一株分离自潮间带污泥细菌Clostridium sp. T7产氢特性分析

产氢菌株Clostridium sp. T7分离自天津海水浴场潮间带的污泥.研究起始pH、碳源、氮源、NaCl质量分数对菌株T7产氢性质的影响.结果表明:菌株T7最适产氢的起始pH是6.0,能够利用蔗糖、葡萄糖和果糖等碳源发酵产氢.菌株T7能够利用牛肉膏和酵母粉为单一氮源产氢,不能利用蛋白胨为氮源进行产氢.NaCl质量分数能影响菌株T7的产氢量,海水培养条件(NaCl质量分数为3%)下,最高产氢量是每摩尔葡萄糖(1.48,±0.05)mol,相比之下,淡水培养条件下其产氢量提高20%.NaCl质量分数在0.4%～7%时,菌株T7都能够产氢,这表明菌株T7有望应用于淡水或高盐有机废水产氢领域.     

马铃薯发酵产氢的菌株培养基改良研究

为了促进马铃薯发酵产氢,研制了改良培养基驯化产氢菌株并使用淀粉生物酶降解马铃薯.将活性污泥煮沸后作为产氢菌株的接种体富集培养,使用Cl-代替SO42-改良传统培养基消除了传统发酵产氢的启动滞留期(约12h),单位最大产氢速率从703.4mL/(g·d)提高到800.5mL/(g·d),最大产氢潜力从205.2mL/g增加到218.2mL/g.通过a淀粉酶和糖化酶处理进一步提高了马铃薯的发酵产氢能力,单位最大产氢速率进一步提高到944.7mL/(g·d),最大产氢潜力进一步提高到267.2mL/g.生物气中氢气的体积分数为43%～69%,无甲烷.     

Klebsiella oxytoca HP1乙醇脱氢酶基因敲除对产氢的影响

Klebsiella oxytcoHP1是从温泉筛选得到的很有潜力的产氢菌株。为了进一步提高产氢能力，应用同源重组技术构建了乙醇途径缺少菌株，并分析和比较了野生菌和工程菌的代谢流量分布。     

有机废水厌氧发酵产氢技术的现状与发展

采用厌氧发酵产氢技术处理有机废水产生氢气,并且实现工业化生产的研究在国内外受到广泛关注。文章归纳分析了国内外的厌氧发酵生物产氢原理、厌氧发酵产氢途径及厌氧发酵产氢影响因素,如产氢菌株、基质产氢潜能和外部环境等因素,并总结厌氧产氢数学模式,提出研究与应用中存在的主要问题,最后对该技术的应用发展前景进行了探讨。     

产氢菌株Clostridium sp.T7的快速筛选

取自潮间带的污泥分别在不同温度下(80、100、121,℃)进行热休克预处理,富集产氢菌群并测定其产氢量,利用变性梯度凝胶电泳(DGGE)分析混合菌群组成.结果表明:3种热处理条件下混合菌群的产氢量都要高于对照未处理菌群.DGGE图谱表明,与80、100,℃热休克处理混合菌群相比,经121,℃热休克处理富集的混合菌群,其电泳条带最少,测序结果发现该混合菌群中包括产氢菌Clostridium sp..从该混合菌群中纯化并鉴定了1株产氢菌株Clostridiumsp.T7(登录号HM104461).培养温度对菌株T7产氢有一定影响,温度在25～55,℃范围内菌株Clostridium sp.T7都能产氢,最适产氢温度是35,℃.     

红树林沉积物中产氢微生物的分离鉴定和性能分析

采用厌氧富集三层平板和螺口试管培养法,根据产气特征从红树林沉积物中筛选分离海水条件下的高效产氢菌株,获得1株兼性厌氧具有较高产氢能力的菌株BH-16.通过形态观察、Biolog鉴定和16S rDNA序列分析,该菌株鉴定为摩氏摩根氏菌BH-16.实验表明:在厌氧培养条件下,菌株BH-16的最适起始pH、NaCl质量浓度和培养温度分别为8.0、0.01 g/mL、37,℃.通过间歇产气实验和气相色谱分析对菌株的产氢能力进行分析,结果表明:菌株BH-16大量产氢发生在细胞指数生长后期和细胞生长平稳期;在海水培养条件下,在起始葡萄糖质量浓度为20 g/L、起始pH为7.2时,菌株的总产氢量和平均产氢速率分别为1,120 mL/L和93.3 mL/(L·h).     

