Dechlorination of trichloroethene in a continuous-flow bioelectrochemical reactor: effect of cathode potential on rate, selectivity, and electron transfer mechanisms

The exciting discovery that dechlorinating bacteria can use polarized graphite cathodes as direct electron donors in the reductive dechlorination has prompted investigations on the development of novel bioelectrochemical remediation approaches. In this work, we investigated the performance of a bioelectrochemical reactor for the treatment of trichloroethene (TCE). The reactor was continuously operated for about 570 days, at different potentiostatically controlled cathode potentials, ranging from -250 mV to -750 mV vs standard hydrogen electrode. The rate and extent of TCE dechlorination, as well as the competition for the available electrons, were highly dependent on the set cathode potential. When the cathode was controlled at -250 mV, no abiotic hydrogen production occurred and TCE dechlorination (predominantly to cis-DCE and VC), most probably sustained via direct extracellular electron transfer, proceeded at an average rate of 15.5 ± 1.2 μmol e(-)/L d. At this cathode, potential methanogenesis was almost completely suppressed and dechlorination accounted for 94.7 ± 0.1% of the electric current (15.0 ± 0.8 μA) flowing in the system. A higher rate of TCE dechlorination (up to 64 ± 2 μmol e(-)/L d) was achieved at cathode potentials lower than -450 mV, though in the presence of a very active methanogenesis which accounted for over 60% of the electric current. Remarkably, the bioelectrochemical reactor displayed a stable and reproducible performance even without the supply of organic carbon sources with the feed, confirming long-term viability.     

Reduction of pH buffer requirement in bioelectrochemical systems

Low pH buffer capacity of waste streams limits further development of bioelectrochemical systems (BES) because accumulation of protons potentially leads to acidification of the anodic biofilm. Here we introduce a system that makes it possible to recover alkalinity in an extra recovery compartment. The system consisted of this extra compartment which was located between anode and cathode compartment. The compartment was separated from the anode by a cation exchange membrane and from the cathode by an anion exchange membrane, which made clean hydrogen production possible. To compensate for the charge movement as a result of the flow of electrons, both cations and hydroxyl ions moved into the new recovery compartment. When a synthetic waste stream was fed through this recovery compartment, both pH and conductivity increased. When this stream is then fed to the anode of the BES, no additional buffer was required to produce the same current (3.5 A/m(2)) at an applied voltage of 1 V.     

Operation of a two-stage fermentation process producing hydrogen and methane from organic waste

A pilot-scale experimental plant for the production of hydrogen and methane by a two-stage fermentation process was constructed and operated using a mixture of pulverized garbage and shredded paper wastes. Thermophilic hydrogen fermentation was established at 60 degrees C in the first bioreactor by inoculating with seed microflora. Following the hydrogenogenic process, methanogenesis in the second bioreactor was conducted at 55 degrees C using an internal recirculation packed-bed reactor (IRPR). After conducting steady-state operations under a few selected conditions, the overall hydraulic retention time was optimized at 8 d (hydrogenogenesis, 1.2 d; methanogenesis, 6.8 d), producing 5.4 m3/m3/d of hydrogen and 6.1 m3/m3/d of methane with chemical oxygen demand and volatile suspended solid removal efficiencies of 79.3% and 87.8%, respectively. Maximum hydrogen production yield was calculated to be 2.4 mol/mol hexose and 56 L/kg COD loaded. The methanogenic performance of the IRPR was stable, although the organic loading rate and the composition of the effluent from the hydrogenogenic process fluctuated substantially. A clone library analysis of the microflora in the hydrogenogenic reactor indicated that hydrogen-producing Thermoanaerobacterium-related organisms in the inoculum were active in the hydrogen fermentation of garbage and paper wastes, although no aseptic operations were applied. We speculate that the operation at high temperature and the inoculation of thermophiles enabled the selective growth of the introduced microorganisms and gave hydrogen fermentation efficiencies comparable to laboratory experiments. This is the first report on fermentative production of hydrogen and methane from organic waste at an actual level.     

