Evaluation of methods for preparing hydrogen-producing seed inocula under thermophilic condition by process performance and microbial community analysis

Five methods for preparation of hydrogen-producing seeds (base, acid, 2-bromoethanesulfonic acid (BESA), load-shock and heat shock treatments) as well as an untreated anaerobic digested sludge were compared for their hydrogen production performance and responsible microbial community structures under thermophilic condition (60 degrees C). The results showed that the load-shock treatment method was the best for enriching thermophilic hydrogen-producing seeds from mixed anaerobic cultures as it completely repressed methanogenic activity and gave the a maximum hydrogen production yield of 1.96 mol H(2) mol(-1) hexose with an hydrogen production rate of 11.2 mmol H(2) l(-1)h(-1). Load-shock and heat-shock treatments resulted in a dominance of Thermoanaerobacterium thermosaccharolyticum with acetic acid and butyric acid type of fermentation while base- and acid-treated seeds were dominated by Clostridium sp. and BESA-treated seeds were dominated by Bacillus sp. The comparative experimental results from hydrogen production performance and microbial community analysis showed that the load-shock treatment method was better than the other four methods for enriching thermophilic hydrogen-producing seeds from anaerobic digested sludge. Load-shock treated sludge was implemented in palm oil mill effluent (POME) fermentation and was found to give maximum hydrogen production rates of 13.34 mmol H(2) l(-1)h(-1) and resulted in a dominance of Thermoanaerobacterium spp. Load-shock treatment is an easy and practical method for enriching thermophilic hydrogen-producing bacteria from anaerobic digested sludge.     

厌氧细菌Acetanaerobacterium elongatum从葡萄糖的产氢特性研究

为了了解影响厌氧发酵产氢细菌Acetanaerobacterium elongatumZ7产氢效率的因素,采用生理学方法对其进行了研究。结果表明:乙醇型发酵菌A.elongatumZ7的最适产氢温度为37℃,最适产氢的起始pH为8.0。该菌发酵葡萄糖和阿拉伯糖产氢的能力较强,氢气产率分别为1.55mol H2/mol葡萄糖和1.50mol H2/mol阿拉伯糖。酵母粉是菌株Z7生长和产氢所必须的生长因子;pH影响菌株的生长和葡萄糖利用率;氢压则影响电子流的分配,从而改变代谢产物乙酸和乙醇的比例;当产氢菌与甲烷菌共培养以维持发酵体系低的氢压时,可使氢的理论产量提高约4倍;培养基中乙酸钠浓度>60mmol/L明显抑制产氢。另外,一个只利用蛋白类物质的细菌能够促进菌株Z7对葡萄糖的利用,进而提供氢产量,为生物制氢的工业化生产提供理论参考。     

Start‐up of Anaerobic Hydrogen Producing Reactors Seeded with Sewage Sludge

The procedure for starting‐up continuously stirred tank reactors (CSTR) for acclimating anaerobic hydrogen‐producing microorganisms with sewage sludge was investigated. Initially, feeding with glucose and sucrose as well as mixing were carried out in semicontinuous mode; hydraulic retention time (HRT) was in an order of 20, 15, 10, 5, 2.5 and 2 days. When the pH declined to its lowest value (pH 5.18), it was adjusted to 6.7 using sodium hydroxide (1 N). At the same time, the semi‐continuous operation was changed to a continuous one. Finally, the pH was continuously regulated at approximately 6.7. The results indicate that this procedure can be used to cultivate seed sludge for hydrogen production from sewage sludge resulting in a large hydrogen production in less than 60 days. When the substrate was glucose, a hydrogen yield of 1.63 mol H₂/mol glucose and a specific hydrogen production rate of 321 mmol H₂/g VSS day at an HRT of 13.3 h was achieved. When the substrate was sucrose with the same HTR, a hydrogen yield of 4.45 mol H2/mol sucrose and a specific hydrogen production rate of 707 mmol H₂/g VSS day was obtained.     

