含有甲烷氧化菌的混合菌群特性研究

为获得高效甲烷氧化微生物体系,从农业土壤中采样,以甲烷作为唯一碳源进行好氧选择性传代培养,得到生长性能稳定、生长优于Methylosinus trichosporium OB3b纯培养的具有甲烷单加氧酶(Methane Monooxygenase,MMO)活性甲烷氧化混合菌。利用MMO的共代谢特性,分别以苯酚和环氧丙烷作为目标对象,考察该混合菌对有机污染物的降解及用于生产有用化学物质的催化特性。结果表明,所得混合菌具有高效降解苯酚能力,对初始浓度为600mg/L的苯酚,经过11h培养,苯酚降解率可达99%。另外,以该混合菌为催化剂可以实现丙烯氧化生产环氧丙烷。通过降低磷酸盐浓度可以有效提高环氧丙烷的积累浓度,最大可至5mmol/L。此外,采用纯种分离方法结合PCR扩增、16SrRNA和MMO功能基因分析技术对混合菌群结构进行解析。结果表明,该混合菌群由Ⅱ型甲烷氧化菌及其它至少4种非甲烷氧化菌组成,它们分别属于Methylosinus trichosporium和Acinetobacter junii、Cupriavidusme tallidurans、Comamonas testosteroni和Stenotrophomonas maltophilia。采用PCR方法从混合菌及纯化菌株M.trichosporiums Y9总DNA中都能扩增得到mmoB、mmoX和pmoA基因片段,表明该甲烷氧化菌同时具有sMMO和pMMO两种形式的MMO。通过对从甲烷氧化混合菌中分离纯化得到的甲烷氧化菌进行PCR产物测序,结果发现其与Methylosinus trichosporium的同源性为99.9%。     

外源电子给体对甲烷单加氧酶催化丙烯环氧化反应活性的影响

以丙烯氧化反应为指标研究了不同外源电子给体对甲烷细菌(Methylomonas sp．GYJ30)休止细胞催化活性的影响。结果表明甲烷、甲醇、甲醛和甲酸盐作为电子给体加入反应中,将甲烷单加氧酶催化丙烯环氧化反应活性分别提高5．3,12．7,10和12．4倍。以甲烷和甲醛作为外源电子给体时提高初始浓度对甲烷单加氧酶具有抑制作用;而以甲醇和甲酸盐作为电子给体时提高初始浓度对甲烷单加氧酶催化活性无明显抑制作用。研究了甲醇作为电子给体时它的代谢、环氧丙烷的积累以及催化反应活性与反应时间的关系     

微水-有机溶剂两相体系中消旋萘普生的酶法拆分

研究了微水-有机溶剂两相体系中固定化脂肪酶催化的萘普生甲酯的立体选择性水解反应。固定化酶活性受载体极性、水含量、有机溶剂的logP值、产物抑制的影响,据此构建了一种可以连续拆分产生(S)(+)萘普生的微水-有机溶剂两相体系。反应在一个具有回路的连续流搅拌反应器中进行,反应器中添加有采用吸附法固定化的脂肪酶,载体为一种弱极性的合成载体,水相连同固定化酶颗粒一起永久保持在反应器中,有机流动相带入底物,带出产物。固定化酶在该50mL反应器中30℃连续操作60d,仅损失活性25%,产生(S)(+)萘普生900mg,产物对映体过量值(ee_p)为95%。     

丙烯酶催化氧化产生环氧丙烷的研究 I.甲烷氧化菌的筛选及其特性

从甘肃玉门油田土样、油样和水样中分别筛选到具有低碳烃催化氧化活性的菌种。以甲醇为碳源培养的菌体没有丙烯酶催化氧化为环氧丙烷的活力。只有以甲烷作为唯一碳源、能源培养发酵的菌体才有高专一性的酶催化氧化活性,并对筛选到的一株具有较高活性的菌株从形态、营养和生长特征等方面进行了研究,经初步鉴定表明为甲烷氧化菌甲基单胞菌属(Methylomonas sp,)。     

中试厌氧氨氧化反应器的启动与调控

研究了中试厌氧氨氧化(Anaerobic ammonium oxidation,Anammox)反应器的启动性能。结果表明,以硝化反硝化污泥、短程硝化污泥、厌氧絮体污泥和厌氧颗粒污泥混合接种,经过255d的运行,可在常温下(5oC～27oC)成功启动中试Anammox反应器,反应器的基质氮去除速率可达1.30kg/(m3·d)。厌氧氨氧化是致碱反应,厌氧氨氧化成为反应器内的主导反应后,进水pH宜控制在厌氧氨氧化适宜范围的偏低水平(6.8左右)。亚硝酸盐既是Anammox菌的基质,也是抑制剂,控制进水亚硝酸盐浓度(13～36mg/L)有助于厌氧氨氧化反应。菌种是生物反应器的功能之源,向中试装置投加少量厌氧氨氧化污泥(投加比2%),可大大加速中试Anammox反应器的启动进程。     