Hydrogen production by fermentation using acetic acid and lactic acid

Microbial hydrogen production from sho-chu post-distillation slurry solution (slurry solution) containing large amounts of organic acids was investigated. The highest hydrogen producer, Clostridium diolis JPCC H-3, was isolated from natural environment and produced hydrogen at 6.03+/-0.15 ml from 5 ml slurry solution in 30 h. Interestingly, the concentration of acetic acid and lactic acid in the slurry solution decreased during hydrogen production. The substrates for hydrogen production by C. diolis JPCC H-3, in particular organic acids, were investigated in an artificial medium. No hydrogen was produced from acetic acid, propionic acid, succinic acid, or citric acid on their own. Hydrogen and butyric acid were produced from a mixture of acetic acid and lactic acid, showing that C. diolis. JPCC H-3 could produce hydrogen from acetic acid and lactic acid. Furthermore, calculation of the Gibbs free energy strongly suggests that this reaction would proceed. In this paper, we describe for the first time microbial hydrogen production from acetic acid and lactic acid by fermentation.     

Novel methanogenic rotatable bioelectrochemical system operated with polarity inversion

A novel membraneless bioelectrochemical system termed rotatable bioelectrochemical contactor (RBEC) was fabricated and evaluated for its ability to recover useful energy (here methane) from a low organic strength wastewater. We studied the operational characteristics of the RBEC by operating it as a three-electrode electrolysis cell. A stack of conductive disks (each subdivided into two half disks), similar to rotating biological contactors, were rotated with one-half disk immersed in the wastewater and the other into the gas headspace. By carrying out regular half rotations (180° rotation) the anode became the cathode and vice versa. This operation resulted in the build-up of a biofilm that could catalyze both an anodic acetate oxidation and a cathode-driven methanogenesis. Methane production rate was directly proportional to the applied electrical energy. Increase in current density (from 0.16 to 4.1 A m(-2)) resulted in a faster COD removal (from 0.2 to 1.38 kg COD m(-3) day(-1)) and methane production (from 0.04 to 0.53 L L(-1) day(-1)). Of the electrons flowing across the circuit, over 80% were recovered as methane. Such methane production was electrochemically driven by the headspace-exposed cathodic half disks, which released the methane directly to the gas-phase. Energy analysis shows that the new design requires less energy for COD removal than what is typically required for oxygen supply in activated sludge processes. Because the system could operate without wastewater recirculation against gravity; additional pH buffer chemicals; ion-exchange membranes or electrochemical catalysts, it has desirable characteristics for process up-scale. Further, the current report shows the first example of a BES with identical biofilm (due to intermittent polarity inversion) on both electrodes.     

Dehalogenation of iodinated X-ray contrast media in a bioelectrochemical system

Iodinated X-ray contrast media (ICM) are only to a limited extent removed from conventional wastewater treatment plants, due to their high recalcitrance. This work reports on the cathodic dehalogenation of the ICM iopromide in a bioelectrochemical system (BES), fed with acetate at the anode and iopromide at the cathode. When the granular graphite cathode potential was decreased from -500 to -850 mV vs standard hydrogen electrode (SHE), the iopromide removal and the iodide release rates increased from 0 to 4.62 ± 0.01 mmol m(-3) TCC d(-1) and 0 to 13.4 ± 0.16 mmol m(-3) TCC d(-1) (Total Cathodic Compartment, TCC) respectively. Correspondingly, the power consumption increased from 0.4 ± 1 to 20.5 ± 3.3 W m(-3) TCC. The Coulombic efficiency of the iopromide dehalogenation at the cathode was less than 1%, while the Coulombic efficiency of the acetate oxidation at the anode was lower than 50% at various granular graphite cathode potentials. The results suggest that iopromide could be completely dehalogenated in BESs when the granular graphite cathode potential was controlled at -800 mV vs SHE or lower. This finding was further confirmed using mass spectrometry to identify the dehalogenated intermediates and products of iopromide in BESs. Kinetic analysis indicates that iopromide dehalogenation in batch experiments can be described by a first-order model at various cathode potentials. This work demonstrates that the BESs have a potential for efficient dehalogenation of ICM from wastewater or environmental streams.     

