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.     

甲烷氧化菌吸附膜反应器中环氧丙烷的连续生物转化

以流化床作为固定化体系 ,在硅藻土颗粒表面构建了混合培养的甲烷氧化细菌的吸附膜。研究发现延迟期后固定化细胞的甲烷单加氧酶活性明显增加。流化床中 90 %以上的甲烷氧化细菌以吸附形式存在。吸附膜浓度为 3.3～3.7 mgdryweightcell gDS。通过批式反应考察了丙烯 甲烷共氧化过程合成环氧丙烷的可能性。研究了甲烷对丙烯环氧化以及丙烯对甲烷氧化细菌生长的影响。通过最佳配比的混合反应气体 (methane :35 % ;propene :20% ;oxygen :45 % )连续循环通入流化床反应器中抽提产物环氧丙烷 ,克服了产物抑制。该生物反应器最初产生环氧丙烷的日产量为 110～ 150μmol d ,连续操作25d ,未观察到环氧丙烷生产能力的明显减小.     

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.     

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     

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．     

Molecular sequencing and analysis of soluble methane monooxygenase gene clusters from methanotroph <Emphasis Type="Italic">Methylomonas</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> GYJ3

Summary A methanotroph Methylomonas sp. GYJ3 was isolated, whose sMMO genes and 16S rDNA were sequenced and analysed, demonstrating that the bacterium might be a type I methanotroph (γ-Proteobacteria) and was closer to Methylomonas sp. KSWIII/KSPIII. This result was consistent with the result previously determined by biochemistry and morphological taxonomy. Sequence comparison among six open reading frames and the deduced amino acid sequences of the sMMO genes from six strains revealed that the strain GYJ3 had highly conserved regions in MMOX with other strains, amounting to 78–99% homology at protein level and 71–97% homology at DNA level. Highly conserved sequences lay in two iron-binding regions. Furthermore, scanning electron microscopy of the strain GYJ3 showed rod shapes with a slightly bent configuration on the even surfaces and with plump bodies.     

甲烷利用细菌降解三氯乙烯的研究

GYJ3菌株细胞微细结构的电镜观察结果表明：它具有Ⅱ型甲烷利用细菌的特征,应归属于Ⅱ型菌。考察了Cu2+浓度、培养气相中甲烷浓度对菌株细胞中甲烷单加氧酶（EC1．14．13．25,简称MMO）活性的影响。结果表明,培养液中Cu2+浓度为1．5μmol／L,培养气相中甲烷：空气比为2∶1时,可溶性甲烷单加氧酶占细胞中MMO总量的95％。研究了GYJ3菌株细胞悬浮液降解三氯乙烯过程。实验结果表明,GYJ3菌株能够降解不同浓度的三氯乙烯,较高浓度的三氯乙烯对降解反应没有明最的抑制作用。加入甲酸盐作为电子给体能够提高三氯乙烯降解反应速率。实验中观察到GYJ3菌株降解三氯乙烯过程中反应速率随着反应的进行而下降,在三氯乙烯降解过程中三氯乙烯氧化产物是导致细胞失活的主要原因。实验室中测定了GYJ3菌株单位重量细胞降解三氯乙烯极限量,它可作为评价细菌降解三氯乙烯能力的重要指标。     

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     

甲烷氧化菌Methylomonas sp.GYJ3中甲烷单加氧酶基因与16S rRNA序列分析

甲烷氧化细菌中的关键酶系甲烷单加氧酶是一个含双核铁的多组份氧化酶,常温、常压下能够催化甲烷转化为甲醇。对甲烷氧化细菌Methylomonas sp.GYJ3中溶解性甲烷单加氧酶基因和16SrDNA进行了测序与分析。利用已知相关基因数据库信息,设计了PCR引物和测序引物,获得了满意的测序结果。全长的溶解性甲烷单加氧酶基因为5690bp,部分16S rDNA的序列长度为1280bp。与已发表的甲烷氧化细菌中甲烷单加氧酶进行了比较,结果表明MMOX组份中氨基酸序列的同一性为78%到99%,基因序列的同一性为71%到97%,6个组份中orfY片段的同一性相对较低。MMOX氨基酸序列的多序列联配表明,MMOX序列具有高度保守性,特别是在双核铁中心区域。16S rDNA进化分析显示Methylomonas sp.GYJ3与γ蛋白细菌是相关联的,基于MMOX氨基酸序列的进化分析证明,与Methylomonas sp.GYJ3最近似的菌株是Ⅰ型甲烷氧化细菌Methylomonas sp.KSWⅢ。综合分析表明,菌株GYJ3属于Ⅰ型甲烷氧化细菌Methylomonas sp.属。这个结果为Ⅰ型甲烷氧化细菌也能表达溶解性甲烷单加氧酶提供了新的证据。羟基化酶的理论等电点是6.28,理论分子量为248874.41Da。     

