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Impacts of Sterilization, Microwave and Ultrasonication Pretreatment On Hydrogen Producing Using Waste Sludge
Impacts of Sterilization, Microwave and Ultrasonication Pretreatment On Hydrogen Producing Using Waste Sludge
Impacts of Sterilization, Microwave and Ultrasonication Pretreatment On Hydrogen Producing Using Waste Sludge
com
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.
1. Introduction Landll is one of the most common methods that are used
in the nal disposal of sludge world wide (Ozsoy et al.,
Biomass (waste sludge, plants, animals, and their 2006). The disposal of sludge generated in urban wastewa-
organic waste products) provides approximately 14% of ter treatment plants is an important environmental prob-
the worlds energy needs. In developing countries, which lem (Solis et al., 2002). Unused, discarded biomass
often have limited supplies of fossil fuels but abundant bio- residues are a potential energy resource, which at present
mass resources, as much as 35% of the energy demand is are not well managed and thus pose signicant environ-
supplied by biomass. However, in developed countries with mental problems (Levin et al., 2007).
still abundant fossil fuel supplies, reliance on biomass as an Recently, some studies are focusing on using the sludge
energy resource is very limited, despite its huge energy to produce hydrogen by anaerobic fermentation. The
potential. Waste-activated sludge from a wastewater treat- hydrolysis of the microbial cell limits the speed of the
ment plant is rich in polysaccharides and proteins and thus anaerobic digestion of sludge and results in a long HRT
it is a potential substrate for producing hydrogen (Wang (hydraulic retention time) (Weemaes and Verstraete,
et al., 2003a). In 2001, about 4220 million t of municipal 1998; Alvarez et al., 2000). To improve anaerobic digestion
wastewater was treated in more than 150 municipal waste- of waste sludge, the most logical approach is to disrupt the
water treatment plants in China, producing about 0.55 microbial cells of sludge. Several pretreatments have been
1.06 million t of dry sludge (Statistical Gazette, 2001). 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
*
Corresponding author. Tel.: +86 731 8823967; fax: +86 731 8823701. the raw sewage sludge, a lot of pre-treatments had been
E-mail address: xmli@hnu.cn (X.-M. Li). 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- (2) Ultrasonication sonication tests were performed
cation (Zhu and Beland, 2006), sterilization (Wang et al., with the help of a cell-breaker at a sonication power
2003a), methanogenic inhibitor (Wang et al., 2003a; Zhu density of 2 w/ml. Original sludge (200 ml) was
and Beland, 2006), freezing and thawing (Wang et al., placed in a 1000 ml beaker with the ultrasonic probe
2003a), ultrasonication (Wang et al., 2003a) to enhance positioned 2 cm submerge the surface of the sludge.
the hydrogen yield. Hydrogen yield was improved to The sonication time was 5 min to release the insoluble
1.4 mgH2/gDS (dissolved sludge) using the boiled sludge organic matters from the solids (Branson, digita son-
and 1.52.1 mmol H2/gCOD with sludge pretreated by ier 450, USA).
sterilization or freezing and thawing (Wang et al., 2003a). (3) Sterilization sample of sludge was sterilized at
Until recently, there was little information about Pseu- 121 C for 20 min (Hirayama Manufacturing Corpo-
domonas to produce hydrogen with waste sludge and the ration, HV-50, Japan).