Bioelectrochemical regulation accelerates facultatively syntrophic proteolysis

Bioelectrochemical systems can affect microbial metabolism by controlling the redox potential. We constructed bioelectrochemical cultures of the proteolytic bacterium, Coprothermobacter proteolyticus strain CT-1, both as a single-culture and as a co-culture with the hydrogenotrophic methanogen, Methanothermobacter thermautotrophicus strain ?H, to investigate the influences of bioelectrochemical regulation on facultatively syntrophic proteolysis. The co-culture and single-culture were cultivated at 55°C with an anaerobic medium containing casein as the carbon source. The working electrode potential of the bioelectrochemical system was controlled at -0.8V (vs. Ag/AgCl) for bioelectrochemical cultures and was not controlled for non-bioelectrochemical cultures. The cell densities of hydrogenotrophic methanogen and methane production in the bioelectrochemical co-culture were 3.6 and 1.5 times higher than those in the non-bioelectrochemical co-culture after 7 days of cultivation, respectively. Contrastingly, the cell density of Coprothermobacter sp. in the bioelectrochemical co-culture was only 1.3 times higher than that in the non-bioelectrochemical co-culture. The protein decomposition rates were nearly proportional to the cell density of Coprothermobacter sp. in the all types of cultures. These results indicate that bioelectrochemical regulation, particularly, affected the carbon fixation of the hydrogenotrophic methanogen and that facultatively syntrophic proteolysis was accelerated as a result of hydrogen consumption by the methanogens growing well in bioelectrochemical co-cultures.     

Concurrent desalination and hydrogen generation using microbial electrolysis and desalination cells

The versatility of bioelectrochemical systems (BESs) makes them promising for various applications, and good combinations could make the system more applicable and economically effective. An integrated BES called microbial electrolysis and desalination cell (MEDC) was developed to concurrently desalinate salt water, produce hydrogen gas, and potentially treat wastewater. The reactor is divided into three chambers by inserting a pair of ion exchange membranes, with each chamber serving one of the three functions. With an added voltage of 0.8 V, lab scale batch study shows the MEDC achieved the highest H(2) production rate of 1.5 m(3)/m(3) d (1.6 mL/h) from the cathode chamber, while also removing 98.8% of the 10 g/L NaCl from the middle chamber. The anode recirculation alleviated pH and high salinity inhibition on bacterial activity and further increased system current density from 87.2 to 140 A/m(3), leading to an improved desalination rate by 80% and H(2) production by 30%. Compared to slight changes in desalination, H(2) production was more significantly affected by the applied voltage and cathode buffer capacity, suggesting cathode reactions were likely affected by the external power supply in addition to the anode microbial activity.     

The relative effectiveness of pH control and heat treatment for enhancing biohydrogen gas production

Hydrogen gas can be recovered from the microbial fermentation of organic substrates at high concentrations when interspecies hydrogen transfer to methanogens is prevented. Two techniques that have been used to limit methanogenesis in mixed cultures are heat treatment, to remove nonsporeforming methanogens from an inoculum, and low pH during culture growth. We found that high hydrogen gas concentrations (57-72%) were produced in all tests and that heat treatment (HT) of the inoculum (pH 6.2 or 7.5) produced greater hydrogen yields than low pH (6.2) conditions with a nonheat-treated inoculum (NHT). Conversion efficiencies of glucose to hydrogen (based on a theoretical yield of 4 mol-H2/mol-glucose) were as follows: 24.2% (HT, pH = 6.2), 18.5% (HT, pH = 7.5), 14.9% (NHT, pH = 6.2), and 12.1% (NHT, pH = 7.5). The main products of glucose (3 g-COD/L) utilization (> or = 99%) in batch tests were acetate (3.4-24.1%), butyrate (6.4-29.4%), propionate (0.3-12.8%), ethanol (15.4-28.8%), and hydrogen (4.0-8.1%), with lesser amounts of acetone, propanol, and butanol (COD basis). Hydrogen gas phase concentrations in all batch cultures reached a maximum of 57-72% after 30 h but thereafter rapidly declined to nondetectable levels within 80 h. Separate experiments showed substantial hydrogen losses could occur via acetogenesis and that heat treatment did not prevent acetogenesis. Heat treatment consistently eliminated the production of measurable concentrations of methane. The disappearance of ethanol produced during hydrogen production was likely due to acetic acid production as thermodynamic calculations show that this reaction is spontaneous once hydrogen is depleted. Overall, these results show that low pH was, without heat treatment, sufficient to control hydrogen losses to methanogens in mixed batch cultures and suggest that methods will need to be found to limit acetogenesis in order to increase hydrogen gas yields by batch cultures.     

Methanogenic communities on the electrodes of bioelectrochemical reactors without membranes

Methane fermentation was successfully carried out in bioelectrochemical reactors without membranes under a working potential of − 0.6 or − 0.8 V (vs. Ag/AgCl) and neutral pH conditions. The hydrogenotrophic methanogens that dominated on the anodic and cathodic electrodes differed from those found on the electrodes in the control reactors without electrochemical reactions.     