丁酸梭菌LMG1213、A69及巴氏梭菌LMG3285~T在不同碳源上生长产氢的研究

丁酸梭菌和巴氏梭菌是两种产氢能力很强的细菌。本文研究了Clostridium butyricum LMG1213、A69及Clostridium pasteurianum LMG3285~T在不同碳源上的生长及产氢。不同的菌株对碳源的利用差异很大,但葡萄糖做碳源时都能很好的生长产氢。     

胶质红假单胞菌J2菌株光合产氢的研究

选择苹果酸为碳源，谷物酸为氮源，对胶质红假单胞菌Ｊ２菌株进行光照分批及连续培养产氢试验，分批试验最大产氢活性为每克干细胞每小时产氢１３．３８ｍｌ。连续试验持续１５天以上，平均产氢速率为每克干细胞每小时产氢１２．０１ｍｌ。此后进行了固定化细胞光合产氢的研究，选择角叉菜聚糖作为固定化载体，结果表明，固定化细胞在产氢活性的保持和反应稳定性等方面明显优于游离细胞，连续培养时，平均产氢速率为每克干细胞每小时     

高产氢耐热菌株的分离鉴定及其放氢特性

从高温环境中分离得到具有高产氢活力的微生物菌株,经16SrRNA基因序列分析及生理生化特性分析,鉴定该菌株为Proteussp.该菌株能以葡萄糖、蔗糖、麦芽糖、乳糖和淀粉等多种有机物为底物催化产氢.该菌株利用葡萄糖产氢的最适条件为:葡萄糖浓度0.1mol/L;菌体细胞密度OD600=0.60;反应系统起始pH值7.0;反应温度40℃.在该条件下菌株的最高产氢活性可达8.05μmolH2/h·mgprot.     

高效选育产氢光合细菌的研究

降解有机废水转化太阳能为氢能是一条理想制氢途径 .为使选育的菌株实用性更强 ,本文选用有机废水主要降解产物—乙酸为唯一氢供体 ,在自然生态环境条件下 ,利用紫色非硫细菌培养基 ,紫色硫细菌培养基和绿硫细菌培养基 ,从不同的水域环境中进行了产氢光合细菌的筛选 .从影响太阳能转化效率主要因素出发 ,本文对分离纯化的 1 5株光合细菌进行形态学特征研究基础上 ,着重进行了最适生长温度、光合色素成分、利用硫化物能力和耐盐能力的测定 ,并将其分为 4个类群 .在对每组细菌初筛基础上 ,本文对选育出的 8个菌株进行了生长动力学测定 ,其中Z ,SP2 ,R3,Y7菌株能利用乙酸快速生长 .对其产氢动力学研究表明 ,这些菌株均具有光合放氢活性 ,其中Z菌株产氢得率最高 .在含有 2 0mmol·L-1 乙酸钠和 5mmol·L-1 谷氨酸钠的培养基中 ,其产氢得率为 3 0 8.9ml·g-1 .这 4个菌株生理生态特性完全不同 ,且能利用乙酸光合放氢 ,在有机废水光合制氢技术中具有重要的潜在应用价值 .     

产酸克雷伯氏菌氢酶分离提纯与特性的初步研究

采用硫酸铵沉淀和柱层析 (DEAE-Sepharose F.F., Sephacryl S-200, TSK-DEAE)初步分离纯化了产酸克雷伯氏菌(Klebsiella oxytoca)HP1可溶性氢酶,研究了温度、pH值、电子载体等对氢酶催化放氢活性的影响.研究表明,可溶性氢酶催化放氢的最适温度为30℃,催化放氢的最适pH值为7.5,甲基紫晶(MV)是氢酶催化放氢的最适人工电子载体,氧对氢酶催化活性有较大的抑制作用.     

Enhanced hydrogen production by insertional inactivation of adhE gene in Klebsiella oxytoca HP1

Ethanol is the main byproduct of anaerobic H2-producing fermentation in Klebsiella oxytoca HP1. Two moles of NAD(P)H are consumed to yield one mole of ethanol that may decrease bacterial hydrogen production. In this article the adhE gene that codes for acetaldehyde dehydrogenase was disrupted for the first time. A homologous recombination vector pTA-Str was constructed in which the adhE gene was disrupted by inserting an aminoglycoside-3'-adenyltransferase (aadA) gene. As expected, the vector includes the insertion 5′-adhE-aadA-adhE-3′. The amplified DNA fragment 5′-adhE-aadA-adhE-3′ from pTA-Str was transformed into K. oxytoca HP1 and one recombinant was obtained. PCR analysis of the resulting genomic DNA indicated the appropriate deletion and insertion. Compared with the H2-production of wild type K. oxytoca HP1, the hydrogen yield of the mutant increased by 16.07% and ethanol concentration decreased by 77.47%, suggesting that inactivation of the adhE gene in K. oxy- toca HP1 is a potential method for enhancing bacterial H2-production.     

Enhanced hydrosen production by insertional inactivation of adhE gene in Klebsiella oxytoca HP1

Ethanol is the main byproduct of anaerobic H2-producing fermentation in Klebsiella oxytoca HP1. Two moles of NAD（P）H are consumed to yield one mole of ethanol that may decrease bacterial hydrogen production. In this article the adhE gene that codes for acetaldehyde dehydrogenase was disrupted for the first time. A homologous recombination vector pTA-Str was constructed in which the adhE gene was disrupted by inserting an aminoglycoside-3＇-adenyltransferase （aadA） gene. As expected, the vector includes the insertion 5＇-adhE-aadA-adhE-3＇. The amplified DNA fragment 5＇-adhE-aadA-adhE-3＇ from pTA-Str was transformed into K. oxytoca HP1 and one recombinant was obtained. PCR analysis of the resulting genomic DNA indicated the appropriate deletion and insertion. Compared with the H2-production of wild type K. oxytoca HP1, the hydrogen yield of the mutant increased by 16.07% and ethanol concentration decreased by 77.47%, suggesting that inactivation of the adhE gene in K. oxytoca HP1 is a potential method for enhancing bacterial H2-production.     