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。     

Selective bio-oxidation of propane to acetone using methane-oxidizing <Emphasis Type="Italic">Methylomonas</Emphasis> sp. DH-1

Propane is the major component of liquefied petroleum gas (LPG). Nowadays, the use of LPG is decreasing, and thus utilization of propane as a chemical feedstock is in need of development. An efficient biological conversion of propane to acetone using a methanotrophic whole cell as the biocatalyst was proposed and investigated. A bio-oxidation pathway of propane to acetone in Methylomonas sp. DH-1 was analyzed by gene expression profiling via RNA sequencing. Propane was oxidized to 2-propanol by particulate methane monooxygenase and subsequently to acetone by methanol dehydrogenases. Methylomonas sp. DH-1 was deficient in acetone-converting enzymes and thus accumulated acetone in the absence of any enzyme inhibition. The maximum accumulation, average productivity and specific productivity of acetone were 16.62 mM, 0.678 mM/h and 0.141 mmol/g cell/h, respectively, under the optimized conditions. Our study demonstrates a novel method for the bioconversion of propane to acetone using methanotrophs under mild reaction condition.     

Molecular analysis of methane monooxygenase from Methylococcus capsulatus (Bath)

Methane monooxygenase (MMO) is the enzyme responsible for the conversion of methane to methanol in methanotrophic bacteria. In addition, this enzyme complex oxidizes a wide range of aliphatic and aromatic compounds in a number of potentially useful biotransformations. In this study, we have used biochemical data obtained from purification and characterization of the soluble MMO from Methylococcus capsulatus (Bath), to identify structural genes encoding this enzyme by oligonucleotide probing. The genes encoding the and subunits of MMO were found to be chromosomally located and were linked in this organism. We report here on the analysis of a recombinant plasmid containing 12 kilobases of Methylococcus DNA and provide the first evidence for the localization and linkage of genes encoding the methane monooxygenase enzyme complex. DNA sequence analysis suggests that the primary structures of the and subunit of MMO are completely novel and the complete sequence of these genes is presented.     

Production of methanol from methane by methanotrophic bacteria

Methanotrophs can oxidize methane to carbon dioxide through sequential reactions catalyzed by a series of enzymes including methane monooxygenase, methanol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase. When suspensions of methanotrophic bacteria of Methylosinus trichosporium IMV 3011 were incubated at 32°C with methane and oxygen, there was an extracellular accumulation of methanol from methane oxidation in response to carbon dioxide addition. Maximal accumulation of methanol was achieved with 40% carbon dioxide in the mixed reaction gases. A continuous experiment was performed in a continuous ultrafiltration reactor. The optimum gas mixture containing 20% (v v^?1) methane, 20% oxygen, 20% nitrogen and 40% carbon dioxide was used to provide substrates and to maintain the transmembrane pressure. The product (methanol) was removed in the eluate buffer. The initial methanol concentration in the eluate buffer was 8.22 μmol L^?1. The bioreactor was operated continuously for 198 h without obvious loss of productivity.     

Continuous acetic acid production by the bioreactor system loading a new ceramic carrier for microbial attachment

Summary We have developed a bioreactor system for aerobic fermentation, using a new ceramic carrier APHROCELL which has a suitable shape for liquid and gas passage. In acetic acid fermentation byAcetobacter cells from ethanol, as a typical example of aerobic fermentation, a productivity of 17.25 g/l h was attained at continuous production of 23 g-acetic acid/l; at an acetic acid concentration around 53 g/l, the productivity was 6.4 g/l h. Thus a marketable vinegar can be obtained continuously by this bioreactor system. Because of the simplicity of the APHROCELL reactor, scale up should be relatively easy.     

