厌氧复合菌系的筛选及含纤维素原料预处理复合菌剂的研究

本文从不同厌氧生境中获得7组(C-2、Y-2、L-2 、NZ、H-3、CZ、L-3)具有纤维素降解能力的复合菌系。经过不断传代、淘汰纤维素降解能力降低的菌系，最后得到一组高效、传代稳定的厌氧纤维素分解复合菌系L-3。该菌系可使滤纸在42 h内溃烂，并能在分解纤维素的同时产氢气。对L-3复合菌系的产酶条件进行了研究，结果表明，在实验范围内该菌系的产酶最适条件为：pH 6.5，温度37 ℃，接种量5 %，最佳碳源为滤纸，最佳氮源为硫酸铵。第10天测得羧甲基纤维素酶(CMCase)、滤纸酶(FPA)、外切葡聚糖酶(C1)、β-葡聚糖苷酶(β-glucodase)的酶活分别为0.216 U/ml、0.101 U/ml、0.132 U/ml、0.002 U/ml，滤纸失重率70.6 %。发酵代谢产物乙醇和丁酸含量分别可达1378 mg/L 、2695 mg/L，发酵产生的气体中氢气含量最高可达70.2 %。DGGE结果表明该菌系主要由14种菌组成，其中有三株菌在发酵前后菌数发生了明显的变化，说明在以滤纸为底物的降解过程中，这三株菌起到了重要作用，对这三株菌进行了分子生物学鉴定，初步定为Clostridium phytofermentans、Clostridium cellulovorans、Desulfovibrio sp。 利用实验室分离得到的纤维素降解菌，最终配制出由10、X-1、X-13、ST-13、L-3组成的好氧-厌氧纤维素降解复合菌剂。以秸秆为发酵底物，菌剂接种量1%，利用复合菌剂预处理后的秸秆，发酵总产气量相对于对照提高了71.62%，甲烷含量最高可达70.08%。 A group of microbial consortia L-3 was isolated from the anaerobic fermentation residue of corn stalk, which could degrade cellulose and produce hydrogen. The CMCase, FPA, C1 and β-glucosidase activity of L-3 could reach to 0.216 U/ml, 0.101 U/ml, 0.132 U/ml and 0.002 U/ml, respectively. In the filter degrading process, the filter paper collapsed in the liquid culture within 42 h and the filter degrading rate could reach to 70.6% in the 13 days, meanwhile, hydrogen was determined and the highest hydrogen content was 70.2%. The optimum cellulase-degrading conditions were filter papaer as the carbon source, (NH4)2SO4 as the nitrogen source, 37 ℃ and pH 6.5 in this experiment. DGGE results showed that the microbial consortia L-3 mainly included 14 strains. The amount of 3 strains were changed during the fermentation. These strains were identified as Clostridium phytofermentans、Clostridium cellulovorans、Desulfovibrio sp by 16S rDNA sequence analysis. The cellulose- degrading microbial agent was composed by 10, X-1, X-13, ST-13, L-3 which were isolated in the laboratory. The straw pretreated by cellulose-degrading microbial agent was used to ferment, the total biogas production increased by 72% comparing to the control. The content of methane could reach to 70.08%。

