一株高温高效产氢细菌的分离及产氢特征研究

实验组采用非选择性培养、选择性培养、液体培养和固体培养一整套培养方法,结合平面厌氧操作技术与培养瓶厌氧技术,从混合产氢培养液中分离得到1株较好的产氢细菌。细菌需氧实验证明:此细菌为厌氧细菌;Gram实验和芽孢染色实验表明:此菌为革兰氏阴性杆菌,无芽孢。在不同的pH值、不同金属离子与温度下,于间歇式条件下测定其产氢量。实验结果表明:此菌株对pH值敏感,其最适产氢pH值为5.5,最适温度在45℃左右,在最适宜条件下产气量在1000 mL/(5 g葡萄糖),产气周期在15 d左右。金属离子影响实验表明:缺少铁和镁离子对此产气菌株的产气量有明显影响,此菌株对铜离子(0.1 g/L)无抗性。     

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。     

产气肠杆菌fhlA基因克隆、表达及重组菌株产氢量

【目的】本研究以产氢细菌产气肠杆菌Enterobacter aerogenes ATCC13408为研究对象,克隆甲酸-氢裂解酶(formate hydrogen lyase,FHL)系统的转录激活蛋白FHL activator(fhlA)基因,构建过表达重组菌株,以提高菌株产氢效率。【方法】利用简并引物和Genome walking技术,克隆fhlA的全长基因,将该基因连接到改造质粒pGEX-4T-2-Cat中,电击转化得到重组菌株,用厌氧发酵方法测定重组细菌的产氢量。【结果】E.aerogenes ATCC13408fhlA ORF全长2073bp,编码一个含690个氨基酸残基的蛋白(GenBank accessionGU188474)。SDS-PAGE和Western blot分析证明fhlA基因在重组菌中得到了融合表达。对重组后菌株的产氢量进行了测定,结果表明:底物产氢潜力由原来的1.23±0.08mol H2/mol葡萄糖提高到了1.48±0.04mol H2/mol葡萄糖,提高了20.36%。【结论】本研究首次克隆了E.aerogenes ATCC13408的fhlA基因,并将该基因在原菌中过量表达。重组后菌株的产氢量得到显著提高,为进一步研究和开发利用E.aerogenes ATCC13408的fhlA基因提供了基础。     

莱氏野村菌产几丁质酶条件及酶学性质研究

对莱氏野村菌(Nomuraea rileyi)菌株CQ031021产几丁质酶条件及酶学性质进行了研究。结果表明:该菌株最适产酶碳源为2.0%(W/V)葡萄糖,氮源为1.2%(W/V)复合氮源(蛋白胨、牛肉膏按1∶1的比例),接种量为孢悬液2mL(1×107个/mL),培养温度28℃,培养液初始pH6.0,培养时间6d;一定浓度的吐温-80对几丁质酶活性有促进作用,而SDS有抑制作用;粗酶液最适反应温度50℃,最适pH6.0,在40℃以下及pH5.5～6.5范围内酶活力较稳定。     

硫和pH对蛋白核小球藻光照产氢的影响

在有无硫及pH5.0-8.0下对蛋白核小球藻(Chlorella pyrenoidosa)光照产氢的影响进行了研究。结果表明,在持续光照(165μmolm-2s-1)条件下,从有硫培养液(TAP培养液)内叶绿素a含量、Fv/Fm值及ΦPSII值的变化表明蛋白核小球藻在pH6.0-7.0时生长最佳,生长旺盛易形成暂时的无氧环境而利于藻产氢。最高的产氢速率和总产氢量出现在pH7.0,分别是0.10mlmg-1chlh-1和1.39ml。从无硫培养液(TAP-S培养液)内叶绿素a含量、Fv/Fm值及ΦPSII值的变化表明蛋白核小球藻生长明显受抑制,形成的无氧环境持久,故产氢持久,总产氢量比有硫培养液内高。蛋白核小球藻在pH5.5培养液内的Fv/Fm值后期高于其他4种pH值的,表明潜在的PSII光化学效率高,在光照条件下产氢电子主要来源于PSII,故pH5.5的无硫培养液内藻的产氢速率和总产氢量最大,分别是0.58mlmg-1chlh-1和10.98ml。说明pH为5.5的无硫培养液是蛋白核小球藻产氢的最佳条件。     

