Photoproduction of H2 and NADPH2 by polyurethane-immobilized cyanobacteria

Summary Cyanobacterial cells were grown in polyurethane foams (polyester or polyvinyl types). Chlorogloea fritschii, Nostoc muscorum and Mastigocladus laminosus remained immobilized in the foams and were used for continuous photoproduction of H₂ from ascorbate with methyl viologen (MV) and hydrogenase or Pt catalysts, for periods in excess of 9 days. Foam-immobilized N. muscorum continuously photoreduced exogenous NADP for at least 24 h in the presence of Fd-NADP reductase with ascorbate as electron donor.     

Increased Nitrogenase-Dependent H2 Photoproduction by hup Mutants of Rhodospirillum rubrum

Transposon Tn5 mutagenesis was used to isolate mutants of Rhodospirillum rubrum which lack uptake hydrogenase (Hup) activity. Three Tn5 insertions mapped at different positions within the same 13-kb EcoRI fragment (fragment E1). Hybridization experiments revealed homology to the structural hydrogenase genes hupSLM from Rhodobacter capsulatus and hupSL from Bradyrhizobium japonicum in a 3.8-kb EcoRI-ClaI subfragment of fragment E1. It is suggested that this region contains at least some of the structural genes encoding the nickel-dependent uptake hydrogenase of R. rubrum. At a distance of about 4.5 kb from the fragment homologous to hupSLM, a region with homology to a DNA fragment carrying hypDE and hoxXA from B. japonicum was identified. Stable insertion and deletion mutations were generated in vitro and introduced into R. rubrum by homogenotization. In comparison with the wild type, the resulting hup mutants showed increased nitrogenase-dependent H₂ photoproduction. However, a mutation in a structural hup gene did not result in maximum H₂ production rates, indicating that the capacity to recycle H₂ was not completely lost. Highest H₂ production rates were obtained with a mutant carrying an insertion in a nonstructural hup-specific sequence and with a deletion mutant affected in both structural and nonstructural hup genes. Thus, besides the known Hup activity, a second, previously unknown Hup activity seems to be involved in H₂ recycling. A single regulatory or accessory gene might be responsible for both enzymes. In contrast to the nickel-dependent uptake hydrogenase, the second Hup activity seems to be resistant to the metal chelator EDTA.     

Photoproduction of h(2) from cellulose by an anaerobic bacterial coculture

Cellulomonas sp. strain ATCC 21399 is a facultatively anaerobic, cellulose-degrading microorganism that does not evolve hydrogen but produces organic acids during cellulose fermentation. Rhodopseudomonas capsulata cannot utilize cellulose, but grows photoheterotrophically under anaerobic conditions on organic acids or sugars. This report describes an anaerobic coculture of the Cellulomonas strain with wild-type R. capsulata or a mutant strain lacking uptake hydrogenase, which photoevolves molecular hydrogen by the nitrogenase system of R. capsulata with cellulose as the sole carbon source. In coculture, the hydrogenase-negative mutant produced 4.6 to 6.2 mol of H(2) per mol of glucose equivalent, compared with 1.2 to 4.3 mol for the wild type.     

Photoproduction of H2 from acetate by syntrophic cocultures of green sulfur bacteria and sulfur-reducing bacteria

The marine green sulfur bacterium Chlorobium vibrioforme strain 1930 produced H₂ and elemental sulfur from sulfide or thiosulfate under N limitation in the light. H₂ production depended on nitrogenase and occurred only in the absence of ammonia. Methionine sulfoximine, an inhibitor of glutamine synthetase, prevented the switch-off by ammonia. In defined syntrophic cocultures of the acetate-oxidizing, sulfur-reducing bacterium Desulfuromonas acetoxidans with green sulfur bacteria, H₂ was produced from acetate via a light-driven sulfur cycle. The sulfur-reducing bacterium could not be replaced by sulfate-reducing bacteria in these experiments. In a coculture of the marine Chlorobium vibrioforme strain 1930 and the sulfur-reducing bacterium Desulfuromonas acetoxidans strain 5071, optimum long-term H₂ production from acetate was obtained with molecular nitrogen as N source, at low light intensity (110 mol · m^-2 · s^-1), in sulfide-reduced mineral medium (2 mM Na₂S) at pH 6.8. Traces of sulfide (10 M) were sufficient to keep the sulfur cycle running. The coculture formed no poly--hydroxyalkanoates (PHA), but 20%–40% polysaccharide per cell dry mass. Per mol acetate added, the coculture formed 3.1 mol of H₂ (78% of the theoretical maximum). Only 8% of the reducing equivalents was incorporated into biomass. The maximum rate of H₂ production was 1300 ml H₂ per day and g cell dry mass.Non-standard abbrevations MOPS 2-(N-morpholino) propane sulfonic acid - MSX Methionine sulfoximine - PHA poly--hydroxyalkanoates     