云南建水酸浆豆腐中乳酸菌的生长特性

为了筛选出优良的乳酸菌进行发酵制作酸浆,对分离自云南建水豆腐酸浆中的五株乳酸菌(SYG01、SYG02、SYG03、SYG04、SYG05)的生长曲线、产酸能力、耐酸能力和耐渗透压能力进行了比较。生长曲线实验表明在相同培养时间内,菌株SYG02繁殖能力最强,其次是菌株SYG03,而菌株SYG05、SYG04、SYG01的生长速率明显较慢;在产酸方面SYG02产酸能力最强,速度最快,SYG03、SYG05和SYG04次之,SYG01最弱。在耐酸方面,在pH4.0的环境下5株菌生长良好,在pH3.0的酸性环境下5株菌虽然能够存活,但活菌数的数量级仅在102~104 CFU/mL,其中菌株SYG02和SYG03比其它三株菌株表现出较好的耐酸能力。在耐渗透压方面,SYG02在8%(w/V)的NaCl中依然表现出较强的耐受性,活菌数的数量级达到107 CFU/mL,其余4株菌在NaCl含量大于6%(w/V)时生长受到明显抑制。并以发酵黄浆水的pH及产酸量为指标对菌株的产酸能力进行比较,发现混合菌株的产酸能力高于单菌株,其中菌株SYG02和菌株SYG03组合发酵的效果最好,在发酵72 h后黄浆水的pH为3.52,产酸量达到6.46 g/L。从而得出菌株SYG02具有良好的生长、产酸和耐渗透压能力,具有良好的应用潜力,对酸浆豆腐的工业化生产有重要的意义。     

Electrochemical reduction of carbon dioxide in an MFC-MEC system with a layer-by-layer self-assembly carbon nanotube/cobalt phthalocyanine modified electrode

Electrochemical reduction of carbon dioxide (CO(2)) to useful chemical materials is of great significance to the virtuous cycle of CO(2). However, some problems such as high overpotential, high applied voltage, and high energy consumption exist in the course of the conventional electrochemical reduction process. This study presents a new CO(2) reduction technique for targeted production of formic acid in a microbial electrolysis cell (MEC) driven by a microbial fuel cell (MFC). The multiwalled carbon nanotubes (MWCNT) and cobalt tetra-amino phthalocyanine (CoTAPc) composite modified electrode was fabricated by the layer-by-layer (LBL) self-assembly technique. The new electrodes significantly decreased the overpotential of CO(2) reduction, and as cathode successfully reduced CO(2) to formic acid (production rate of up to 21.0 ± 0.2 mg·L(-1)·h(-1)) in an MEC driven by a single MFC. Compared with the electrode modified by CoTAPc alone, the MWCNT/CoTAPc composite modified electrode could increase the current and formic acid production rate by approximately 20% and 100%, respectively. The Faraday efficiency for formic acid production depended on the cathode potential. The MWCNT/CoTAPc composite electrode reached the maximum Faraday efficiency at the cathode potential of ca. -0.5 V vs Ag/AgCl. Increasing the number of electrode modification layers favored the current and formic acid production rate. The production of formic acid was stable in the MFC-MEC system after multiple batches of CO(2) electrolysis, and no significant change was observed on the performances of the modified electrode. The coupling of the catalytic electrode and the bioelectrochemical system realized the targeted reduction of CO(2) in the absence of external energy input, providing a new way for CO(2) capture and conversion.     

酸汤子发酵工艺的优化及其品质分析

目的 优化酸汤子的发酵工艺，比较传统发酵和人工接种发酵酸汤子的品质差异。方法 以有机玉米面粉为主要原料，以从传统发酵的酸汤子中分离纯化的植物乳杆菌和酿酒酵母按1:2 (V:V)进行酸汤子人工接种发酵，以感官评价为指标，通过单因素和响应面试验优化，研究了液料比、pH、发酵时间和发酵温度对酸汤子品质的影响。结果 实验结果表明，获得适宜的酸汤子发酵工艺为：液料比2:1，pH 6.9，发酵温度37℃，发酵时间16 h, 感官综合评价得分为92.9分。在缩短发酵时间的同时，与市售菌剂发酵的酸汤子、传统发酵的酸汤子相比，总酸含量分别提高了1.65倍、2.63倍；熟断条率分别降低2.65%、5.37%。结论 在最优条件下制作的酸汤子味香而酸甜，顺滑爽口。与传统发酵相比，采用人工接种的酸汤子在外观品质、适口性及营养方面均得到很大提升，本研究为酸汤子工业化生产提供实验参考数据。     