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 elongatum Z7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. elongatum Z7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。     

初始pH值对产氢产乙酸/耗氢产乙酸两段耦合工艺定向生产乙酸的影响

以葡萄糖为底物,以经加热预处理并活化过的厌氧污泥为种泥,研究了初始pH值对产氢产乙酸/耗氢产乙酸两段耦合工艺厌氧发酵定向生产乙酸的影响。实验考察了7个初始pH值(5、6、7、8、9、10、11)条件下的底物降解、产物产生和发酵过程pH值的变化。结果表明:产氢产乙酸段初始pH值的变化不仅影响本阶段产酸,而且影响耗氢产乙酸段产酸。初始pH=5时主要进行乙醇型发酵;pH=6和7时主要进行丁酸型发酵;pH=8时混合酸型发酵类型逐渐占优势,pH=8~11时均以乙酸为主要产物,耦合系统生产乙酸最优初始pH值为10。在初始pH=8~11范围内,产氢产乙酸段初期的乙醇浓度一般较高,但到后期因乙醇被微生物进一步代谢转化成乙酸而使其含量下降。     

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.     

秸秆蓝藻混合发酵产沼气过程中微生物群落分析

采用实验室自制秸秆蓝藻混合厌氧反应装置进行沼气发酵实验,利用16S rRNA基因克隆文库的方法,对不同产气阶段的细菌和古菌的优势菌群进行多样性研究。结果表明:(1)不同产气阶段细菌优势种类存在差异,供试秸秆沼气反应器阶段细菌种类较为丰富,分属于6个门:产气初始阶段优势菌群为变形菌门(Proteobacteria),相对丰度为51.76%;产气高峰阶段优势菌群为厚壁菌门(Firmicutes),相对丰度为47.13%;产气结束阶段优势菌群为厚壁菌门(Firmicutes),相对丰度为28.89%;此外,还包括绿弯菌门、螺旋体门、绿菌门的细菌。(2)古菌种类明显少于细菌,均属于甲烷微菌纲(Methanomicrobia)、甲烷杆菌纲(Methanobacteria)和热原体纲(Thermoplasmata)。秸秆蓝藻沼气系统微生物群落结构的阐明具有一定的意义,可为秸秆沼气工程调控提供科学依据。     

正交实验优选光合细菌混合菌群产氢的最佳条件

对光合细菌混合菌群产氢影响因子进行了实验研究。通过单因素实验和正交实验, 系统考察了碳源、氮源、碳源浓度、氮源浓度、初始pH值、光照方式、接种量等因素对产氢量的影响, 实验得出最佳工艺条件为: 采用3号菌群, 碳源为葡萄糖, 碳源浓度为3 g/L, 氮源为尿素, 氮源浓度为9 g/L, 接种量为10%, pH值为8.5, 光照方式为12 h光照-12 h黑暗交替光照, 培养温度为30°C。菌种、碳源、碳源浓度、氮源是影响产氢量的重要因素。     

Optimization of key process variables for enhanced hydrogen production by Enterobacter aerogenes using statistical methods

The individual and mutual effects of glucose concentration, temperature and pH on the hydrogen production by Enterobacter aerogenes were investigated in a batch system. A Box-Behnken design and response surface methodology (RSM) were employed to determine the optimum condition for enhanced hydrogen production. The hydrogen production rate was investigated by simultaneously changing the three independent variables, which all had significant influences on the hydrogen production rate. The maximum hydrogen production rate of 425.8 ml H(2)(g dry cell h)(-1) was obtained under the optimum condition of glucose concentration 118.06 mM, temperature 38 degrees C and pH 6.13. The experimental results showed that the RSM with the Box-Behnken design was a useful tool for achieving high rate of hydrogen production by E. aerogenes.     