reason that the same sludge using dierent pretreatment
and releasing similar nutrients had dierent hydrogen yield 2.3. The inoculum
and lag time (the time phase from the beginning of inocu-
lation to the beginning of gas production) was not clear. In A Pseudomonas sp. GZ1 (EF551040) was used as seed
this study, the waste sludge pretreated by microwave, ultra- bacteria, which was isolated from the anaerobic granule
sonication, and sterilization was used to produce hydrogen sludge collected from Yueyang paper mill. The applied
by a new strain of Pseudomonas sp. GZ1 (EF551040) and puried procedures include: (1) Heat pre-treating at 80 C
the variety of organic matters released from microbe in for 2 h to inactivate the methanogenic bacteria; (2) Tritu-
waste sludge such as protein, carbohydrate, SCOD (soluble rating the granule sludge using sterilized apparatus. (3) Iso-
chemical oxygen demand) and VFA (volatility fatty acid) lating and purifying the incubated sludge on solid culture
was discussed to investigate the impact of microwave, medium rst by Hungate method (Hungate, 1969) and
ultrasonication and sterilization pretreatment on waste about three days later transferred the colony to liquid cul-
sludge to produce hydrogen, and the possible reason was ture medium. These procedures were repeated for three
also analyzed in detail. The objective of this study was to times. (4) The preliminary hydrogen production was tested,
discuss the impacts of pretreatment on hydrogen produc- and the strains with the highest hydrogen yields were
tion, which would make possible to further enhance the selected as the inoculum in this study. The strain was ana-
hydrogen production using waste sludge. 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
2. Methods showed that strain GZ1 was a member of the Pseudomonas
family.
2.1. Substrate
2.4. Fermentation and testing
Wastewater sludge was taken from the recycled stream
of the secondary treatment stage of a Municipal Wastewa- Substrate (100 ml) was mixed with 5 ml of inoculum sus-
ter Treatment Plant in Changsha which handles 140,000 m3 pension and was anaerobically (blow N2 for 10 min) incu-
wastewater daily. The collected sludge settled about one bated at 35 C in 250 ml serum bottles with stirring of
week to yield solids content of 15.42 g/L SS and 8.29 g/L 125 r/min and was not added other nutrients. The bottles
VSS. The pH value of the sludge was about 7.57. The were capped with silica gel stoppers. Under identical condi-
TCOD (total chemical oxygen demand) and SCOD (solu- tion three fermentation bottles were measured and their
ble chemical oxygen demand) of raw waste sludge was average of data was reported to prevent any possible errors
12,133 mg/L and 587 mg/L, respectively. introduced by sampling procedure. The COD, protein, car-
bohydrate, pH and VFA were measured during fermenta-
2.2. Pre-treatment tion. The concentration of H2 was tested frequently
during fermentation and the H2 production was also
Three pre-treatments were applied to waste sludge. recorded during the whole examination. The samples were
These pre-treatment not only released insoluble organic extracted every 37 h.
matters into water to increase the eciency of fermenta- Volatile fatty acids (VFA) were measured with a gas
tion, but also inactivated methanogenic bacteria in the sub- chromatograph (Agilent Technologies 6890N, USA)
strate to reduce their consumption of hydrogen. The equipped with a ame-ionization detector (FID) and the
pretreated sludge was the substrate used in the hydrogen automatic liquid sampler. The gas chromatograph (GC)
fermentation tests. was equipped with a capillary free fatty acid phase (polar-
ity) column (DB-FFAP, 30 m 0.25 mm 0.25 lm). The
(1) Microwave the sludge was heated in the microwave temperatures of the injector and detector were 250 and
for 2 min at a power of 560 w (Galanz, P7023TP-K7, 300 C, respectively. The initial temperature of oven was
China). 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 organic matters from waste sludge (Table 1). Protein, car-
was used as carrier gas with a ow rate of 2.6 ml/min. A bohydrate and SCOD were all increased considerably,
GC-TCD (Agilent Technologies 6890N, USA), equipped but the change of TCOD, SS and VSS were inconspicuous.
