产氢菌的分离鉴定及其产氢特性研究

从河底污泥中分离到一株产氢菌,通过16S rDNA序列和系统发育分析,鉴定为克雷伯氏菌属,命名为Klebsiella sp. HQ-3.运用单因子试验考察了摇瓶发酵培养基中碳源、氮源、无机盐等因素对克雷伯氏菌HQ-3发酵产氢的影响,并对其产氢特性做了初步研究.结果表明,克雷伯氏HQ-3在葡萄糖浓度为24 g/L,蛋白胨为18 g/ L,KH2PO4为 8g/ L时具有较高产氢量,其最佳产氢初始pH为8.0左右.     

发酵产氢菌株的筛选及影响因素研究

采用双层平板法从污泥池中筛选出一株产氢较高的发酵菌株,经生理生化鉴定表明,分离菌株初步鉴定为消化链球菌属(Peptostreptococcus).研究静态培养条件下葡萄糖、pH、温度及和酵母膏对菌株产氢的影响及不同发酵时间段的产氢情况.结果表明,在葡萄糖浓度20.0 g/L,pH 7.0,温度37 ℃和酵母膏2.0 g/L时,产氢量达21.07 mmol/L,为初始培养条件下的4.14倍.同时,在24～36 h时间段产氢率达到最高,为0.44 mmol/(L·h),并且在60 h时产氢量达到最大累计产氢量的89.2%.     

产氢菌的复合诱变选育及突变株HCM-23的产氢特性

以厌氧产氢细菌Clostridium sp. H-61为原始菌株, 先后经亚硝基胍(NTG)、紫外(UV)诱变, 选育得到1株高产突变株HCM-23。在葡萄糖浓度为10 g/L的条件下, 其产氢量为3024 mL/L, 比原始菌株提高了69.89%; 其最大产氢速率为33.19 mmol H2/g DW·h, 比原始菌株(19.74 mmol H2/g DW·h)提高了68.14%。经过多次传代试验, 稳定性良好。其发酵末端产物以乙醇和乙酸为主, 属于典型乙醇型发酵代谢类型。其最适产氢初始pH为6.5, 最适生长温度为36℃, 以蔗糖为最佳碳源。与原始菌株相比, 突变株HCM-23的产氢特性发生了改变, 如生长延滞期延长, 可利用无机氮源等。     

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.     

产氢菌Enterobacter sp.和Clostridium sp.的分离鉴定及产氢特性

在高温水体中分离得到2株具有较高产氢活性的微生物菌株Z-16和C-32。根据两菌株的16SrDNA序列分析,初步鉴定菌株Z-16为Enterobactersp.,菌株C-32为Clostridiumsp.。研究了起始pH值、反应温度、碳源等对菌株放氢活性的影响。菌株Z-16的最适产氢条件为:反应系统起始pH7·0,反应温度35℃,以蔗糖为产氢底物。在最适条件下,菌株Z-16的氢转化率为2·68molH2/mol蔗糖。菌株C-32的最适产氢条件为:反应系统起始pH8·0,反应温度35℃,以麦芽糖为产氢底物。在最适条件下,菌株C-32的氢转化率为2·71molH2/mol麦芽糖。以葡萄糖为碳源时,菌株Z-16和菌株C-32的氢转化率分别为2·35和2·48molH2/mol葡萄糖。     

一个新的产氢细菌的鉴定及产氢特性的研究

利用Hungate滚管技术从福建省漳州垃圾处理厂厌氧消化器的颗粒污泥中分离到一株产氢的细菌L15。菌株L15为严格厌氧的革兰氏阳性杆菌,菌体大小为0.5μm～0.7μm×2.5μm～5.0μm,以侧生鞭毛运动。在孢肉培养基上产生端生的卵圆形芽孢。温度生长范围15℃～45℃（最适温度30℃～37℃）;pH 范围5.0～8.4（最适pH 6.3～6.8）。该菌株不水解明胶和七叶灵,不还原硫酸盐,牛奶变酸但不凝固,发酵多糖和少数的单糖、双糖和寡糖;发酵葡萄糖的最终产物为乙酸、丁酸、H2和CO2。G+C含量为298mol%。16S rDNA序列分析表明,该菌株属于梭菌的簇Ⅰ,与Clostridium paraputrificum较为接近（相似性为97.1%）。通过生理特征和16S rDNA序列的同源性分析,表明菌株L15应是梭菌属簇Ⅰ中的一个新种,命名为Clostridium defluvii。菌株L15保藏在中国普通微生物菌种保藏中心,保藏号为AS1.3489。菌株L15的最佳产氢温度为34℃、pH为7.0。当葡萄糖浓度为0.4%时,氢气产率可达到1.41mol H2/mol 葡萄糖。该菌可利用下列底物产酸产氢,括号内为产氢率(底物浓度1%)：果糖(1.00mol H2/mol)、麦芽糖(2.17mol H2/mol)、蔗糖(1.69mol H2/mol)、菊糖(4.70mol H2/mol)、糖原(5.49mmol H2/g)、淀粉(7.34mmol H2/g)。     

