Effects of different pretreatment methods on fermentation types and dominant bacteria for hydrogen production

international journal of hydrogen energy 33 (2008) 4318–4324

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Effects of different pretreatment methods on fermentation types and dominant bacteria for hydrogen production Nan-Qi Rena,*, Wan-Qian Guoa, Xiang-Jing Wangb, Wen-Sheng Xiangb, Bing-Feng Liua, Xing-Zu Wanga, Jie Dinga, Zhao-Bo Chena a

State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, Heilongjiang, China Research Center of Life Science and Biotechnology, Northeast Agricultural University, Harbin 150030, China

b

article info

abstract

Article history:

In order to enrich hydrogen producing bacteria and to establish high-efficient communities

Received 4 May 2008

of the mixed microbial cultures, inoculum needs to be pretreated before the cultivation.

Received in revised form 10 June

Four pretreatment methods including heat-shock pretreatment, acid pretreatment, alka-

2008

line pretreatment and repeated-aeration pretreatment were performed on the seed sludge

Accepted 10 June 2008

which was collected from a secondary settling tank of a municipal wastewater treatment

Available online 8 August 2008

plant. In contrast to the control test without any pretreatment, the heat-shock pretreatment, acid pretreatment and repeated-aeration pretreatment completely suppressed the

Keywords:

methanogenic activity of the seed sludge, but the alkaline pretreatment did not. Employing

Hydrogen production

different pretreatment methods resulted in the change in fermentation types as butyric-

Pretreatment

acid type fermentation was achieved by the heat-shock and alkaline pretreatments,

Fermentation type

mixed-acid type fermentation was achieved by acid pretreatment and the control, and

Microbial community

ethanol-type fermentation was observed by repeated-aeration pretreatment. Denaturing

Mixed culture

gradient gel electrophoresis (DGGE) profiles revealed that pretreatment method substan-

Denaturing gradient gel electropho-

tially affected the species composition of microbial communities. The highest hydrogen

resis (DGGE)

yield of 1.96 mol/mol-glucose was observed with the repeated-aeration pretreatment method, while the lowest was obtained as the seed sludge was acidified. It is concluded that the pretreatment methods led to the difference in the initial microbial communities which might be directly responsible for different fermentation types and hydrogen yields. ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

Hydrogen energy, as an alternative energy resource, has drawn more and more interests currently. In fact, hydrogen may be produced directly from acidogenic fermentation of the two-phase anaerobic process [1–4]. However, fermentative hydrogen production from organic substrates also has some limitation, principally the relatively low yields of hydrogen obtained until now [4,5]. Thus, many studies were conducted

trying to achieve higher hydrogen production yields through various efforts [6–11]. Although hydrogen can be produced efficiently through acidogenic fermentation in lab-scale efforts, many factors should be taken into consideration for a full-scale biological hydrogen production process. One of the difficulties is to get a large amount of anaerobic hydrogen producing biomass economically and easily [12,13]. Furthermore, quick recovery from process upsets in full-scale applications may also require large quantities of

* Corresponding author. Tel./fax: þ86 451 86282008. E-mail address: [email protected] (N.-Q. Ren). 0360-3199/$ – see front matter ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2008.06.003