Increased Nitrogenase-Dependent H(2) Photoproduction by hup Mutants of Rhodospirillum rubrum

Transposon Tn5 mutagenesis was used to isolate mutants of Rhodospirillum rubrum which lack uptake hydrogenase (Hup) activity. Three Tn5 insertions mapped at different positions within the same 13-kb EcoRI fragment (fragment E1). Hybridization experiments revealed homology to the structural hydrogenase genes hupSLM from Rhodobacter capsulatus and hupSL from Bradyrhizobium japonicum in a 3.8-kb EcoRI-ClaI subfragment of fragment E1. It is suggested that this region contains at least some of the structural genes encoding the nickel-dependent uptake hydrogenase of R. rubrum. At a distance of about 4.5 kb from the fragment homologous to hupSLM, a region with homology to a DNA fragment carrying hypDE and hoxXA from B. japonicum was identified. Stable insertion and deletion mutations were generated in vitro and introduced into R. rubrum by homogenotization. In comparison with the wild type, the resulting hup mutants showed increased nitrogenase-dependent H(2) photoproduction. However, a mutation in a structural hup gene did not result in maximum H(2) production rates, indicating that the capacity to recycle H(2) was not completely lost. Highest H(2) production rates were obtained with a mutant carrying an insertion in a nonstructural hup-specific sequence and with a deletion mutant affected in both structural and nonstructural hup genes. Thus, besides the known Hup activity, a second, previously unknown Hup activity seems to be involved in H(2) recycling. A single regulatory or accessory gene might be responsible for both enzymes. In contrast to the nickel-dependent uptake hydrogenase, the second Hup activity seems to be resistant to the metal chelator EDTA.     

Homoacetogenic Fermentation of Cellulose by a Coculture of Clostridium thermocellum and Acetogenium kivui

Interrelationships between methanogens and fermentative or hydrolytic bacteria are well documented; however, such cocultures do not allow a complete fermentation shift to a peculiar metabolite. We describe here a new stable association between Clostridium thermocellum and Acetogenium kivui which converts 1 mol of cellulose (anhydroglucose equivalent) into 2.7 mol of acetate.     

H2-CO2-Dependent Anaerobic O-Demethylation Activity in Subsurface Sediments and by an Isolated Bacterium

The ability of microorganisms in sediments from the Atlantic Coastal Plain to biodegrade methoxylated aromatic compounds was examined. O-demethylation activity was detected in deep (121- and 406-m) sediments, as well as in the surface soil. A syringate-demethylating consortium, containing at least three types of bacteria, was enriched from a deep-sediment sample in a medium containing syringate as the sole organic carbon source and with a N₂-CO₂ atmosphere. An isolate which demethylated syringate was obtained from the enrichment on an agar medium incubated under a H₂-CO₂ but not a N₂-CO₂ or N₂ atmosphere. O demethylation of syringate of this isolate was dependent on the presence of both H₂ and CO₂ in the gas phase. The metabolism of syringate occurred in a sequential manner: methylgallate accumulated transiently before it was converted to gallate. Mass balance analysis suggests that the stoichiometry of the reaction in this isolate proceeds in accordance with the following generalized equation: C₇H₃O₃(OCH₃)_n^- + nHCO₃^- + nH₂ → C₇H₃O₃(OH)_n^- + nCH₃COO^- + nH₂O.     