with a capillary column packed with CARBONPLT It was shown that the pretreatments could destroy cellular
(30 m 0.53 mm 3 lm) at the temperature of 80 C wall, and release dissoluble matters. So pretreatments were
(oven), 150 C (injector), 200 C (detector), measured the advantageous for hydrogen production. In this study, the
hydrogen and methane concentrations in the gas phase. lag time was 3 h using ultrasonicated sludge, the shortest
The ow rate of N2 used as carrier gas was 1 ml/min. comparing to that using other pretreated sludge. But the
The TCOD (total chemical oxygen demand) of the sludge hydrogen production was the lowest of all. The maximal
was measured by the microwave digestion method (model yield of hydrogen was 4.68 ml/gTCOD. The largest hydro-
MS-3 Microwave Digestion System for COD Determina- gen production was observed using sterilization pretreat-
tion, China). The SCOD of the sludge sample was obtained ment. The maximal yield of hydrogen was 15.02 ml/
after ltering through a 0.45 lm membrane. SS and VSS gTCOD. But the lag time was 15 h. The microwave pre-
were determined by standard method (He, 1998). The pH treated sludge was then used for bio-hydrogen production,
was measured with a digital pH-meter (pH315i, WTW with a maximal hydrogen yield of 11.44 ml/gTCOD, and
82362 Weilheim, Germany). Protein and carbohydrate the lag time was 10 h (Fig. 1). However, hydrogen accumu-
were determined by their respective standard method lated in the headspace of bottles was then consumed by
(Ren et al., 2005). The concentrations of Zn, Cu, Pb and hydrogen consumers in the following fermentation time,
Cd in waste sludge were determined by atomic absorption which was observed in all the pretreated sludge. A common
spectrophotometry analysis (AAS-700, Perkin Elmer, phenomenon, higher hydrogen yield and slower hydrogen
USA). The accumulative volume of hydrogen produced consumption was observed in the tests. Although, the
(H) over the time course during the batch tests was tted hydrogen yield with microwave pretreated sludge was less
with the Gompertz equation (Lay et al., 1999) than sterilizations, the lag time was also shorter. These
results showed that the hydrogen production could be
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 addition, the estimated parameters of P, Rm and k were
hydrogen production rate (ml/h), k is the lag phase time quite consistent with these experimental data (Lay et al.,
(h), and e is 2.718281828. 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
3. Results and discussions
phase was more than 70%. Results of hydrogen production
in this study were compared with other results in the liter-
3.1. Hydrogen production
atures as shown in Table 2. It was shown that the Pseudo-
monas sp. GZ1 was an excellent hydrogen production
In this study, the inuence of dierent pretreatment on
bacteria using waste sludge.
the hydrogen production using waste sludge by anaerobic
fermentation was investigated. Sterilization, microwave
and ultrasonication were all eective for solubilizing 3.2. Hydrolyzing organic matters releasing from waste
bio-sludge
Table 1
The character of raw and pretreated waste sludge used in this studya Anaerobic hydrogen fermentation needs carbohydrate-
Parameter Sterilization Microwave Ultrasonication
rich organic wastes as its substrate (Lay et al., 2003).
Organic matters contained in the sewage sludge mainly
Proteinr (mg/L) 25.40 85.88 0
Proteinp (mg/L) 410.52 597.01 72.07
consist of protein, which is dierent from glucose or
Carbohydrater (mg/L) 18.72 14.26 0
Carbohydratep (mg/L) 202.65 450.51 101.96
hydrogen yield (ml/gTCOD)
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
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
protein and carbohydrate could be partly consumed by Huang, C.H., Lin, H.Y., Tsai, Y.Y., Hsie, Y.K., 2000. The preliminary
Pseudomonas sp. GZ1. with dierent substrates and cultivations. The Twenty-fth Wastewa-
ter Technology Conference, Yunlin, Taiwan.
The toxic metals in sludge could be extracted after ultra- Hungate, R.E., 1969. Methods in Microbiology. Academic Press Inc.,
sonication and microwave pretreatment, which was delete- New York.
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sludge oc could be changed by ultrasonication, and this Shirakashi, T., 1994. Reocculation of sewage activated sludge
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The study was supported by the Natural Science Foun- Levin, D.B., Zhu, H.G., Beland, M., Cicek, N., Holbein, B.E., 2007.
dation of China (Grant Number: 50478054); the Interna- Potential for hydrogen and methane production from biomass residues
tional Science and Technology Cooperation Key Item in Canada. Biores. Technol. 98, 645660.
Li, C.L., Fang, H.H.P., 2007. Inhibition of heavy metals on fermentative
(Grant Number: 2004DFA06200); the Changsha Science hydrogen production by granular sludge. Chemosphere 67, 668673.
and Technology Program Key Item (Grant Number: Liao, P.H., Wong, W.T., Lo, K.V., 2005. Release of phosphorus from
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