international journal of hydrogen energy 33 (2008) 4318–4324

readily available hydrogen producing seeds [14]. Previous studies reported several pretreatment methods to enrich hydrogen producing bacteria. Heat-shock pretreatment has been most widely used in literature [14–20] for eliminating the non-spore forming methanogenic bacteria, since hydrogen producing bacteria, like most Clostridium, can form protective spores under extremely strict living environment. The control condition of the heatshock pretreatment in literature was ranging from 80 to 121  C, and exposure time between 15 and 120 min. Repeated heatshock pretreatment [21] and two-stage cultivation heat-shock pretreatment [14] were also reported using sucrose as medium. Other researches have successfully prepared the hydrogen producing seed by treating the sludge by acid at the pH value of 2– 4 [22–24]. Zhang [25,26] also applied the method of combined heat-shock and acid-shock treating of the original sludge used for hydrogen production. Cai [27] has performed an extensive study on the pretreatment of sewage sludge by alkaline pretreatment and found that maximum hydrogen occurred at initial pH of 11.0. Zhu [14] and Mu [18] also evaluated the alkaline effects on hydrogen yield. Other chemicals of methanogen inhibitors like BESA and iodopropane were also tested to prepare hydrogen production seeds at different concentrations [14]. A few researchers have reported the preparation of hydrogen producing bacteria by aeration pretreatment. The aeration condition parameters vary with the complete aeration time ranging from 30 to 60 min [14, 28] and incomplete aeration time of 5–7 d [2,3,29]. As a result, different hydrogen production yields were achieved due to different aeration time and the corresponding gas amount. There are also some studies which indicate that the parameters control can inhibit methanogenesis in some cases, like low pH [30] and short HRT operation [31]. However, methanogenic activity was not completely repressed in other cases as reported by Yu [32]. Comparing with the former pretreatment methods, in this research, better hydrogen yields were obtained by a method of batch incomplete repeated-aeration. It appears from the above-mentioned researches that proper pretreatment has effectively inhibited methanogens and thus enhanced hydrogen production. Up to now, most studies on the pretreatment effects have focused on hydrogen production, substrate utilization and product formation. The microbial population responsible for hydrogen production after different pretreatment has not been examined yet. Accordingly, this paper compared heatshock, acid, alkaline and repeated-aeration pretreatment methods on the efficiency of hydrogen producing bacteria enrichment. The resulting microbial communities were also monitored by DGGE. An untreated sludge as the control was examined in parallel for comparison. The main goals of the present research were to suggest an effective method for hydrogen producing bacteria enrichment for a certain microbial community and to provide a feasible and economical means for guidance in a full-scale operation.

2.

Materials and methods

2.1.

Original sludge and enrichment medium

The original sludge was taken from the secondary settling tank of Wenchang Municipal Wastewater Treatment Plant in

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Harbin, China. The MLVSS was 8.25 g/L. This sludge was regarded as the control sludge compared with the pretreated sludge. The carbon source is glucose (10 g/L). The medium added for sludge culture was described in detail according to previous study [9].

2.2.

Pretreatment methods

2.2.1.

Heat-shock pretreatment

The heat-shock pretreatment was conducted by sterilizing the original sludge in a sterilization pot at 121  C for 20 min.

2.2.2.

Acid pretreatment

The acid pretreatment was conducted by adjusting the pH of the sludge to 3.0 with 1 N HCl and maintained for 24 h firstly, and then back to pH 6.8 with the addition of 1 N NaOH [22].

2.2.3.

Alkaline pretreatment

The alkaline pretreatment was conducted by adjusting the pH of the sludge to 11.0 with 1 N NaOH and maintained for 24 h firstly, and then back to pH 6.8 with the addition of 1 N HCl [27].

2.2.4.

Repeated-aeration pretreatment

The repeated-aeration pretreatment was used in our group for some time. The sludge was aerated by controlling the DO (<0.5 mg/L) and feeding glucose for 12 h, and then settled and removed the liquid. This method ensures the sludge mixed culture in an anoxic environment. This method is not reported in detail in literature before. The acid, alkaline and repeated-aeration pretreatments were all conducted at room temperature ofabout 25  C. The inoculation amount was 20 mL.

2.3.

Experimental equipments

The experiment was carried out in triplicate in 150 mL Erlenmeyer flasks as a batch reactor placed in an air-bath shaker. The presented value in this study was average values of the triplication. The mixture was cultivated under 35  0.5  C, and the stirring rate was 120 rpm/min to provide release of gas and better contact between substrate and bacteria. All the batch culture was conducted under anaerobic conditions with the anaerobic culture technique proposed by Bryant [33]. The experimental equipments were shown in Fig. 1.

2.4.

Analytical methods

The procedures described in Standard Methods [34] were used to determine pH. The composition of biogas (H2 and CH4) was analyzed by a SP-2305 gas chromatograph (Shandong Lunan Instrument Factory, China) equipped with a thermal conductivity detector and a 2-m stainless column packed with Porapak TDS-01 (60/80 mesh). The operational temperatures at the column oven and the detector were both 150  C. Nitrogen was used as the carrier gas at a flow rate of 70 ml/ min. The liquid samples taken from the batch culture reactor were centrifuged at 4000 rpm for 15 min, and then the

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international journal of hydrogen energy 33 (2008) 4318–4324

Gas sample draw N2 outlet

Reactor

Gas-measuring tube

Equilibrium flask

Air-bath shaker

3.

Results and discussion

3.1.

Hydrogen and methane production

Fig. 2(A) shows the accumulated hydrogen yield inoculated with different pretreated sludge samples and the control sample during the fermentation period. After a certain period of

A

250

Accumulated hydrogen yield (mL)

Sample inlet and N2 inlet

EMBL to search for similar sequences using the BLAST algorithm.