响应面法优化黄浆水发酵生产单细胞蛋白的工艺参数研究

以大豆黄浆水为原料,选用白地霉、扣囊拟内孢霉、解脂假丝酵母和产朊假丝酵母（2∶1∶1∶2）作为发酵菌株发酵黄浆水。在单因素试验的基础上,通过响应面设计法,对黄浆水发酵生产单细胞蛋白（SCP）的工艺进行研究。结果表明,影响SCP产量的因素大小顺序为初始pH、发酵时间、装液量;最佳发酵工艺参数为碳氮比5、初始pH 6、装液量140 mL/250 mL、接种量6%、发酵温度28 ℃、发酵时间42 h;经过优化后的SCP产量达到3.28 g/100 mL。     

分段调控pH值对阴沟肠杆菌 WL1318发酵棉秆水解糖液产氢的影响

为有效调控阴沟肠杆菌（Enterobacter cloacae）WL1318发酵棉秆水解糖液产氢的过程,研究了分段调控pH值对该菌株发酵棉秆水解糖液产氢过程中发酵液pH值、生物氢合成、棉秆水解糖液中葡萄糖和木糖利用、菌株生长的影响。结果表明,分段调控pH值避免了发酵液pH值的急剧下降。分段调控pH值处理的产氢潜力P均高于未调控处理的,48 h时调控pH值能使发酵72 h的日均产氢量较未调控处理的提高约1.5倍;24 h调控pH值和48 h调控pH值处理的累积产氢量相较未调控处理的分别提高约15%和30%。分段调控pH值对阴沟肠杆菌WL1318发酵棉秆水解糖液产氢过程中的菌体生长有较大影响,使其在调控时间点后达到活菌数峰值,且菌体生长OD600 nm值在调控时间点后均高于未调控处理的。     

E．harbinense YUAN-3厌氧发酵影响因素的初步研究

产乙醇杆菌YUAN-3是一种具有良好自凝集特性的高效产氢菌株,已经引起了国内外研究者们的广泛关注。为了提高其发酵性能,有利推进利用该菌株发酵产氢的产业化进程,通过发酵罐静态试验的手段,从接种时间、接种浓度、培养基初始pH值等方面优化了YUAN-3厌氧发酵的条件。结果表明,在种子活化3天后进行接种发酵的效果良好;在5L的发酵罐中,YUAN-3的最优接种浓度是6％;而且,当培养基的初始pH值为6．40时,该厌氧发酵体系的氢气产量最高。因此得出结论,接种时间、接种浓度、培养基初始pH值等对于YUAN-3发酵的氢气产量都具有重要影响。     

Enhanced biohydrogen production from sewage sludge with alkaline pretreatment

Batch tests were carried out to analyze influences of the alkaline pretreatment and initial pH value on biohydrogen production from sewage sludge. Experimental results of the impact of different initial pH on biohydrogen production showed that both the maximal hydrogen yield occurred and that no methane was detected in the tests of at the initial pH of 11.0. The final pH decreased at the initial pH of 7.0-12.5 but increased atthe initial pH of 3.0-6.0, probably due to the combination of solubilized protein from sludge and the formation of volatile fatty acids (VFAs) and ammonia during biohydrogen fermentation. The performance of biohydrogen production using the raw sludge and the alkaline pretreated sludge was then compared in batch fermentation tests atthe initial pH of 11.0. The hydrogen yield was increased from 9.1 mL of H2/g of dry solids (DS) of the raw sludge to 16.6 mL of H2/g of DS of the alkaline pretreated sludge. No methane and less carbon dioxide (0.8% of control) were present in the biohydrogen production from the alkaline pretreated sludge. These results clearly showed that biohydrogen production could be enhanced and maintained stable by the combination of the high initial pH and alkaline pretreatment. The mechanism of biohydrogen production from sewage sludge at high initial pH was therefore investigated because the results of this study were differentfrom previous studies of biohydrogen production. Results showed that protein was the major substrate for biohydrogen production from sewage sludge and that Eubacterium multiforme and Paenibacillus polymyxa were the dominant bacteria in biohydrogen production from alkaline pretreated sludge at an initial pH of 11.0. The combination of alkaline pretreatment and high initial pH could not only maintain a suitable pH range for the growth of dominant hydrogen-producing anaerobes but also inhibit the growth of hydrogen-consuming anaerobes. In addition, the changes in pH value, oxidation-reduction potential, VFAs and soluble COD during hydrogen fermentation were also discussed.     