微生物絮凝剂连续化生产及其稳定性研究

利用多孔聚酯泡沫为载体,进行微生物絮凝剂产生菌的固定化及连续生产絮凝剂的研究。研究发现,利用多孔聚酯泡沫可吸附固定XN1菌丝细胞,且能较长时间保持高的活性。固定化XN1菌在三相流化床反应器中连续发酵运行13d无发现菌膜脱落现象,且发酵液絮凝活性均维持在90%以上,说明利用多孔聚酯泡沫颗粒作为固定化载体,连续生产絮凝剂的方法是可行的。另外,研究还发现,该菌所产生絮凝剂具有较高的热稳定性,在反应器中室温下保存数日仍可维持较高的絮凝活性。     

利用硝酸盐和亚硝酸盐同步富集厌氧甲烷氧化微生物的比较实验

【背景】反硝化厌氧甲烷氧化(Denitrifying anaerobic methane oxidation,DAMO)是以硝酸盐或亚硝酸盐为电子受体以甲烷为电子供体的厌氧氧化过程,对认识全球碳氮循环、削减温室气体排放和开发废水脱氮新技术等方面具有重要意义。【目的】认识以硝酸盐和亚硝酸盐为电子受体的DAMO微生物富集过程和结果的差异性。【方法】在序批式反应器(Sequencing batch reaetor,SBR)内接种混合物,分别以硝酸盐和亚硝酸盐为电子受体连续培养800 d,定期检测反应器基质浓度变化、计算转化速率;利用16S rRNA基因系统发育分析研究功能微生物的多样性,利用实时荧光定量PCR技术定量测定功能微生物。【结果】以亚硝酸盐为电子受体的1、3号反应器富集到了DAMO细菌,未检测到DAMO古菌;以硝酸盐为电子受体的2号反应器富集到了DAMO细菌和古菌的混合物;3个反应器的脱氮速率经过初始低速期、快速提升期,最终达到稳定,但2号快速提升期开始时间比1、3号晚了80 d左右,达到稳定的时间更长,稳定最大速率为1、3号的44.7%、40.3%。【结论】硝酸盐和亚硝酸盐对富集产物有决定性影响;以硝酸盐为电子受体富集得到的DAMO古菌和细菌协同体系可以长期稳定共存,DAMO古菌可能是协同体系中脱氮速率的限制性因素。     

固定化米根霉发酵制L-乳酸

采用海藻酸钙包埋法固定化米根霉（Ｒｈｉｚｏｐｕｓｏｒｙｚａｅ）,菌体在颗粒表面形成一层菌丝膜,有利于氧气和其它营养物质的传递;三相流化床生物反应器结构简单、动力消耗低、反应器内物质混合均匀、氧传递量大于固定化米根霉的需氧量,非常适合好氧的固定化米根霉发酵。利用它进行重复使用固定化米根霉的间歇发酵或连续发酵制备Ｌ 乳酸,整个过程一般可持续两周以上。固定化米根霉的产酸速率达１６～１８ｇ／Ｌ ｂｅａｄ．ｈｒ,得率为７０～８０％,反应器生产能力约为传统搅拌罐的３倍。采用海藻酸钙包埋法固定化米根霉在三相流化床生物反应器中进行发酵可以有效地提高Ｌ 乳酸的生产效率,具有良好的工业应用前景。     

固定化米根霉发酵制L—乳酸

采用海藻酸钙包埋法固定化米根霉,菌体在颗粒表面形成一层菌丝膜,有利于氧气和其它营养物质的传递;三相流化床生物反应器结构简单,动力消耗低,反应器内物质混合均匀、氧传递量大于固定化米根霉的需氧量,非常适合好氧的固定化米根霉发酵。     

复合固定化光合细菌及其处理养鱼水的效果

利用海藻酸钠和沸石,将含有球形红假单胞菌、荚膜红假单胞菌、沼泽红假单胞菌、万尼氏红微菌等菌体的复合光合细菌固定化,研究其对养鱼水的氮磷去除效果.比较了两种不同包埋材料固定化光合细菌处理养鱼废水的效果,对固定化光合细菌去除废水中氮磷的工艺条件进行了优化、并通过生物反应器连续处理养鱼水分析了处理后水质的效果.通过2 种固定化工艺的比较,确定了2 %沸石+2 %海藻酸钠(CA )的凝胶剂组合作为固定材料,其颗粒内生物活性最高.复合固定化光合细菌处理养鱼水的最佳条件为:厌氧光照条件下,颗粒粒径3 mm ,包埋比1 :5 ,颗粒投加量5 mg·L-1 ,4d 后养鱼水中NH4+-N 、PO43-和CODMn 的去除率分别为74.4%、84.26%、78.92%.此外,通过连续试验可以看出,固定化光合细菌具有明显的去除氨氮、磷酸盐的作用,其在净化养鱼水质方面具有非常明显的优越性.     