高效产氢菌株Enterococcus sp. LG1的分离及产氢特性

采用Hungate厌氧培养技术分别从厌氧污泥、好氧污泥及河底泥中分离出12株厌氧产氢细菌,并对其中的Enterococcus sp.LG1(注册号:EU258743)进行了研究.结果表明,该株细菌为专性厌氧菌,经革兰氏染色结果为阴性.通过16S rDNA碱基测序和比对证实,该菌株是目前尚未报道过的1个新菌种,初步确定其细菌学上的分类地位.同时,以灭菌预处理的污泥为底物培养基,对该菌的产氢能力及污泥发酵过程中底物性质变化(SCOD、可溶性蛋白质、总糖和pH值等)进行了探讨.实验结果显示,产氢茵Enterococcus sp.LG1的发酵过程中只有H2和CO2产生,无CH4产生.产气量最高为36.48 mL/g TCOD,氢气含量高达73.5%,为已报道文献中以污泥为底物发酵制氢中之最高.根据污泥发酵产物分析得知,该菌的发酵类行为典型的丁酸型发酵.     

Improvement of biohydrogen production by Enterobacter cloacae IIT-BT 08 under regulated pH

The present study investigates the effect of pH and intermediate products formation on biological hydrogen production using Enterobacter cloacae IIT-BT 08. Initial pH was found to have a profound effect on hydrogen production potential, while regulating the pH 6.5 throughout the fermentation was found to increase the cumulative hydrogen production rate and yield significantly. Modified Gompertz equation was used to fit the cumulative hydrogen production curves to obtain the hydrogen production potential P, the hydrogen production rate R and lag phase λ. At regulated pH 6.5, higher H(2) yield (3.1molH(2)mol(-1) glucose), specific hydrogen production potential (798.1mL/g) and specific rate of H(2) production (72.1mLL(-1)h(-1)g(-1)) were obtained. The volatile fatty acid profile showed butyrate, ethanol and acetate as the major end metabolites of fermentation under the operating pH conditions tested; however, their pattern of distribution was pH dependent. At the optimum pH of 6.5, the acetate to butyrate ratio (A/B ratio) was found to be higher than that at any other pH. The study also investigates the effect of sodium ions on biohydrogen production potential. It was also found that sodium ion concentration up to 250mM enhanced the hydrogen production potential; however, any further increase in the metal ion concentration had an inhibitory effect.     

Feasibility of hydrogen production in thermophilic mixed fermentation by natural anaerobes

The biological sludge from an animal wastewater treatment plant was treated to enrich hydrogen-producing mixed bacteria, and effects on hydrogen yield were investigated during anaerobic fermentation at 55 degrees C. Enrichment of hydrogen-producing bacteria was conducted at pH adjustment of inocula to 3 and 5 with and without additional heat treatment (NHT and HT). The enriched mixed bacteria were cultivated at initial pHs of 5, 6, and 7 with synthetic organic wastewater containing different levels of nitrogen (2.0 and 0.8 g/l as total nitrogen) under static batch conditions. The main effects of heat treatment and enrichment pH were significant on hydrogen production. There was no significant effect of different nitrogen concentrations on hydrogen production. The methane-free biogas contained hydrogen levels of up to 64% for a fermentative condition that showed maximum hydrogen evolution (at culture pH 5 after enrichment at pH 5 with HT). The dominating intermediate metabolites were acetate, n-butyrate, and ethanol. Yields of produced hydrogen were significantly dependent upon levels of n-butyrate.     

Microbial hydrogen production with Bacillus coagulans IIT-BT S1 isolated from anaerobic sewage sludge

Bacillus coagulans strain IIT-BT S1 isolated from anaerobically digested activated sewage sludge was investigated for its ability to produce H(2) from glucose-based medium under the influence of different environmental parameters. At mid-exponential phase of cell growth, H(2) production initiated and reached maximum production rate in the stationary phase. The maximal H(2) yield (2.28 mol H(2)/molglucose) was recorded at an initial glucose concentration of 2% (w/v), pH 6.5, temperature 37 degrees C, inoculum volume of 10% (v/v) and inoculum age of 14 h. Cell growth rate and rate of hydrogen production decreased when glucose concentration was elevated above 2% w/v, indicating substrate inhibition. The ability of the organism to utilize various carbon sources for H(2) fermentation was also determined.     