200

Fig. 1 – Schematic of the batch reactor.

150

100

50

0 0

10

20

30

40

50

Time (h) control acid pretr. repeat-aeration pretr.

B Accumulated methane yield (mL)

supernatants were filtered through 0.45 mg cellulose acetate membranes for the analysis of the volatile fatty acids (VFAs) and ethanol. The concentrations of the VFAs and ethanol were determined using a model GC-122 gas chromatograph (Model GC-122, Shanghai Analytical Apparatus Corporation, China) with a flame ionization detector and a 2-m stainless (5-mm inside diameter) column packed with Porapak GDX103 (60/80 mesh). The operational temperatures of the injection port, the column and the detector were 220, 190 and 220  C, respectively. Nitrogen was used as carrier gas at a flow rate of 50 ml/min [35]. The microbial communities of the different pretreated sludge samples and the control sludge sample were evaluated and compared by analyzing the denaturing gradient gel electrophoresis (DGGE) profiles of 16S rDNA fragments. DNA in the sludge samples was extracted, followed by 16S rDNA fragments amplified by polymerase chain reaction (PCR), and then separated by DGGE. A freeze-thaw method was used in the extraction of bacterial DNA from the anaerobic sludge [35]. The primers used for DGGE were 338F, 50 ACT CCT ACG GGA GGC AGC AG-30 (Escherichia coli 16S rRNA position of 338–357), and 534R, 50 -ATT ACC GCG GCT GCT GG-30 (E. coli 16S rRNA position of 518–534). The samples were then amplified in a Perkin–Elmer Applied Biosystems (Foster City, California) Gen Amp PCR System 9700 programmed as follows. Initial denaturation of DNA for 5 min at 94  C; 30 cycles of 1 min at 94  C, 30 s at 60  C, and 30 s at 72  C; decreasing 0.1  C per cycle to 57  C, and extension of incomplete products for 8 min at 72  C. PCR products were examined by electrophoresis on a 2% (w/v) agarose gel containing ethidium bromide (0.5 mg/mL). The DGGE was performed following the method described by the previous study [36]. Then, the predominant bands in each lane were excised from DGGE gel, and DNA was recovered and reamplified. The amplified products were then analyzed by agarose gel electrophoresis, and the 16S rDNA was cloned and sequenced. Sequencing was performed by Bioasia Biological Technology Service (Shanghai, China) using ABI PRISM Big Dye Terminator cycle sequencing ready reaction kits (Perkin–Elmer) and an ABI PRISM 377XL DNA sequencer. The corresponding results were then submitted to Genbank and

heat-shock pretr. base pretr.

2.4

2.0

1.6

1.2

0.8

0.4

0.0 0

5

10

15

20

25

30

Time (h) control acid pretr. repeat-aeration pretr.

heat-shock pretr. base pretr.

Fig. 2 – Accumulated (A) hydrogen production and (B) methane production. Data represent averages from triplicate assays and the standard deviations were lower than 5% in all cases.

international journal of hydrogen energy 33 (2008) 4318–4324

lag time, five sludge samples all began to produce hydrogen gradually. The maximum hydrogen yield was 224.5 mL by repeated-aeration pretreatment, while samples by heat-shock pretreatment, alkaline pretreatment, acid pretreatment and the control achieved their maximum hydrogen yield of 189.5 mL, 134.1 mL, 51.9 mL and 180.4 mL, respectively. The rank of hydrogen yield was repeated-aeration pretreatment > heat-shock > control > alkaline > acid. Zhu’s [14] results were rather inconsistent with this study, with the rank of alkaline > control > aeration > heat-shock > acid. Mu [18] compared the three methods of pretreatment and indicated that alkaline pretreatment got the lowest hydrogen yield of 0.48 mol/mol-glucose while the heat pretreatment got the highest. Kawagoshi [37] observed no difference between non-heat-treated and heat-treated digested sludge in hydrogen production. All the results above lack some details. It is evident that other factors, such as inoculated source, exposure time and so on, also affected the hydrogen production ability besides pretreatment methods. On the other hand, the hydrogen production and the lag time were quite different in various pretreatments. The longest lag time was 14 h by alkaline pretreatment sludge, followed by 13 h by acid pretreatment, 11 h by heat-shock pretreatment, 3 h for the control and 2 h by repeated-aeration pretreatment. These were consistent with other studies [7,13,38] using mixed culture. Fig. 2(B) shows the accumulated methane yield using five kinds of inoculated sludge in batch cultures. It was found that only the alkaline pretreatment sludge and the control sludge generated methane during the early cultivation period of the bacteria. This is consistent with Cai’s research [27] in digesting sewage sludge. No methane was detected in the rest of the samples, repeated-aeration pretreatment, heatshock and acid pretreatments, during the whole fermentation process. Compared to Terentiew’s [28] and Zhu’s [14] work, repeated-aeration pretreatment method has efficiently enhanced hydrogen production by different exposure time and amount to the initial sludge.

