ultrasonica-
tion inhibited hydrogen production yield (4.68 ml/
gTCOD). In the aspects of lag time, ultrasonication could
accelerate produce hydrogen velocity, about 3 h lag time.
The lag time of using sterilization and microwave was
15 h and 10 h, respectively. Pretreatment on waste sludge
could release SCOD, protein and carbohydrate. During
fermentation, the ratio of SCOD/TCOD increased further,
 L. Guo et al. / Bioresource Technology 99 (2008) 36513658
protein and carbohydrate could be partly consumed by
Pseudomonas sp. GZ1.
The toxic metals in sludge could be extracted after ultra-
sonication and microwave pretreatment, which was delete-
rious for hydrogen production bacteria. The structure of
sludge oc could be changed by ultrasonication, and this
change could increase the production of VFA, which could
restrain the hydrogen production. Sterilized sludge could
adsorb toxic metals and less VFA was produced during fer-
mentation, so it was advantageous for producing
hydrogen.
Acknowledgements
The study was supported by the Natural Science Foun-
dation of China (Grant Number: 50478054); the Interna-
tional Science and Technology Cooperation Key Item
(Grant Number: 2004DFA06200); the Changsha Science
and Technology Program Key Item (Grant Number:
K051132-72); Program for New Century Excellent Talents
in University Foundation (Grant Number: 2004 NCENT-
0770).
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