厌氧产氢菌的分离筛选、诱变选育及其产氢特性的研究

本文从四川绵竹酒厂、成都市龙泉长安垃圾填埋场以及四川大学荷花池底的厌氧污泥中先后分离得到63株厌氧产氢菌，其中H-8、H-61、HC-10等16株产氢细菌产氢能力较高，HC-10的产氢能力最高，最大产氢量和最大产氢速率分别达到2840 ml H2/L培养基和25.39 mmol H2/g drycell·h，对HC-10进行生理生化鉴定和分子生物学鉴定，判定其为clostridium sp.，对HC-10的产氢条件进行了研究，结果表明，该菌的最适生长温度为35 ℃，最适生长初始pH为7，以葡萄糖为最佳碳源，以蛋白胨为最佳氮源，不利用无机氮源，其产氢发酵液相产物以乙醇和乙酸为主，其发酵类型属于乙醇型发酵。此外，以酒糟废液作为底物，进行了菌株HC-10的生物强化试验，研究表明，投加了HC-10的强化系统其产氢量比对照高出40.32%。 同时为了获得厌氧产氢菌的高效突变株，分别以产氢菌H-8和H-61为原始菌株进行微波诱变处理，对微波诱变参数进行了优化，考察了突变株的遗传稳定性、产氢特性及耐酸性。菌株H-8经过微波诱变得到5株高产氢突变株HW7、HW33、HW181、HW184、HW195，经多次传代表明HW195是稳定的高产突变株。突变株HW195具有较好的耐酸性，在pH值为2.8时仍能生长。通过间歇发酵实验，其最大产氢量和最大产氢速率分别达到2460 mL/L培养基和27.97 mmol H2/g drycell·h，比原始菌分别提高了50.75%和41.7%。菌株H-61经过微波诱变后选育得到的突变株HW-18，其最大产氢量和最大产氢速率分别达到2190 mL/L培养基和25.86 mmol H2/g drycell·h，比原始菌分别提高了23.03%和31.00%。 为了对比各种诱变方式对产氢菌产氢能力的影响，以厌氧产氢菌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培养基，比原始菌株提高了69.89%；其最大产氢速率为33.19 mmol H2/g drycell·h，比原始菌株提高了68.14%。经过多次传代实验，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。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。 From anaerobic activated sludge, 16 strains of hydrogen producing bacteria were newly isolated. One of them named as HC-10 had the highest hydrogen producing capability, under the batch fermentative hydrogen production condition, the maximal hydrogen yield and hydrogen production rate was 2840 mL/L culture and 25.39 mmol H2/g drycell·h. It was identified as clostridium sp.HC-10 by 16S rDNA sequence analysis. Various parameters for hydrogen production, including substrates, initial pH and temperature, have been studied. The optimum condition for hydrogen producing of strain HC-10 were achieved as: initial pH 7.0, temperature 35 ℃, glucose as the favorite substrate, Moreover, using distiller's solubles wastewater as substrate, HC-10 strain was added in the biohydrogen producing system to research the bioaugmentation effection. The results showed that the hydrogen production of bioaugmentation system was 40.32% higher than the noaugmentation system. An anaerobic, hydrogen producing strain H-8 was irradiated by microwave to optimize the microwave mutagenesis condition, and to test the heredity, hydrogen-producing potential and aciduric of the mutants. An aciduric mutant named as HW195 with steady hydrogen-producing capability was obtained, which can grow at pH 2.8. Its capability of hydrogen production was tested in the batch culture experiments. The maximum hydrogen yield and hydrogen production rate was 2460 mL/L culture and 29.97 mmol H2/g drycell·h, which was 50.7% and 41.7% higher than those of the initial strain, respectively. When used the strain H-61 as original strain, a mutant named as HW18 was obtained. The maximum hydrogen yield and hydrogen production rate was 2190 mL/L culture and 25.86 mmol H2/g drycell·h, which was 23.03% and 31.00% higher than those of the initial strain, respectively. The results demonstrated that microwave mutagenesis could be used in the field of hydrogen producing microorganism. The hydrogen producing strain H-61 was used as an original strain which was induced by NTG and UV for increasing and the hydrogen production capability. One of the highest efficient H2-producing mutants was named as HCM-23 with its stable hydrogen production capability. which was tested in the batch culture experiments. With the condition of 10 g/L glucose, its cumulative hydrogen yield and hydrogen production rate was 3024 mL/L culture and 33.19 mmol H2/g drycell·h, 69.89％and 68.14％ higher than that of the original strain, respectively. The terminal liquid product compositions showed that the mutant HCM-23 fermentation was ethanol type, while the original strain H-61 fermentation was butyric acid type. Varieties of parameters of hydrogen production fermentation were studied, including time, carbon source, nitrogen source, glucose concentration, glucose utilization, initial pH and incubation temperature had been studied, indicated the optimum condition of hydrogen production for the mutant HCM-23 as initial pH6.5, temperature 36 ℃, and the favorite substrate was sucrose. The hydrogen production characters of the mutant and the original strain were different, such as, the growth lag phase and the utilization of inorganic nitrogen source, etc. This work shows a good application potential of NTG-UV combined mutation in the biohydrogen production. And the hydrogen production mechanism and metabolic pathway should be explored furthermore.

三株嗜热厌氧纤维素分解菌的分离纯化和鉴定

从成都市洪河垃圾场采样分离到三株嗜热纤维素分解细菌，均为严格厌氧的革兰氏阴性杆菌，有较高的纤维素降解活性，能直接转化为纤维素产酒精。其中TCl菌株为周生鞭毛运动，不产生芽孢，大小为0.9×2.4～5.4um,表明菌落为白色，边缘整齐的圆形，最适温度在55℃，最适PH大约7.0，发酵纤维素或纤维而糖产生乙酸、丁酸。该菌属于拟杆菌料(Bacteroidaceae)，不能归入已有的任何一属，很可能是一个新的属。TC3和TC4为单端丛生鞭毛运动，大小为0.6×2.4～15um，形成卵圆形或圆形的端生芽孢，使菌体膨大。TC3的菌落为灰白色、透镜状，TC4的菌落为黄色，透镜状，TC4在液体培养基中产生非水溶性黄色素。最适温度均为55℃，最适PH值TC3为8.0，TC4为7.5。TC3、TC4属于梭菌属，与所有已报道的同类菌株均有显著差异，因此认为它们可能是梭菌属 (clostridium)中的新种。

砷还原菌群对砷的还原作用及菌群的多样性分析

从砷污染土壤中富集砷抗性细菌,在厌氧环境中进行培养,观察其对砷的还原能力。结果表明:在21h之内,As(V)就被完全还原为As(Ⅲ);培养72h后,培养基中出现黄色沉淀,采用X射线衍射分析(XRD)和扫描电镜-能谱分析(SEM-EDS)技术对沉淀进行分析表明,沉淀主要是以3种晶型存在的硫化砷(AsS);培养150h后,大约有65%的As以上述沉淀的方式从溶液中移除。此外,本文还采用了构建16SrDNA文库的方式对该体系中的微生物种群进行分析,利用RFLP技术对16SrDNA片段进行分型,共得到72个操作单元类型(OTU),其中6个OTU占了库容的51%,从这6个OTU中各选取1个克隆进行测序,结果表明,富集到的砷还原细菌属于喜热菌属(Caloramator)、梭菌属(Clostridium)和杆菌属(Bacillus)。