Hydrogen production of a salt tolerant strain Bacillus sp. B2 from marine intertidal sludge

To isolate a salt tolerant hydrogen-producing bacterium, we used the sludge from the intertidal zone of a bathing beach in Tianjin as inoculum to enrich hydrogen-producing bacteria. The sludge was treated by heat-shock pretreatment with three different temperature (80, 100 and 121°C) respectively. A hydrogen-producing bacterium was isolated from the sludge pretreated at 80°C by sandwich plate technique and identified using microscopic examination and 16S rDNA gene sequence analysis. The isolated bacterium was named as Bacillus sp. B2. The present study examined the hydrogen-producing ability of Bacillus sp. B2. The strain was able to produce hydrogen over a wide range of initial pH from 5.0 to 10.0, with an optimum at pH 7.0. The level of hydrogen production was also affected by the salt concentration. Strain B2 has unique capability to adapt high salt concentration. It could produce hydrogen at the salt concentration from 4 to 60‰. The maximum of hydrogen-producing yield of strain B2 was 1.65 ± 0.04 mol H₂/mol glucose (mean ± SE) at an initial pH value of 7.0 in marine culture conditions. Hydrogen production under fresh culture conditions reached a higher level than that in marine ones. As a result, it is likely that Bacillus sp. B2 could be applied to biohydrogen production using both marine and fresh organic waste.     

Selection of a Photosynthetic Bacterium Suitable for Hydrogen Production in Outdoor Cultures among Strains Isolated in the Seoul,Taegu, Sendai and Bangkok Areas

Very efficient hydrogen producing photosynthetic bacteria, strains SL1, SL3, SL16 and TG28 newly isolated in Korea, and strain KM113 newly isolated in the Sendai area, were found to be Rhodopseudomonas spp. To examine the stability of cell suspensions of the cultures for hydrogen production, which is closely associated with light absorption, we conducted larger scale cultures under periodic illumination (12-hr intervals) without stirring at 30°C using strains SL1 and Rhodopseudomonas sphaeroides B5, the latter was isolated in the Bangkok area. Both strains gave homogeneous cell suspensions throughout the incubation period and larger amounts of hydrogen were produced in a shorter period of time by both cultures than obtained with Rhodopseudomonas sp. TN3, an isolate from the Sendai area which was reported previously. With the cells of the new isolates and strains TN3 and B5 grown on glutamate-malate medium at 30°C, we measured hydrogen production at 20, 30 and 40°C in the same medium. Among them, strains SL1, SL16 and KM113 showed the highest hydrogen production activity at 30°C. The maximum hydrogen production rates with these strains were over 130 µ1/hr/mg dry cells, but at 40°C, the highest activity (138 µl/hr/mg dry cells) was obtained with strain B5. Since strain B5 also showed good activities at 20 and 30°C, we suggest that this strain might be suitable for hydrogen production in outdoor cultures.     

Metabolism of 1-naphthyl-N-methyl carbamate (carbaryl) by bacterial isolates from honey bees and the effect of bacterial inoculations on carbaryl tolerance in bees

I.D. SHARMA AND A. NATH. 1996. The bacteria which inhabit the body of foragers of Apis cerana indica F. were evaluated for degradation of carbaryl (1-naphthyl- N -methyl carbamate). Citrobacter sp., Enterobacter aerogenes and an unidentified isolate could utilize carbaryl as sole carbon and energy source. The enrichment cultures of bacteria could degrade carbaryl 0.019–0.082 in nutrient broth and 0.111–7.895 mg μg^-1 bacterial dry wt in basal salts medium of 1 mmol 1^-1 carbaryl. 1-Naphthol was the major product of carbaryl degradation. The dip and injection inoculations of foragers ( Apis cerana indica F. and Apis mellifera L.) with these bacteria resulted in a significant increase in tolerance to carbaryl. The application of mixed culture to bees induced higher tolerance than the individual isolates. This is the first report of the use of bacteria to decontaminate a living system.     