利用克雷伯氏菌进行同步糖化发酵产氢的研究

氢作为一种清洁的能源引起了人们的普遍重视.实验以产酸克雷伯氏菌(Klebsiella oxytoca)HP1为产氢菌株,以稻草粉为产氢底物,进行同步糖化发酵(Simultaneous Saccharification and Fermentation,SSF)产氢.对影响同步糖化发酵产氢的单因子进行试验,选取对氢产率影响较大的因子:温度、pH、纤维素酶用量等进行L9(34)正交试验.结果表明同步糖化发酵产氢的最佳条件为:温度40℃,pH6.5,纤维素酶用量为20FPAU/g稻草粉,摇床转速100r/min,发酵时间42h.在该条件下的最大氢产率为110.6mL/g稻草粉,稻草粉的氢转化率为22%.进行了10L放大发酵产氢试验,最大氢产率为122.3mL/g稻草粉,氢转化率为24.3%.与分步糖化发酵(Separate Hydrolysis and Fermentation,SHF)产氢相比,氢产率提高34.4%.研究表明,利用同步糖化发酵工艺可以提高生物制氢的产量和得率.     

一株苯酚降解产酸克雷伯菌的分离与鉴定

从活性污泥中分离出一降解苯酚菌株，经16s rRNA基因测序确定属于产酸克雷伯菌。研究了产酸克雷伯菌降解苯酚动力学行为，显示出该菌株具有显著的降解苯酚能力。     

通气提高Klebsiella oxytoca HP1发酵产氢

研究了Klebsiella oxytoca HP1在不同通气条件下的生长和放氢特性．批式发酵实验结果表明：通氩气放氢效果最好，通二氧化碳放氢效果最差．提高氩气通气速率能显著提高产氢量。0．25L／Lmin条件下的产氢量达到1155mL是不通气时的2．41倍，但通气也影响延迟期的长短。氩气通量应该与微生物生长阶段相匹配．代谢产物分析表明：K．oxytoca HP1呈混合酸发酵特性，主要代谢产物有乙酸、乙醇、乳酸和2，3-丁二醇，氩气通气速率明显影响丙酮酸节点的代谢途径和通量，从高通气条件到不通气条件，代谢主要途径按乙酸途径→乙醇途径→乳酸途径的次序转变，通气速率是K．oxytoca HP1发酵产氢的重要调控因子．     

产酸克雷伯氏菌的吸附固定及其产氢研究

利用曲霉(Aspergillussp.XF101)所形成的菌丝球吸附产氢细菌产酸克雷伯氏菌(Klebsiella oxytocaHP1),当曲霉培养时间为50 h,菌丝球直径为1.3~1.8 mm,菌悬液pH值为5.0~6.0,吸附温度为30℃,吸附时间为1.5 h时,可获得最佳吸附效果,吸附率可达99%以上.利用菌丝球固定产氢菌可进行连续产氢,其最大产氢速率为18 mmol/L.h,平均产氢速率为1.7 mmol/L.h,持续产氢时间为15 d.     

Construction and characterization of double mutants in nitrogenase of<Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Two mutants in nitrogenase of Klebsiella pneumoniae are constructed by site-directed mutagenesis and gene replacement procedure, which express the nitrogenases with Lysine and Glutamine substituting for α-Glutamine 190 and α-Histidine 194 respectively (Kp-Q α190 K and Kp-Hα194 Q). The above two substitutions are respectively introduced into a nifV mutant (expressing a citrate-containing nitrogenase) and sequentially two double mutants are obtained (Kp-Q α190 K-nifV and Kp-H α194 Q-nifV). All four mutants exhibit strict Nif phenotype under the N2-fixation condition and fail to grow diazotrophically. Altered nitrogneases are effectively depressed and the C2H2 reduction analysis shows that the double substitutions in Kp-Q α190 K-nifV abolish cell C2H2 reduction activity, but Kp-H α194 Q-nifV cells maintain a C2H2 reduction activity at 10% of that of wild type. Whole cell C2D2 reduction by all four mutants in comparison to the wild type and nifV mutant is also detected. The results show that only single α-Gln^194 substitution does not perturb the stereospecificity of protonation of C2D2. These results indicate that the α-Glutamine 190 and its combination with homocitrate are essential to the catalytic activity of nitrogenase and it is proposed that α-Glutamine 190 and its combination with homocitrate are involved in the proton and/or electron transfer to FeMoco. The nitrogenases from these double mutants will be useful in further analysis of the entry of the proton and/or electron to FeMoco and the substrate binding sites.