Biocathodic nitrous oxide removal in bioelectrochemical systems

Anthropogenic nitrous oxide (N(2)O) emissions represent up to 40% of the global N(2)O emission and are constantly increasing. Mitigation of these emissions is warranted since N(2)O is a strong greenhouse gas and important ozone-depleting compound. Until now, only physicochemical technologies have been applied to mitigate point sources of N(2)O, and no biological treatment technology has been developed so far. In this study, a bioelectrochemical system (BES) with an autotrophic denitrifying biocathode was considered for the removal of N(2)O. The high N(2)O removal rates obtained ranged between 0.76 and 1.83 kg N m(-3) net cathodic compartment (NCC) d(-1) and were proportional to the current production, resulting in cathodic coulombic efficiencies near 100%. Furthermore, our experiments suggested the active involvement of microorganisms as the catalyst for the reduction of N(2)O to N(2), and the optimal cathode potential ranged from -200 to 0 mV vs standard hydrogen electrode (SHE) in order to obtain high conversion rates. Successful operation of the system for more than 115 days with N(2)O as the sole cathodic electron acceptor strongly indicated that N(2)O respiration yielded enough energy to maintain the biological process. To our knowledge, this study provides for the first time proof of concept of biocathodic N(2)O removal at long-term without the need for high temperatures and expensive catalysts.     

Efficient reduction of nitrobenzene to aniline with a biocatalyzed cathode

Nitrobenzene (NB) is a toxic compound that is often found as a pollutant in the environment. The present removal strategies suffer from high cost or slow conversion rate. Here, we investigated the conversion of NB to aniline (AN), a less toxic endproduct that can easily be mineralized, using a fed-batch bioelectrochemical system with microbially catalyzed cathode. When a voltage of 0.5 V was applied in the presence of glucose, 88.2 ± 0.60% of the supplied NB (0.5 mM) was transformed to AN within 24 h, which was 10.25 and 2.90 times higher than an abiotic cathode and open circuit controlled experiment, respectively. AN was the only product detected during bioelectrochemical reduction of NB (maximum efficiency 98.70 ± 0.87%), whereas in abiotic conditions nitrosobenzene was observed as intermediate of NB reduction to AN (decreased efficiency to 73.75 ± 3.2%). When glucose was replaced by NaHCO(3), the rate of NB degradation decreased about 10%, selective transformation of NB to AN was still achieved (98.93 ± 0.77%). Upon autoclaving the cathode electrode, nitrosobenzene was formed as an intermediate, leading to a decreased AN formation efficiency of 71.6%. Cyclic voltammetry highlighted higher peak currents as well as decreased overpotentials for NB reduction at the biocathode. 16S rRNA based analysis of the biofilm on the cathode indicated that the cathode was dominated by an Enterococcus species closely related to Enterococcus aquimarinus.     

Effect of inoculum conditioning on hydrogen fermentation and pH effect on bacterial community relevant to hydrogen production

The effect of conditioning for a variety of inoculums on fermentative hydrogen production was investigated. In addition, the effects of pH condition on hydrogen fermentation and bacterial community were investigated. The effect of conditioning on hydrogen production was different depending on the inoculum types. An appreciable hydrogen production was shown with anaerobic digested sludge and lake sediment without conditioning, however, no hydrogen was produced when refuse compost and kiwi grove soil were used as inoculums without conditioning. The highest hydrogen production was obtained with heat-conditioned anaerobic digested sludge, almost the same production was also obtained with unconditioned digested sludge. The pH condition considerably affected hydrogen fermentation, hydrogen gas was efficiently produced with unconditioned anaerobic sludge when the pH was controlled at 6.0 throughout the culture period and not when only the initial pH was adjusted to 6.0 and 7.0. Hydrogen production decreased when the culture pH was only adjusted at the beginning of each batch in continuous batch culture, and additionally, bacterial community varied with the change in hydrogen production. It was suggested that Clostridium and Coprothermobacter species played important role in hydrogen fermentation, and Lactobacillus species had an adverse effect on hydrogen production.