Effect of mineral media on trichloroethylene oxidation by aquifer methanotrophs

The effect of growth in different mineral media on subsequent oxidation of trichloroethylene (TCE) by type I and type II aquifer methanotrophs was evaluated. Mixed culture MM1, containing a type II methanotroph, and a type I pure culture tentatively identified as aMethylomonas sp., were enriched and isolated from an uncontaminated groundwater aquifer. The second-order rate coefficients (k/K_s) for TCE oxidation by these cultures varied by more than an order of magnitude when the cultures were grown in different mineral media. The presence of a chelator (NaEDTA) in one of these media, termed Whittenbury, significantly enhanced rates of TCE oxidation by all the cultures tested. When pregrown in this mineral medium, the resting cells of the pure cultureMethylomonas sp. MM2 exhibited second-order TCE oxidation rates as great as 0.78 liter/mg·day, whereas when pregrown in Whittenbury lacking the chelator, the rates did not exceed 0.018 liter/mg·day. The rate of TCE oxidation byMethylomonas sp. MM2 pregrown in another mineral medium formulation, devoid of chelators (termed Fogel), was intermediate in value (0.26 liter/mg·day), and adding EDTA to this medium did not affect the rate. Adding 1.6 μM copper to both Whittenbury and Fogel mineral media reduced the TCE oxidation rates about an order of magnitude; subsequent addition of 84 μM EDTA partially alleviated this effect. The maximal rate coefficients (k) for TCE oxidation byMethylomonas sp. MM2 were significantly higher, and the half saturation coefficients (K_s) for TCE significantly lower, following growth in the presence of EDTA. Stationary phase TCE oxidation rates as great as 2.3 liter/mg·day were achieved whenMethylomonas sp. MM2, grown in Whittenbury medium was provided formate as a source of reducing power. Omitting EDTA from Whittenbury medium also significantly reduced the methane oxidation rate and the growth yield. Copper addition did not significantly affect the methane oxidation rate or growth yield. The internal membrane structures ofMethylomonas sp. MM2 evaluated by transmission electron microscopy showed the presence of internal membranes, the ultrastructure of which was the same regardless of growth medium or TCE oxidation rate. The methane monooxygenase responsible for TCE oxidation inMethylomonas sp. MM2 under the conditions of this study appears to be associated with the particulate fraction.     

A novel methanotroph in the genus <Emphasis Type="Italic">Methylomonas</Emphasis> that contains a distinct clade of soluble methane monooxygenase

Aerobic methane oxidation is a key process in the global carbon cycle that acts as a major sink of methane. In this study, we describe a novel methanotroph designated EMGL16-1 that was isolated from a freshwater lake using the floating filter culture technique. Based on a phylogenetic analysis of 16S rRNA gene sequences, the isolate was found to be closely related to the genus Methylomonas in the family Methylococcaceae of the class Gammaproteobacteria with 94.2–97.4% 16S rRNA gene similarity to Methylomonas type strains. Comparison of chemotaxonomic and physiological properties further suggested that strain EMGL16-1 was taxonomically distinct from other species in the genus Methylomonas. The isolate was versatile in utilizing nitrogen sources such as molecular nitrogen, nitrate, nitrite, urea, and ammonium. The genes coding for subunit of the particulate form methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), and methanol dehydrogenase (mxaF) were detected in strain EMGL16-1. Phylogenetic analysis of mmoX indicated that mmoX of strain EMGL16-1 is distinct from those of other strains in the genus Methylomonas. This isolate probably represents a novel species in the genus. Our study provides new insights into the diversity of species in the genus Methylomonas and their environmental adaptations.     