Evaluation of metabolism using stoichiometry in fermentative biohydrogen

We first constructed full stoichiometry, including cell synthesis, for glucose mixed-acid fermentation at different initial substrate concentrations (0.8-6 g-glucose/L) and pH conditions (final pH 4.0-8.6), based on experimentally determined electron-equivalent balances. The fermentative bioH2 reactions had good electron closure (-9.8 to +12.7% for variations in glucose concentration and -3 to +2% for variations in pH), and C, H, and O errors were below 1%. From the stoichiometry, we computed the ATP yield based on known fermentation pathways. Glucose-variation tests (final pH 4.2-5.1) gave a consistent fermentation pattern of acetate + butyrate + large H2, while pH significantly shifted the catabolic pattern: acetate + butyrate + large H2 at final pH 4.0, acetate + ethanol + modest H2 at final pH 6.8, and acetate + lactate + trivial H2 at final pH 8.6. When lactate or propionate was a dominant soluble end product, the H2 yield was very low, which is in agreement with the theory that reduced ferredoxin (Fd(red)) formation is required for proton reduction to H2. Also consistent with this hypothesis is that high H2 production correlated with a high ratio of butyrate to acetate. Biomass was not a dominant sink for electron equivalents in H2 formation, but became significant (12%) for the lowest glucose concentration (i.e., the most oligotrophic condition). The fermenting bacteria conserved energy similarly at approximately 3 mol ATP/mol glucose (except 0.8 g-glucose/L, which had approximately 3.5 mol ATP/mol glucose) over a wide range of H2 production. The observed biomass yield did not correlate with ATP conservation; low observed biomass yields probably were caused by accelerated rates of decay or production of soluble microbial products.     

Direct fermentation of fodder maize, chicory fructans and perennial ryegrass to hydrogen using mixed microflora

This study examined the feasibility of producing hydrogen by direct fermentation of fodder maize, chicory fructooligosaccharides and perennial ryegrass (Lolium perenne) in batch culture (pH 5.2-5.3, 35 degrees C, heat-treated anaerobically digested sludge inoculum). Gas was produced from each substrate and contained up to 50-80% hydrogen during the peak periods of gas production with the remainder carbon dioxide. Hydrogen yields obtained were 62.4+/-6.1mL/g dry matter added for fodder maize, 218+/-28mL/g chicory fructooligosaccharides added, 75.6+/-8.8mL H(2)/g dry matter added for wilted perennial ryegrass and 21.8+/-8mL H(2)/g dry matter added for fresh perennial ryegrass. Butyrate, acetate and ethanol were the main soluble fermentation products. Hydrogen yields of 392-501m(3)/hectare of perennial ryegrass per year and 1060-1309m(3)/hectare of fodder maize per year can be obtained based on the UK annual yield per hectare of these crops. These results significantly extend the range of substrates that can be used for hydrogen production without pre-treatment.     

Enhanced hydrogen and 1,3‐propanediol production from glycerol by fermentation using mixed cultures