3.2.

Distribution of soluble fermentation products

Fig. 3 shows the distribution of the main soluble fermentation products of VFAs and ethanol associated by different

Concentration (mg/L)

2000

1600

acetic butyric ethanol

propionic valeric

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pretreatment methods. The highest VFAs and ethanol generation occurred by repeated-aeration pretreatment, followed by alkaline pretreatment, the control, heat-shock pretreatment and acid pretreatment. Acetic acid was found to be the most dominant in all samples. For the control and the acid pretreatment, mixed-acid type fermentation, based on the concentration ratio of the total soluble products, was observed with acetic acid of 46.1% and 45.4%, propionic acid of 8.5% and 34.2%, butyric acid of 19.1% and 15.4%, and ethanol of 25.6% and 4.7%, respectively. For the heat-shock and alkaline pretreatments, butyric-acid type fermentation formed with the acetic acid of 46.1% and 45.4% and butyric acid of 19.1% and 15.4%, respectively. This result was quite consistent with other studies [18]. As for repeated-aeration pretreatment sludge sample, the typical ethanol-type fermentation [28,39] was achieved with the acetic acid (48.4%) and ethanol (40.7%) [40]. These results also indicated that different microbial communities might form from the initial stimulation by the different restrict pretreatment conditions.

3.3.

Microbial community

The different pretreatment sludge samples and the control sample were separated by DGGE as shown in Fig. 4, and the specific bands were chosen for cloning and sequencing. It was shown that different pretreatments led to significant microbial population shift of the seed sludge. It is demonstrated from Table 1 that different initial microbial populations formed because of various initial environmental conditions with different pretreatments. On the other hand, the growth characteristics of the various microbial populations also led to the difference in microbial populations. For example, the Ethanoligenens harbinens might be responsible for the high hydrogen yield treated by repeated-aeration pretreatment [42]. Whereas large amount of the Propionibacterium propionicus was detected by acid pretreatment, resulting in low hydrogen productivity. P. propionicus was likely to grow in lower acclimation pH value [43]. In fact, the dominant microbial populations are directly responsible for hydrogen production and the soluble products formation. This might well explain the view of different fermentation types and hydrogen productivities by other researchers [18, 24–26]. Some researchers [13] reported that they would do some effort to imitate ethanol-type fermentation and were unsuccessful. The reason was because they adopted heat-shock pretreatment method and only got butyrate type fermentation.

1200

3.4.

Evaluation of different pretreatment methods

800

400

0 control

heat-shock

acid

base

repeatedaeration

Different pretreatment approaches Fig. 3 – VFA and alcohol production with different pretreatment methods. Data represent averages from triplicate assays and the standard deviations were lower than 5% in all cases.

For acid pretreatment, the results in this study were consistent with most researchers [14,18]. Although the acid pretreatment would successfully suppress the methanogenic activity, the activity of hydrogen producers was also inhibited. Mohan’s group [41] found that the initial pH of 6.0 was the best pH value for hydrogen production. However, according to our results, the hydrogen production capability was still the worst even the initial pH was 6.8. Alkaline pretreatment did not completely suppress the methanogen activity. This result was also found by Zhu’s

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international journal of hydrogen energy 33 (2008) 4318–4324

Table 1 – Sequences of predominant DGGE bands of 16S rDNA Band number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Fig. 4 – Variation of DGGE profiles with different pretreatment methods. Lane A represents for the initial sludge taken from the wastewater treatment facilities; lane B to band F was taken at the end of the experiment when biogas generation stopped. Lane B represents for control sludge sample; lane C for heat-shock pretreatment; lane D for acid pretreatment; lane E for alkaline pretreatment; lane F for repeated-aeration pretreatment. Those specific bands were labelled with numbers 1–15, and were executed and sequenced.

study [14]. Previous studies [27] showed that when pH was set at 11.0, alkaline-tolerant hydrogen producing bacteria would reach the peak activity. Thus, the hydrogen productivity should only depend on large population of such alkalinetolerant bacteria. The heat-shock method has been applied mostly in preparing hydrogen producing seeds. However, the limitation of such a method was also proposed by some researchers