Oxidation of propene and 1-butene byMethylococcus capsulatus andMethylosinus trichosporium

Summary Methane-grown cells ofMethylococcus capsulatus andMethylosinus trichosporium readily oxidized propene and various isomers of butene to their respective epoxides. When examined in a proton NMR spectrum using tris([3-trifluoromethylhydroxymethylene]-d-camphorato), europium III derivative as an optically active chemical shift reagent, the products propylene oxide and 1,2-epoxybutane were found to contain equal amounts of both isomers. Methane-grown cells of both bacteria had considerable levels of reducing equivalents to catalyze the epoxidation of gaseous olefins. Cells depleted of reductants catalyzed the oxidation in the presence of low levels of methanol or formaldehyde with a stoichiometry of about 2:1. The rates of epoxidation of propene and 1-butene in a continuous reactor were 2–3-times that of a batch-wise reaction; the epoxidation activity, however, was lost within 3 h. The inactivation was attributed to the reactivity of the accumulated epoxides in the reactor. Propene and 1-butene oxidation by both bacteria were drastically inhibited by the respective products. Thus, the major problem in the application of microorganisms for production of epoxides from gaseous olefins is the rapid separation of the reactive products.     

Enhanced biodegradation of methylhydrazine and hydrazine contaminated NASA wastewater in fixed-film bioreactor

The aerobic biodegradation of National Aeronautics and Space Administration (NASA) wastewater that contains mixtures of highly concentrated methylhydrazine/hydrazine, citric acid and their reaction product was studied on a laboratory-scale fixed film trickle-bed reactor. The degrading organisms, Achromobacter sp., Rhodococcus B30 and Rhodococcus J10, were immobilized on coarse sand grains used as support-media in the columns. Under continuous flow operation, Rhodococcus sp. degraded the methylhydrazine content of the wastewater from a concentration of 10 to 2.5 mg/mL within 12 days and the hydrazine from 0.8 to 0.1 mg/mL in 7 days. The Achromobacter sp. was equally efficient in degrading the organics present in the wastewater, reducing the concentration of the methylhydrazine from 10 to 5 mg/mL within 12 days and that of the hydrazine from 0.8 to 0.2 mg/mL in 7 days. The pseudo first-order rate constants of 0.137 day^-1 and 0.232 day^-1 were obtained for the removal of methylhydrazine and hydrazine, respectively, in wastewater in the reactor column. In the batch cultures, rate constants for the degradation were 0.046 and 0.079 day^-1 for methylhydrazine and hydrazine respectively. These results demonstrate that the continuous flow bioreactor afford greater degradation efficiencies than those obtained when the wastewater was incubated with the microbes in growth-limited batch experiments. They also show that wastewater containing hydrazine is more amenable to microbial degradation than one that is predominant in methylhydrazine, in spite of the longer lag period observed for hydrazine containing wastewater. The influence of substrate concentration and recycle rate on the degradation efficiency is reported. The major advantages of the trickle-bed reactor over the batch system include very high substrate volumetric rate of turnover, higher rates of degradation and tolerance of the 100% concentrated NASA wastewater. The results of the present laboratory scale study will be of great importance in the design and operation of an industrial immobilized biofilm reactor for the treatment of methylhydrazine and hydrazine contaminated NASA wastewater.     

甲烷单加氧酶的催化性能和活性中心结构

甲烷单加氧酶是甲烷利用细菌代谢甲烷过程中的重要酶系,它能够催化烷烃羟基化和烯烃环氧化反应;还能催化降解氯代烃类,可用于环境中氯代烃类化合物污染的治理,是具有广泛应用前景的生物催化剂．甲烷单加氧酶是含有μ－氧桥双核铁催化活性中心的蛋白,它的研究对分子氧的活化、化学催化剂的设计具有重要意义．文章介绍了甲烷单加氧酶催化性能和机理的最新研究进展．     

Continuous production of citric acid from sugarcane molasses using a combination of submerged immobilized and surface stabilized cultures of Aspergillus niger KCU 520

Summary A high performance fermentation process for the continuous production of citric acid from sugarcane molasses by using the combination of submerged calcium alginate-immobilized and surface-stabilized cultures of Aspergillus niger KCU 520 in a continuous flow horizontal bioreactor is described. The citric acid productivity was dependent on the dilution rate with an optimum value of 0.015/h. Presaturation of fermentation medium with sterile air, in addition to surface aeration, before feeding into the bioreactor enhanced the citric acid productivity. The highest productivity, citric acid product concentration and yield obtained were 1.7 kg M^–3h^–1, 110kg M^–3 and 91% respectively. The cultures were continuously used for 30 days without any apparent loss in citric acid productivity.