Continuous biocatalytic synthesis of epoxypropane using a biofilm reactor

Mixed culture methanotrophic attached biofilms immobilized on diatomite particles in a three-phase fluidized bed reaction system were developed. Methane monooxygenase (MMO) activity on diatomite particles increased as soon as the lag phase ended. More than 90% of the MMO activity in the fluidized bed was attached. A biofilm concentration of 3.3c3.7mg dry weight cell (dwc) per g dry solid (DS) was observed. Batch experiments were performed to explore the possibility of producing epoxypropane by a propene–methane co-oxidation process. The effect of methane on the epoxidation of propene and the effect of propene on the growth of methanotroph was also studied. In continuous experiments, optimum mixed gas containing 35 methane, 20 propene and 45% oxygen were continuously circulated through the fluidized bed reactor to deliver substrates and extract product. Initial epoxypropane productivity was 110–150 μmol/day. The bioreactor operated continuously for 53 days without obvious loss of epoxypropane productivity.     

Epoxypropane biosynthesis by Methylomonas sp. GYJ3: batch and continuous studies

Methylomonas sp. GYJ3 is a methanotrophic bacterium containing methane monooxygenase (MMO), which catalyses the epoxidation of propene to epoxypropane. In this study, the cell suspension of Methylomonas sp. GYJ3 has been used for epoxypropane biosynthesis from propene. When propene is epoxidized, the product epoxypropane is not further metabolized and accumulates extracellularly. Unfortunately, continuous production of epoxypropane is usually difficult due to exhaustion of reductant and the accumulation of toxic products. Hence, in order to address these problems, batch experiments were performed to explore the possibility of producing epoxypropane by a co-oxidation process. Methane was chosen as the most suitable electron-donating co-substrate since it did not result in molecular toxicity and provided abundant reductant for epoxidation. It was found that the maximum production of epoxypropane occurred in an atmosphere of 30% methane. Batch experiments also indicated that continuous removal of product was necessary to overcome the inhibition of epoxypropane. In continuous experiments, optimum mixed gaseous substrates were continuously circulated through the stirred tank bioreactor to remove product from the cell suspension. Initial epoxypropane productivity was 268 mol/day. The bioreactor has been allowed to operate continuously for 12 days without obvious loss of epoxypropane productivity, and more than 96% of initial MMO activity was retained.     

Epoxypropane biosynthesis by whole cell suspension of methanol-growth Methylosinus trichosporium IMV 3011

Methanotrophs containing methane monooxygenase (MMO) can catalyze the epoxidation of propene to epoxypropane. Methane cannot support dense biomass growth due to its low aqueous solubility. Low growth rate is important limiting factor for the application of methanotrophs. Methanol can act as growth substrate, but direct addition of methanol is toxic to most methanotrophs. The MMO activity during growth on methanol is also uncertain. In this paper, methanol-adapted Methylosinus trichosporium IMV 3011 was successfully cultivated at high cell densities using methanol as sole carbon source. A biomass density of 1.68 g dry weight cell l^?1 was achieved and cells contained almost 80% of the MMO activity measured for cells grown with methane. It has been found that methanol can also act as the electron-donating substrate to regenerate the NADH and drive epoxypropane synthesis. The effect of methanol supply on the epoxidation capacity of Methylosinus trichosporium IMV3011 was studied in batch reactor. 0.016% methanol concentration was found to give the highest propene epoxidation capacity.     

单相和两相发酵体系中Methylomonas Z201细胞的生长和环氧丙烷？…

研究了单相和两相发酵体系中甲基单胞菌Ｚ２０１细胞的生长和环氧丙烷的合成。在单相发酵体系中,底物丙烯和产物环氧丙烷抑制细胞生长,水相中环氧丙烷的浓度达到１．３ｍｍｏｌ／Ｌ。在两相发酵体系中,十六烷作为生长底物甲烷以及反应底物丙烯和分子氧的“储器”,减小了丙烯对细胞生长的抑制作用,水相和十六烷相中环氧丙烷的浓度分别达到１．７ｍｍｏｌ／Ｌ和２．６ｍｍｏｌ／Ｌ。同休止细胞相比,单相和两相发酵体系中辅酶ＮＡ     