The conversion of glycerol into high value products, such as hydrogen gas and 1,3‐propanediol (PD), was examined using anaerobic fermentation with heat‐treated mixed cultures. Glycerol fermentation produced 0.28 mol‐H₂/mol‐glycerol (72 mL‐H₂/g‐COD) and 0.69 mol‐PD/mol‐glycerol. Glucose fermentation using the same mixed cultures produced more hydrogen gas (1.06 mol‐H₂/mol‐glucose) but no PD. Changing the source of inoculum affected gas production likely due to prior acclimation of bacteria to this type of substrate. Fermentation of the glycerol produced from biodiesel fuel production (70% glycerol content) produced 0.31 mol‐H₂/mol‐glycerol (43 mL H₂/g‐COD) and 0.59 mol‐PD/mol‐glycerol. These are the highest yields yet reported for both hydrogen and 1,3‐propanediol production from pure glycerol and the glycerol byproduct from biodiesel fuel production by fermentation using mixed cultures. These results demonstrate that production of biodiesel can be combined with production of hydrogen and 1,3‐propanediol for maximum utilization of resources and minimization of waste. Biotechnol. Bioeng. 2009; 104: 1098–1106. © 2009 Wiley Periodicals, Inc.     

Coproduction of acetaldehyde and hydrogen during glucose fermentation by Escherichia coli

Escherichia coli K-12 strain MG1655 was engineered to coproduce acetaldehyde and hydrogen during glucose fermentation by the use of exogenous acetyl-coenzyme A (acetyl-CoA) reductase (for the conversion of acetyl-CoA to acetaldehyde) and the native formate hydrogen lyase. A putative acetaldehyde dehydrogenase/acetyl-CoA reductase from Salmonella enterica (SeEutE) was cloned, produced at high levels, and purified by nickel affinity chromatography. In vitro assays showed that this enzyme had both acetaldehyde dehydrogenase activity (68.07 ± 1.63 μmol min(-1) mg(-1)) and the desired acetyl-CoA reductase activity (49.23 ± 2.88 μmol min(-1) mg(-1)). The eutE gene was engineered into an E. coli mutant lacking native glucose fermentation pathways (ΔadhE, ΔackA-pta, ΔldhA, and ΔfrdC). The engineered strain (ZH88) produced 4.91 ± 0.29 mM acetaldehyde while consuming 11.05 mM glucose but also produced 6.44 ± 0.26 mM ethanol. Studies showed that ethanol was produced by an unknown alcohol dehydrogenase(s) that converted the acetaldehyde produced by SeEutE to ethanol. Allyl alcohol was used to select for mutants with reduced alcohol dehydrogenase activity. Three allyl alcohol-resistant mutants were isolated; all produced more acetaldehyde and less ethanol than ZH88. It was also found that modifying the growth medium by adding 1 g of yeast extract/liter and lowering the pH to 6.0 further increased the coproduction of acetaldehyde and hydrogen. Under optimal conditions, strain ZH136 converted glucose to acetaldehyde and hydrogen in a 1:1 ratio with a specific acetaldehyde production rate of 0.68 ± 0.20 g h(-1) g(-1) dry cell weight and at 86% of the maximum theoretical yield. This specific production rate is the highest reported thus far and is promising for industrial application. The possibility of a more efficient "no-distill" ethanol fermentation procedure based on the coproduction of acetaldehyde and hydrogen is discussed.     

Kinetics of hydrogen production with continuous anaerobic cultures utilizing sucrose as the limiting substrate

In this study, local sewage sludge was acclimated to establish H2-producing enrichment cultures, which were used to convert sucrose to H2 with continuously stirred anaerobic bioreactors. The steady-state behaviors of cell growth, substrate utilization, and product formation were closely monitored. Kinetic models were developed to describe and predict the experimental results from the H2-producing cultures. Operation at dilution rates (D) of 0.075-0.167 h(-1) was preferable for H2 production, resulting in a H2 concentration of nearly 0.02 mol/l. The optimal hydrogen production rate was 0.105 mol/h occurring at D=0.125 h(-1). The major volatile fatty acid produced was butyric acid (HBu), while acetic acid and propionic acid were also produced in lesser quantities. The major solvent product was ethanol, whose concentration was only 15% of that of HBu, indicating that the metabolic flow favors H2 production. The proposed model was able to interpret the trends of the experimental data. The maximum specific growth rate (mu(max)), Monod constant (Ks), and yield coefficient for cell growth (Y(x/s)) were estimated as 0.172 h(-1), 68 mg COD/l, and 0.1 g/g, respectively. The model study also suggests that product formation in the continuous hydrogen-producing cultures was essentially a linear function of biomass concentration.     