木糖发酵产氢菌的筛选及其生长产氢特性研究

利用改进的Hungate厌氧技术, 从牛粪堆肥中分离出一株能有效利用木糖发酵产氢的中温菌HR-1。通过16S rRNA系统发育树分析表明, 菌株 HR-1 与丙酮丁醇梭菌Clostridium acetobutylicum ATCC 824 相似性最高为96%, 结合生理生化和生长特性分析表明, HR-1是梭菌属Clostridium的一个新种, 命名为Clostridium sp. HR-1。菌株HR-1为单胞生长的规则杆状菌(0.3 mm ~0.6 mm)×(1.4 mm~2.3 mm), 革兰氏染色为阴性, 无荚膜、无鞭毛、表面光滑、无明显凸起, 专性厌氧菌。HR-1可在10°C~45°C, pH 4.0~10.0条件下生长; 37°C和pH 8.0分别为其最适生长条件。发酵PYG的主要发酵产物有氢气、二氧化碳、乙酸、丁酸及少量乙醇。HR-1可以利用有机氮源和无机氮源生长并产氢, 酵母提取物是其最佳产氢氮源。HR-1在木糖浓度为3 g/L和初始pH 6.5条件下, 其比产氢量为1.84 mol-H2/mol-木糖, 最大比产氢速率为10.52 mmol H2/h·g-细胞干重。HR-1可以亦利用葡萄糖、半乳糖、纤维二糖、甘露糖和果糖等碳源生长并发酵产氢, 发酵葡萄糖时比产氢量为2.36 mol-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。     

Acetate as a carbon source for hydrogen production by photosynthetic bacteria

Hydrogen is a clean energy alternative to fossil fuels. Photosynthetic bacteria produce hydrogen from organic compounds by an anaerobic light-dependent electron transfer process. In the present study hydrogen production by three photosynthetic bacterial strains (Rhodopseudomonas sp., Rhodopseudomonas palustris and a non-identified strain), from four different short-chain organic acids (lactate, malate, acetate and butyrate) was investigated. The effect of light intensity on hydrogen production was also studied by supplying two different light intensities, using acetate as the electron donor. Hydrogen production rates and light efficiencies were compared. Rhodopseudomonas sp. produced the highest volume of H2. This strain reached a maximum H2 production rate of 25 ml H2 l(-1) h(-1), under a light intensity of 680 micromol photons m(-2) s(-1), and a maximum light efficiency of 6.2% under a light intensity of 43 micromol photons m(-2) s(-1). Furthermore, a decrease in acetate concentration from 22 to 11 mM resulted in a decrease in the hydrogen evolved from 214 to 27 ml H2 per vessel.     

Use of carbon and energy balances in the study of the anaerobic metabolism of Enterobacter aerogenes at variable starting glucose concentrations

The anaerobic metabolism of Enterobacter aerogenes was studied in batch culture at increasing initial glucose levels (9.0< S(o) <72 g l(-1)). The ultimate concentrations of fermentation products were utilized to check a metabolic flux analysis based on simple carbon mass and energy balances that promise to be suitable for the study of different fermentation processes, either under aerobic or anaerobic conditions. The stoichiometric coefficients of products collected at increasing starting glucose concentrations under anaerobic conditions suggest: (a) little influence of starting glucose level on the formation of the main fermentation products (2,3-butanediol and ethanol); (b) possible inhibition of 2,3-butanediol and lactate formations by increased ethanol concentration; (c) consequent increase in carbon flux through the remaining metabolic pathways with increased molar productions of succinate, acetate and hydrogen; (d) relative constancy of the molar production of ATP and CO(2).     

Isolation and Identification of Camptothecin from Cells of Camptotheca acuminata Suspension Cultures

Ten strains of non-sulfur purple photosynthetic bacteria were isolated from soil and water samples gathered in Bangkok and its surrounding area. The isolated strains from Thailand were divided into two groups, Al to A4 and BI to B6. They were identified as Rhodopseudomonas gelatinosa and Rhodopseudomonas sphaeroides, respectively. All strains grew well either at 30°C or 40°C, but failed to grow at 45°C. Strains belonging to group A had weak activities of nitrogenase (acetylene reduction) and hydrogen production, while strains of group B showed much higher activities than group A. The activities of nitrogenase and hydrogen production of isolates in Thailand were compared with those of isolates in Japan. The activities of isolated strains in Thailand at 40°C were almost equal to those at 30°C or even higher. On the other hand, both hydrogen production and the nitrogenase activity of isolates in Japan decreased significantly at 40°C as compared to the activities at 30°C. These results suggest an intrinsic thermostability in hydrogen production by the non-sulfur purple photosynthetic bacteria of Thailand. Among isolated strains in Thailand, strain B5 was the most active in nitrogenase and hydrogen production, and its activity was significantly higher than strain TN3 at 40°C. TN3 had been selected as the most active strain among isolates in the Sendai area.