单相和两相发酵体系中Methylomonas Z201细胞的生长和环氧丙烷的合成

研究了单相和两相发酵体系中甲基单胞菌（Methylomonas）Z201细胞的生长和环氧丙烷的合成。在单相发酵体系中,底物丙烯和产物环氧丙烷抑制细胞生长,水相中环氧丙烷的浓度达到13mmol／L。在两相发酵体系中,十六烷作为生长底物甲烷以及反应底物丙烯和分子氧的“储器”,减小了丙烯对细胞生长的抑制作用,水相和十六烷相中环氧丙烷的浓度分别达到1．7mmol／L和2．6mmol／L。同休止细胞相比,单相和两相发酵体系中辅酶NADH的原位再生使生长细胞的操作稳定性显著提高,尤为两相体系为甚。     

甲基单胞菌Methylomonas sp.GYJ3中甲烷单加氧酶羟基化酶组分的纯化和性质

Methylomonassp．GYJ3菌株中经DEAE-SepharoseCL-6B阴离子交换层析和SephacrylS300凝胶层析分离纯化出甲烷加氧酶羟基化酶组分．经HPLC分析,纯度大于90％,分子量为240kD,纯化倍数为3．9,比活为225nmol环氧丙烷每分钟毫克蛋白．SDS-PAGE表明,羟基化酶由三个亚基组成,亚基分子量为56、43、27kD．ICPAES测定羟基化酶的Fe含量为2.1molFe每摩尔蛋白．HPLC法用于甲烷单加氧酶羟基化酶组分的纯化,纯化的羟基化酶组分比活为541nmol（环氧丙烷）每分钟毫克蛋白,是两步LC法纯化的羟基化酶的两倍,Fe含量为3．78molFe每摩尔蛋白．催化性质研究表明羟基化酶能够被化学还原剂还原为还原态羟基化酶,还原态的羟基化酶单独存在时表现出MMO活性,说明它是MMO活性中心,天然态的羟基化酶单独存在时无MMO活性,加入粗酶液中MMO活性明显增加,说明GYJ3菌中MMO是一个复合酶系．     

Cultivation of Methylosinus trichosporium OB3b: III. production of particulate methane monooxygenase in continuous culture

Continous culture experiments with the obligatory methanotroph, Methylosinus trichosporium OB3b, were conducted to study the whole-cell methane monooxygenase (MMO) and nitrogenase activities in a nitrate minimal salts medium under oxygen-limited conditions with methane as the carbone source. The important variables investigated were the feed medium concentrations of copper and nitrate, CO(2) addition, the agitation speed, and the dilution rate. M. trichosporium OB3b required quantitative amounts of copper (2.6 x 10(-4) g Cu/g dry cell Wt) for the exclusive production of particulate MMo during continous culture growth. When the feed medium nitrate concentration was varied in the range of 5-50 mM, the whole-cell specific pMMO activity exhibited a maximum at 40 mM. The elimination of external CO(2) gassing decreased pMMO activity by more than 30%. The steady-state cell density increased continuously over a 300-700 rpm range of agitation speed, whereas, the pMMO activity became maximal at 400 rpm. Also, the pMMO activity increased with the dilution rate up to 0.06 h(-1) and remained constant thereafter. Maximal continuous pMMO productivity was, thus, achieved in Higgin's medium containing 10 muM Cu, 80 muM Fe, and 40 mM nitrate with an agitation speed of 500 rpm and a dilution rate of 0.06 h(-1). Nitrogenase activity, on the other hand, increased over a feed medium copper concentration of 2-15 muM, falling sharply at 20 muM, and it exhibited a minimum at 20 mM when the feed medium nitrate concentration was varied. (c) 1992 John Wiley & Sons, Inc.     

Chemical modification of the hydroxylase of soluble methane monooxygenase gives one form of the protein with significantly increased thermostability and another that functions well in organic solvents

Proteolysis of the hydroxylase component of soluble methane monooxygenase (MMO) with trypsin yielded a protein which retained 50% activity in a standard MMO assay. In an H₂O₂-driven assay, in which H₂O₂ replaced two of the protein components, NADH and O₂ used in the standard assay, the proteolysed hydroxylase retained full activity for ethane, propane and propene, but had a 2–3 fold increase with methane as substrate. Several crosslinking reagents have been tested for their ability to stabilise the proteolysed form of the hydroxylase. Using polyoxyethylene bis(imidazolyl carbonyl) (M_r 3350) as the crosslinking agent, increased thermostability of the hydroxylase was observed. Activated methoxypolyethylene glycol (M_r 5000) was used to modify the hydroxylase which was now soluble in organic solvents as well as water and could be activated by H₂O₂. The glycol-modified hydroxylase functioned well in organic solvents in the catalysis of propene oxidation.     

Application of fluidized carrier to bacterial sulphate-reduction in industrial wastewaters purification

Summary Comparative laboratory investigation of two types bioreactors with iron as a carrier of biofilm was made. One was a packed bed reactor and the another was fluidized bed. The results showed that maximum productivity of carrier in fluidized bed bioreactor {7,97 g/m²·d} is two times higher than productivity of carrier in packed bed one {3,45 g/m²·d}.