Nearest 16S rDNA sequence

Similarity (%)

Propionibacterium granulosum cryptic plasmid PG01 (AY150274) Ethanoligenens harbinens YUAN-3 (AY295777) Acidovorax facilis strain LMG 2193 (EU024133) Clostridium tyrobutyricum stain MPP-41 (DQ911273) Clostridium tyrobutyricum stain MPP-41 (DQ911273) Clostridium tyrobutyricum stain MPP-41 (DQ911273) Clostridium tyrobutyricum stain MPP-41 (DQ911273) Clostridium vincentii CGS6 (AY540110) Clostridium vincentii CGS6 (AY540110) Enterobacter aerogenes strain Aq16 (EU554442) Ethanoligenens harbinens YUAN-3 (AY295777) Bacteroides vulgatus ATCC8482 (CP000139) Bacteroides vulgatus ATCC8482 (CP000139) Clostridium longisporum strain DSM8431 (X76164) Clostridium longisporum strain DSM8431 (X76164)

95 100 98 95 95 95 95 95 95 96 100 96 96 97 97

[11,14,44] for killing methanogens simultaneously with microbial community reduction. Moreover, many non-spore hydrogen producers have been destroyed, resulting in relatively lower hydrogen production efficiency. However, from our results and other reports, heat-shock pretreatment is still better than acid pretreatment and base pretreatment. The repeated-aeration pretreatment could ensure higher microbial diversity. Meanwhile, the most significant advantages of this method were found that it could enrich hydrogen producing bacteria without killing the non-spore forming bacteria and to kill the methanogens by raising the oxidation–reduction potential. Furthermore, the seed had a complex microbial communities compared with others as evidenced by population analysis results in Fig. 4. The repeated-aeration pretreatment method enabled large amount of the acidogenic bacteria to survive and to keep their activity in an anoxic environment with simultaneously killing the methanogens which is obligate anaerobic archaeobacteria. Almost all researchers thought that the inoculated bacteria for hydrogen production should be pretreated for killing methanogens and optimizing hydrogen producing bacteria. Although heat-shock pretreatment is better and selected by most researchers, it still has some limitations. As in the above discussion, the heat-shock pretreatment methods experience the risk of less community diversities and mainly leave spore forming bacteria alive, such as Clostridium. However, many hydrogen producers are non-spore forming bacteria, and the hydrogen production efficiency might probably be affected.

international journal of hydrogen energy 33 (2008) 4318–4324

Such problems will not arise by repeated-aeration pretreatment. This method could guarantee the microbial diversity, and the community structure was more complex and stable. Especially, repeated-aeration pretreatment resulted in a novel hydrogen producing genus, Ethanoligenens predominant which outperformed others to a certain extent [42]. Meanwhile, from our experience of 247 m3-H2/d full-scale demonstration (data not shown), the repeated-aeration pretreatment method was easier to realize enriching and harvesting a large amount of hydrogen producing bacteria with ethanol-type fermentation. Thus, further study should assess the feasibility and enhancing effects in the continuous flow tests, and evaluate the community diversity and succession.

4.

Conclusions

Based on the results obtained, the present study concluded that hydrogen producing bacteria can be enriched by several pretreatment methods directly from the sludge of the municipal wastewater treatment plant. The highest hydrogen production yield of 1.96 mol/mol-glucose was achieved by repeatedaeration pretreatment, and the hydrogen yield efficiency was in the order of repeated-aeration pretreatment > heatshock > control > alkaline > acid. The methanogenic activity had been effectively suppressed by the heat-shock, acid and repeated-aeration pretreatments. However, the alkaline pretreatment and the control tests were unsuccessful at methanogenesis repression. Ethanol-type fermentation was observed using repeated-aeration pretreatment. And butyric-acid type fermentation was achieved by heat-shock and alkaline pretreatments, while mixed-acid type fermentation was achieved by acid pretreatment and the control. Based on DGGE and 16S rDNA analysis, it was concluded that the microbial community differences had led to the variation of hydrogen production yields and metabolic products. Based on the comparison results, repeated-aeration pretreatment method was recommended in a full-scale hydrogen production plant for its excellent hydrogen producing bacteria enrichment ability and operation feasibility.

Acknowledgements This research was supported by the National Natural Science Fund of China (No. 30470054) and National Natural Science Key Fund of China (No. 50638020).

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