高效产氢细菌新种--Ethanologenbacterium sp.X-1的分离鉴定及其产氢效能

为获得高效产氢发酵细菌 ,采用改进的厌氧Hungate培养技术 ,从生物制氢反应器CSTR中分离一株产氢细菌X 1。对该株细菌进行了形态学特征、生理生化指标、16SrDNA和 16S 2 3SrDNA间隔区序列分析等研究。结果表明与最相近的种属Clostridiumcellulosi和Acetanaerobacteriumelongatum等的 16SrRNA基因序列同源性为 94 %以下。16S 2 3SrRNA间隔区基因序列比对分析显示保守区域仅为tRNAAla和tRNAIle序列 ,其它可变部位没有同源性区域 ,鉴定为新属Ethanologenbacteriumsp .。该株细菌为专性厌氧杆菌 ,代谢特征为乙醇发酵 ,葡萄糖发酵产物主要为乙醇、乙酸、H2 和CO2 。在pH4 0和 36℃条件下最大产氢速率是 2 8 3mmolH2 (gdrycell·h)。经鉴定和产氢效能分析表明该菌株是一新属的高效产氢细菌     

产氢菌Enterobacter sp.和Clostridium sp.的分离鉴定及产氢特性

在高温水体中分离得到2株具有较高产氢活性的微生物菌株Z-16和C-32。根据两菌株的16S rDNA序列分析,初步鉴定菌株Z-16为Enterobacter sp.,菌株C-32为Clostridium sp.。研究了起始pH值、反应温度、碳源等对菌株放氢活性的影响。菌株Z-16的最适产氢条件为：反应系统起始pH7.0,反应温度35℃,以蔗糖为产氢底物。在最适条件下,菌株Z-16的氢转化率为2.68mol H₂/mol蔗糖。菌株C-32的最适产氢条件为：反应系统起始pH 8.0,反应温度35℃,以麦芽糖为产氢底物。在最适条件下,菌株C-32的氢转化率为2.71mol H₂/mol 麦芽糖。以葡萄糖为碳源时,菌株Z-16和菌株C-32的氢转化率分别为2.35和2.48mol H₂/mol葡萄糖。     

产氢菌Enterobacter sp.和Clostridium sp.的分离鉴定及产氢特性

在高温水体中分离得到2株具有较高产氢活性的微生物菌株Z-16和C-32。根据两菌株的16S rDNA序列分析,初步鉴定菌株Z-16为Enterobacter sp.,菌株C-32为Clostridium sp.。研究了起始pH值、反应温度、碳源等对菌株放氢活性的影响。菌株Z-16的最适产氢条件为：反应系统起始pH7.0,反应温度35℃,以蔗糖为产氢底物。在最适条件下,菌株Z-16的氢转化率为2.68mol H₂/mol蔗糖。菌株C-32的最适产氢条件为：反应系统起始pH 8.0,反应温度35℃,以麦芽糖为产氢底物。在最适条件下,菌株C-32的氢转化率为2.71mol H₂/mol 麦芽糖。以葡萄糖为碳源时,菌株Z-16和菌株C-32的氢转化率分别为2.35和2.48mol H₂/mol葡萄糖。     

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。     

产氢产乙酸菌ZR-1 的分离鉴定及产酸特性

采用改良的亨盖特厌氧操作技术, 从有机废水污泥中分离到一株耐低温高效产氢产乙酸菌ZR-1。经过对其形态学观察、生理生化特征研究及16S rRNA 序列比对, 初步鉴定为梭状芽胞杆菌属的乙二醇梭菌(Clostridium glycolicum)。通过单因子实验, 在厌氧条件下对该菌株的培养温度、pH、最适底物、金属离子的影响等产酸条件进行了优化。结果表明该菌株最适生长温度37 °C,最佳培养基初始pH 值8.5, 最适发酵底物丁酸盐, Mn²⁺对其产酸有一定的激活作用。最适培养条件下丁酸盐降解率达到12.7%, H₂ 含量达到了28.73%。     