Fermentative H2 production in an upflow anaerobic sludge blanket reactor at various pH values

Fermentative H(2) production in an upflow anaerobic sludge blanket reactor (UASB) at various pH values was investigated in this study. Experimental results show that the H(2) partial pressure in biogas, H(2) production rate and H(2) yield were all pH-dependent, in the range of 0.25-0.52 atm, 42-145 ml-H(2) l(-1) h(-1) and 0.47 to 1.61 mol-H(2)mol-glucose(-1), respectively. The maximum pH for the H(2) partial pressure was observed at pH 7.50. However, the optimum H(2) production rate and H(2) yield were observed at pH 6.50-7.50. In this UASB reactor, acetate, propionate, butyrate, i-butyrate, valerate, caporate and ethanol were present in the effluent as main aqueous products, and the dominant fermentation was butyrate-type at various pHs. The metabolic pathways and thermodynamics of H(2) production were also analyzed. Both H(2) production performance and fermentation pathways in this H(2)-producing UASB reactor were significantly affected by the pH value.     

Comparison of different mixed cultures for bio-hydrogen production from ground wheat starch by combined dark and light fermentation

Composition of the mixed culture was varied in combined dark-light fermentation of wheat powder starch in order to improve hydrogen gas formation rate and yield. Heat-treated anaerobic sludge and pure culture of Clostridium beijerinckii (DSMZ 791 ^T ) were combined with two different light fermentation bacteria of Rhodobacter sphaeroides (RS-NRRL and RS-RV) in order to select a more suitable mixture resulting in high hydrogen yield and formation rate. A combination of the anaerobic sludge and RS-NRRL yielded the highest cumulative hydrogen (CHF = 140 ml), the highest yield (0.36 mol H₂ mol⁻¹ glucose) and specific hydrogen formation rate (2.5 ml H₂ g⁻¹ biomass h⁻¹). During dark fermentation (70 h) hydrogen was produced simultaneously by the dark and light fermentation bacteria using glucose from hydrolyzed starch. However, only light fermentation bacteria produced hydrogen from VFA’s derived from dark fermentation after a long adaptation period.     

Acclimation Strategy of a Biohydrogen Producing Population in a Continuous‐Flow Reactor with Carbohydrate Fermentation

Poor startup of biological hydrogen production systems can cause an ineffective hydrogen production rate and poor biomass growth at a high hydraulic retention time (HRT), or cause a prolonged period of acclimation. In this paper a new startup strategy was developed in order to improve the enrichment of the hydrogen‐producing population and the efficiency of hydrogen production. A continuously‐stirred tank reactor (CSTR) and molasses were used to evaluate the hydrogen productivity of the sewage sludge microflora at a temperature of 35 °C. The experimental results indicated that the feed to microorganism ratio (F/M ratio) was a key parameter for the enrichment of hydrogen producing sludge in a continuous‐flow reactor. When the initial biomass was inoculated with 6.24 g of volatile suspended solids (VSS)/L, an HRT of 6 h, an initial organic loading rate (OLR) of 7.0 kg chemical oxygen demand (COD)/(m³ × d) and an feed to microorganism ratio (F/M) ratio of about 2–3 g COD/(g of volatile suspended solids (VSS) per day) were maintained during startup. Under these conditions, a hydrogen producing population at an equilibrium state could be established within 30 days. The main liquid fermentation products were acetate and ethanol. Biogas was composed of H₂ and CO₂. The hydrogen content in the biogas amounted to 47.5 %. The average hydrogen yield was 2.01 mol/mol hexose consumed. It was also observed that a special hydrogen producing population was formed when this startup strategy was used. It is supposed that the population may have had some special metabolic pathways to produce hydrogen along with ethanol as the main fermentation products.     