A new dinuclear molybdenum(V)-sulfur complex containing citrate ligand: synthesis and characterization of K2.5Na2NH4[Mo2O2S2(cit)(2)].5H(2)O

The complex, K2.5Na2NH4[Mo2O2S2(cit)(2)]. 5H(2)O (1), was obtained by crystallization from a solution of (NH4)(2)MoS4, potassium citrate (K(3)cit) and hydroxyl sodium in methanol and water under an atmosphere of pure nitrogen at ambient temperature. The crystals are triclinic, space group , a = 7.376 (3)Angstrom, b = 14.620 (2) Angstrom, c = 14.661 (1) Angstrom, alpha = 71.10 (1)degrees, beta = 81.77 (1)degrees, gamma = 78.27(2)degrees, R = 0.0584 for 2545 observed (I > 2 sigma (I)) reflections. Single crystal structure analysis reveals that citrate ligand coordinated to molybdenum atom through two carboxylato oxygens and one deprotonated hydroxyl oxygen together with two bridging sulfur atoms and a terminal oxygen atom completes distorted coordination octahedron around each molybdenum atom. Principal dimensions are: Mo = O-t, 1.707 Angstrom (av); Mo-S-b, 2.341 Angstrom (av); Mo-O-(hydroxyl), 2.021 Angstrom (av); Mo-O(alpha-carboxyl), 2.1290 Angstrom (av) and Mo-O(beta-carboxyl), 2.268(av) Angstrom. IR spectrum is in agreement with the structure.

Synthesis and characterization of a new double-cubane Mo-Fe-S cluster compound, [NEt(4)](3)[Mo2Fe6S8(mu-OMe)(3)(SPh)(3)Cl-3]

The new double-cubane cluster compound [NEt(4)](3)[Mo2Fe6S8(mu-OMe)(3)(SPh)(3)Cl-3] is synthesized from (NH4)(2)MoS4, FeCl3, Fe powder, S powder, NaSPh and NEt(4)Br in MeOH-DMF, its crystal structure is determined by X-ray crystallography, and results of XPS indicate a valence state of +4 for Mo.

Enrichment of the hydrogen-producing microbial community from marine intertidal sludge by different pretreatment methods

To determine the effects of pretreatment on hydrogen production and the hydrogen-producing microbial community, we treated the sludge from the intertidal zone of a bathing beach in Tianjin with four different pretreatment methods, including acid treatment, heat-shock, base treatment as well as freezing and thawing. The results showed that acid pretreatment significantly promoted the hydrogen production by sludge and provided the highest efficiency of hydrogen production among the four methods. The efficiency of the hydrogen production of the acid-pretreated sludge was 0.86 +/- 0.07 mol H-2/mol glucose (mean +/- S.E.), whereas that of the sludge treated with heat-shock, freezing and thawing, base method and control was 0.41 +/- 0.03 mol H-2/mol glucose, 0.17 +/- 0.01 mol H-2/mol glucose, 0.11 +/- 0.01 mol H-2/mol glucose and 0.20 +/- 0.04 mol H-2/mol glucose, respectively. The result of denaturing gradient gel electrophoresis (DGGE) showed that pretreatment methods altered the composition of the microbial community that accounts for hydrogen production. Acid and heat pretreatments were favorable to enrich the dominant hydrogen-producing bacterium, i.e. Clostridium sp., Enterococcus sp. and Bacillus sp., However, besides hydrogen-producing bacteria, much non-hydrogen-producing Lactobacillus sp. was also found in the sludge pretreated with base, freezing and thawing methods. Therefore, based on our results, we concluded that, among the four pretreatment methods using acid, heat-shock, base or freezing and thawing, acid pretreatment was the most effective method for promoting hydrogen production of microbial community. (C) 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

Corrosion of carbon steel influenced by anaerobic biofilm in natural seawater

The bacteria in the anaerobic biofilm on rusted carbon steel immersed in natural seawater were characterized by culturing and molecular biology techniques. Two types of anaerobic bacterium, sulfate-reducing bacteria (SRB) Desulfovibrio caledoniensis and iron-reducing bacteria Clostridium sp. uncultured were found. The compositions of the rust layer were also analyzed and we found that iron oxide and sulfate green rust were the major components. To investigate the corrosion mechanisms, electrochemical impedance spectra was obtained based on the isolated sulfate-reducing bacteria and mixed bacteria cultured from rust layer in laboratory culture conditions. We found that single species produced iron sulfide and accelerated corrosion, but mixed species produced sulfate green rust and inhibited corrosion. The anaerobic corrosion mechanism of steel was proposed and its environmental significance was discussed. (c) 2008 Elsevier Ltd. All rights reserved.