联合生物制氢方法及发展趋势

为了在生物制氢过程中最大限度提高产氢量和产氢速率,增大底物的利用率以及更好地发挥菌种间的协同作用,联合生物制氢技术成为近年来人们关注的焦点。综述了目前国内外几种联合生物制氢方法的研究现状。并从产氢机理的角度对几种联合制氢技术进行了分析比较,重点强调光合发酵和暗发酵联合生物制氢技术具有广泛的发展前景,并指出其存在的问题和未来的发展趋势。     

产氢新菌Ethanoligenens sp. B49发酵糖蜜制氢条件

研究1株产氢细菌Ethanoligenens sp.B49利用废糖蜜为基本原料进行生物制氢的条件,及外加氮素营养物对废糖蜜生物制氢的影响.结果表明,在10.3～20.6 g·L-1的化学需氧量(COD)范围内,经过驯化的Ethanoligenens sp.B49细胞具有较好的生物利用能力,细胞生长量和产氢能力随着废糖蜜COD的提高而增加.当废糖蜜COD超过20.6 g·L-1时Ethanoligenens sp.B49的细胞生长受到抑制,同时产氢能力下降,COD超过41.2 g·L-1时细胞基本不具有生长和产氢能力.Ethanoligenens sp.B49利用废糖蜜产氢的最佳COD为20.6 g·L-1.在20.6 g·L-1COD条件下外加有机氮源可以促进Ethanoligenens sp.B49利用废糖蜜制氢的能力,促进作用顺序为酵母粉＞牛肉膏＞蛋白胨＞脲素.添加4 g·L-1的酵母粉时,Ethanoligenens sp.B49细胞具有最好的生长活性和产氢能力.优化营养条件后,单位体积产氢量从44.82 mmol·L-1提高到78.97 mmol·L-1,提高了76.2%.     

绿藻光合生物制氢技术进展

氢能作为可再生、环境友好的能源,已成为营造可持续发展的经济节约型社会的理想能源。绿藻因能利用光能分解水产氢,被称为最有应用前景的方法之一。本文将综述绿藻光合产氢的原理,介绍该生物制氢技术的研究现状和最新进展,并对其发展趋势做以展望。     

Carrier modification and its application in continuous photo‐hydrogen production using anaerobic fluidized bed photo‐reactor

Poor hydrogen production performance and low biomass limit the practical application of photo‐fermentation. To improve the immobilization capability of bacteria and hydrogen production performance, activated carbon fibers (ACFs) were modified by acidic, alkaline, and neutral solutions. The modified ACFs were further used in the anaerobic fluidized bed photo‐reactor (AFBPR) to explore its continuous operation characteristics. Results showed that among the three reagents, nitric acid was the most efficient for ACF modification, and the maximum yield and production rate of hydrogen increased between about 33.6% and 65.8% compared to the control. Furthermore, with the optimal influent glutamate concentration (10 mmol L^?1) and light intensity (4000 lux), the AFBPR gave efficient and stable performance with hydrogen yield of 2.26 mol H₂ mol^?1 acetate and hydrogen production rate of 25.8 mL L^?1 h^?1. The results showed the potential of using the AFBPR with HNO₃‐modified ACF carriers for the large‐scale production of bio‐hydrogen.     

Continuous fermentative hydrogen production from a wheat starch co-product by mixed microflora

For the transition to the hydrogen economy, hydrogen must be produced sustainably, e.g., by the fermentation of agricultural material. Continuous fermentative production of hydrogen from an insoluble substrate in nonsterile conditions is yet to be reported. In this study hydrogen production using mixed microflora from heat-treated digested sewage sludge in nonsterile conditions from a particulate co-product of the wheat flour industry (7.5 g L(-1) total hexose) at 18- and 12-hour hydraulic retention times, pH 4.5 and 5.2, 30 degrees C and 35 degrees C was examined. In continuous operation, hydrogen yields of approximately 1.3 moles hydrogen/mole hexose consumed were obtained, but decreased if acetate or propionate levels rose, indicating metabolism shifted towards hydrogen consumption by homoacetogenesis or propionate producers. These shifts occurred both at pH 4.5 and 5.2. Sparging the reactor with nitrogen to reduce hydrogen in the off-gas from 50% to 7% gave stable operation with a hydrogen yield of 1.9 moles hydrogen /mole hexose consumed over an 18-day period.     