Metabolic pathways of hydrogen production in fermentative acidogenic microflora

Biohydrogen production from organic wastewater by anaerobically activated sludge fermentation has already been extensively investigated, and it is known that hydrogen can be produced by glucose fermentation through three metabolic pathways, including the oxidative decarboxylation of pyruvic acid to acetyl-CoA, oxidation of NADH to NAD+, and acetogenesis by hydrogen-producing acetogens. However, the exact or dominant pathways of hydrogen production in the anaerobically activated sludge fermentation process have not yet been identified. Thus, a continuous stirred-tank reactor (CSTR) was introduced and a specifically acclimated acidogenic fermentative microflora obtained under certain operation conditions. The hydrogen production activity and potential hydrogen-producing pathways in the acidogenic fermentative microflora were then investigated using batch cultures in Erlenmeyer flasks with a working volume of 500 ml. Based on an initial glucose concentration of 10 g/l, pH 6.0, and a biomass of 1.01 g/l of a mixed liquid volatile suspended solid (MLVSS), 247.7 ml of hydrogen was obtained after a 68 h cultivation period at 35 +/- 1 degrees C. Further tests indicated that 69% of the hydrogen was produced from the oxidative decarboxylation of pyruvic acid, whereas the remaining 31% was from the oxidation of NADH to NAD+. There were no hydrogen-producing acetogens or they were unable to work effectively in the anaerobically activated sludge with a hydraulic retention time (HRT) of less than 8 h.     

Effect of some environmental parameters on fermentative hydrogen production by Enterobacter cloacae DM11

Fermentative hydrogen production was carried out by Enterobacter cloacae DM11, using glucose as the substrate. The effects of initial substrate concentration, initial medium pH, and temperature were investigated. Results showed that at an initial glucose concentration of 1.0% (m/v), the molar yield of hydrogen was 3.31 mol (mol glucose)(-1). However, at higher initial glucose concentration, both the rate and cumulative volume of hydrogen production decreased. The pH of 6.5 +/- 0.2 at a temperature of 37 degrees C was found most suitable with respect to maximum rate of production of hydrogen in batch fermentation. Activation enthalpies of fermentation and that of thermal deactivation of the present process were estimated following a modified Arrhenius equation. The values were 47.34 and 118.67 kJ mol(-1) K(-1), respectively. The effect of the addition of Fe(2+) on hydrogen production was also studied. It revealed that the presence of iron (Fe(2+)) in the media up to a concentration of 20 mg L(-1) had a marginal enhancing effect on total hydrogen production. A simple model developed from the modified Gompertz equation was applied to estimate the hydrogen production potential, production rate, and lag-phase time in a batch process, based on the cumulative hydrogen production curves, using the software program Curve Expert 1.3.     

Transcriptional control of hydrogen production during mixed carbon fermentation by hydrogenases 4 (hyf) and 3 (hyc) in Escherichia coli

Escherichia coli molecular hydrogen (H(2)) production was studied during mixed carbon (glucose and glycerol) fermentation at pH 6.5. Wild type cells in the assays supplemented with glucose produced H(2) at ~2 fold lower level than cells grown on glucose only. When compared to the wild type, H(2) production in the assays added with glucose was decreased by ~2 fold in fhlA, hyfG and double fhlA hyfG mutants and by ~1.5 fold in hyaB, hybC, and double hyaB hybC mutants. However, in the assays with glycerol, no measurable H(2) production was detected. Taken together, these results suggest that during mixed carbon fermentation, H(2) could be produced with low efficiency via Hyd-3 and Hyd-4. This is a novel finding for Hyd-4 activity at pH 6.5. The insignificant decrease of H(2) production in the strains with defects in Hyd-1 and Hyd-2 was probably due to an interaction between the Hyd enzymes and their organization in the bacterial membrane. In the glucose assays, H(2) production in the wild type cells was inhibited ~2 fold by 0.3mM N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the F(0)F(1)-ATPase. This inhibition was the same for fhlA and hyfG fhlA mutants but not hyaB, hybC, hyfG or hyaB hybC mutants. The results indicate that the FhlA protein coded by the fhlA gene might interact with the F(0)F(1)-ATPase. We propose that this interaction is mediated by mixed carbon fermentation.     