Biological hydrogen production in a UASB reactor with granules. I: Physicochemical characteristics of hydrogen-producing granules

Hydrogen-producing granules with an excellent settling ability were cultivated in an upflow anaerobic sludge blanket reactor treating a sucrose-rich synthetic wastewater. The physicochemical characteristics of granules were evaluated in this study. The mature granules had a diameter ranging from 1.0 to 3.5 mm and an average density of 1.036 +/- 0.005 g/mL, whereas they had good settling ability and a high settling velocity of 32-75 m/h. The low ratio of proteins/carbohydrates for the extracellular polymeric substances (EPS) in the granules suggests that carbohydrates rather than proteins, might play a more important role in the formation of the H(2)-producing granules. The contact angle of the mature granules, 54 +/- 2 degrees , was larger than that of the seed sludge (38 +/- 2 degrees ), indicating that the microbial cells in the H(2)-producing granules had higher hydrophobicity. The granules had fractal nature with a fractal dimension of 1.78. Their porosities were in the range of 0-0.70, and increased with increasing granule size. The ratios between the observed and predicted settling velocities by Stokes' law were in a range of 1.00-1.50, and the fluid collection efficiency of the granules ranged from 0 to 0.19, indicating that their permeabilities were lower and that there was little advective flow through their interior. Experimental results also suggest that molecular diffusion appeared to play an important role in the mass transfer through the H(2)-producing granules.     

Physical and hydrodynamic properties of flocs produced during biological hydrogen production

Dense flocs readily form in continuous culture bioreactors used for hydrogen production, but the fractal and hydrodynamic properties of these flocs have not been previously analyzed. We therefore examined the size distribution, fractal dimension, and hydrodynamic properties of flocs formed in a continuous flow, well-mixed reactor treating synthetic wastewater at a fixed condition of a 4.5 h hydraulic detention time (23 degrees C, pH 5.5). The reactor was operated for a total of 3 months at three different organic loading rates (27, 53, and 80 g-COD/L-d) with influent glucose concentrations of 5, 10, and 15 g-COD/L. At all three loading rates the removal of glucose was nearly complete (98.6-99.4%) and biomass was produced in proportion to the organic loading rate (0.86 +/- 0.11, 2.40 +/- 0.26, and 4.59 +/- 1.55 g/L of MLVSS in the reactor). Overall conversion efficiencies of glucose to hydrogen, evaluated on the basis of a maximum of 4 mol-H2/mol-glucose, increased with organic loading rates in the order 17.7%, 23.1%, and 25.6%. The gas contained 56.1 +/- 4.9% hydrogen, with the balance as carbon dioxide. No methane gas was detected. Under these conditions, flocs were produced with mean sizes that increased with organic loading, in the order 0.12 cm (5 g-COD/L), 0.35 cm (10 g-COD/L), and 0.58 cm (15 g-COD/L). As the average floc size increased, the flocs became on average denser and less fractal, with fractal dimensions increasing from 2.11 +/- 0.17 to 2.48 +/- 0.13. Floc porosities ranged from 0.75-0.96, and resulted in aggregate densities that allowed little intra-aggregate flow through the floc. As a result, average settling velocities were not appreciably larger than those predicted by Stokes' law for spherical, impermeable flocs. Our results demonstrate that dense, relatively impermeable flocs are produced in biohydrogen reactors that have settling properties in reasonable agreement with Stokes' law.     

Microbial production of hydrogen and ethanol from glycerol-containing wastes discharged from a biodiesel fuel production plant in a bioelectrochemical reactor with thionine

H(2) and ethanol production from glycerol-containing wastes discharged from a biodiesel fuel production plant by Enterobacter aerogenes NBRC 12010 was demonstrated in bioelectrochemical cells. Thionine as an exogenous electron transfer mediator was reduced by E. aerogenes, and was re-oxidized by a working electrode applied at +0.2 V against a Ag/AgCl reference electrode by a potentiostat (electrode system). At the initial glycerol concentration of 110 mM, 92.9 mM glycerol was consumed in the electrode system with 2 mM thionine after 48 h. On the other hand, the concentration of glycerol consumed was only 50.3 mM under the control conditions without thionine and the electrodes (normal fermentation). There are no differences in the yields of H(2) and ethanol against glycerol consumed between the control conditions and the conditions with the electrode system. A pH of 6.0 was suitable for the H(2) production in the range between pH 6 and pH 7.5 in the electrode system. At pH values of 7.0 and 7.5, H(2) production decreased and formate was remarkably produced in the reaction solution. The rates of both glycerol consumption and the H(2) and ethanol production increased as the thionine concentration and the surface area of the working electrode increased. After 60 h, 154 mM of the initial 161 mM glycerol concentration in the wastes was consumed in the electrode system, which is a 2.6-fold increase compared to the control experiment. Biotechnol. Bioeng. 2007;98: 340-348. (c) 2007 Wiley Periodicals, Inc.