The isolation and microbial community analysis of hydrogen producing bacteria from activated sludge

AIMS: To profile the fractions of bacteria in heat-treated activated sludge capable of producing hydrogen and subsequently to isolate those organisms and confirm their ability to produce hydrogen. METHODS AND RESULTS: Profiling the community composition of the microflora in activated sludge using 16S rRNA gene-directed polymerase chain reaction-denaturing gradient gel electrophoresis suggested that a majority of bacteria were various Clostridium species. This was confirmed by clone library analysis, where 80% of the cloned inserts were Clostridium sp. A total of five isolates were established on solid media. Three of them, designated as W1, W4 and W5, harboured the hydrogenase gene as determined by PCR and DNA sequence analysis (99% similarity). These isolates were similar to Clostridium butyricum and Clostridium diolis as determined by 16S rRNA gene sequence. A maximum hydrogen production yield of 220 ml H(2) g(-1) glucose was achieved by W5, which was grown on improved mineral medium by batch fermentation without pH adjustment and nitrogen sparging during fermentation. Accumulation of malic acid and fumaric acid during hydrogen fermentation might lead to higher hydrogen yields for W4 and W5. W1 is the first reported Clostridium species that can tolerate microaerobic conditions for producing hydrogen. CONCLUSION: Clostridium species in heat-treated activated sludge were the most commonly identified bacteria responsible for hydrogen production. Specific genetic markers for strains W1, W4 and W5 would be of great utility in investigating hydrogen production at the molecular level. Two previously described primer sets targeting hydrogenase genes were shown not to be specific, amplifying other genes from nonhydrogen producers. SIGNIFICANCE AND IMPACT OF THE STUDY: Clostridium species isolated from heat-treated activated sludge were confirmed as hydrogen producers during dark hydrogen fermentation. The isolates will be useful for studying hydrogen production from wastewater, including the process of gene regulation and hydrogenase activity.     

一个新的高温产氢菌及产氢特性的研究

利用Hungate滚管技术从西藏山南地区热泉淤泥中分离到一株高温产氢的厌氧发酵细菌T42。菌株T42革兰氏染色反应为阴性,但KOH裂解试验证实其为革兰氏阳性杆菌。菌体大小为0.7μm~0.9μm×3.2μm~7μm,不运动,不产芽孢。其生长温度范围为32℃~69℃,最适生长温度为60℃~62℃,生长pH范围为5.0~8.8,最适生长pH为7.0~7.5,代时30min。有机氮源是T42菌株的必需生长因子。菌株T42利用淀粉、纤维二糖、蔗糖、麦芽糖、糊精、果糖、糖原和海藻糖等底物生长并发酵产氢,发酵葡萄糖的终产物为乙酸、乙醇、H2和CO2。G C含量为31.2mol%。系统发育分析表明菌株T42与Thermobrachium celere和Caloramator indicus位于同一分支,生理生化特征也表明菌株T42应是Thermobrachium属的一个新菌株,在中国普通微生物菌种保藏中心的保藏号为AS1.5039。菌株T42的最佳产氢初始pH为7.2,最佳产氢温度为62℃,其氢转化率为1.06mol H2/mol葡萄糖,最大产氢速率为24.0mmol H2/gDW/h。20mmol/L的Mg2 和2mmol/L的Fe2 可分别提高菌株T42的产氢量20%和23.3%,而Ni2 对其产氢无明显的作用。当菌株T42和热自养甲烷热杆菌(Methanothermobacter thermautotrophicus)Z245共培养时,由于降低了氢分压,使其葡萄糖利用率和氢产量分别提高1倍和2.8倍,发酵产物乙酸和乙醇的比例也从1提高到1.7。