细菌纤维素发酵培养基的优化及超微观结构分析

为了提高细菌纤维素的产量, 本研究对一株氧化葡糖杆菌菌株J2液体发酵生产细菌纤维素的培养基进行了优化, 并对其代谢的细菌纤维的超微观结构进行了观察。运用Plackett-Burman试验设计法对8个相关影响因素的效应进行了评价, 筛选出了有显著效应的3个因素: 酵母膏、ZnSO4、无水乙醇, 其他5个因素的影响未达到显著水平(P>0.05)。然后采用Box-Behnken的中心组合试验设计和响应面分析方法(RSM)确定了上述三个因素的最佳浓度, 并且以棉纤维为对照, 运用扫描电镜观察了细菌纤维素的超微观结构, 结果表明: 菌株J2利用优化后的发酵培养基生产细菌纤维素的产量为11.52 g/100 mL, 是优化前的1.35倍, 电镜照片显示细菌纤维素微纤维丝直径<0.1 mm, 比棉纤维细很多, NaOH处理可以除去纤维网络结构中的菌体。     

细菌纤维素发酵培养基的优化及超微观结构分析

为了提高细菌纤维素的产量, 本研究对一株氧化葡糖杆菌菌株J2液体发酵生产细菌纤维素的培养基进行了优化, 并对其代谢的细菌纤维的超微观结构进行了观察。运用Plackett-Burman试验设计法对8个相关影响因素的效应进行了评价, 筛选出了有显著效应的3个因素: 酵母膏、ZnSO4、无水乙醇, 其他5个因素的影响未达到显著水平(P>0.05)。然后采用Box-Behnken的中心组合试验设计和响应面分析方法(RSM)确定了上述三个因素的最佳浓度, 并且以棉纤维为对照, 运用扫描电镜观察了细菌纤维素的超微观结构, 结果表明: 菌株J2利用优化后的发酵培养基生产细菌纤维素的产量为11.52 g/100 mL, 是优化前的1.35倍, 电镜照片显示细菌纤维素微纤维丝直径<0.1 mm, 比棉纤维细很多, NaOH处理可以除去纤维网络结构中的菌体。     

沼泽红假单胞菌乙酸光合放氢研究

依据光合细菌生长代谢特性和有机废水降解主要产物类型,11种有机物被用于沼泽红假单胞菌（Rhodopseudomonas palustris）Z菌株的光合产氢研究,其中,乙酸反应体系产氢活性最高。在此基础上,研究了该菌株的生长与产氢动力学行为,探求了影响该菌株光合放氢的主要限制性影响因素。结果表明,该菌株产氢与生长部分相关。种子培养基和菌龄对产氢活性有明显影响。细胞最适产氢和生长所需要的光照强度和温度基本一致。当种子来源于硫酸铵高菌龄预培养物或谷氨酸钠对数期预培养物时,该菌株产氢活性显著增加,产氢延滞期明显缩短。氧浓度和接种量对产氢活性也有显著影响。供氢体和氮源浓度直接决定细胞的生长与光放氢活性。在低于70 mmol/L乙酸钠和15 mmol/L谷氨酸钠时,产氢活性随底物浓度的增加而增强。谷氨酸钠浓度高于15mmol/L时,由于游离NH₄⁺的出现,产氢活性受到抑制,但却明显刺激细胞的生长。在标准状况下,该菌株的最大产氢速率可达19.4 mL·L^-1·h^-1。     

Anaerobic Oxidation of n-Dodecane by an Addition Reaction in a Sulfate-Reducing Bacterial Enrichment Culture

We identified trace metabolites produced during the anaerobic biodegradation of H₂₆- and D₂₆-n-dodecane by an enrichment culture that mineralizes these compounds in a sulfate-dependent fashion. The metabolites are dodecylsuccinic acids that, in the case of the perdeuterated substrate, retain all of the deuterium atoms. The deuterium retention and the gas chromatography-mass spectrometry fragmentation patterns of the derivatized metabolites suggest that they are formed by C—H or C—D addition across the double bond of fumarate. As trimethylsilyl esters, two nearly coeluting metabolites of equal abundance with nearly identical mass spectra were detected from each of H₂₆- and D₂₆-dodecane, but as methyl esters, only a single metabolite peak was detected for each parent substrate. An authentic standard of protonated n-dodecylsuccinic acid that was synthesized and derivatized by the two methods had the same fragmentation patterns as the metabolites of H₂₆-dodecane. However, the standard gave only a single peak for each ester type and gas chromatographic retention times different from those of the derivatized metabolites. This suggests that the succinyl moiety in the dodecylsuccinic acid metabolites is attached not at the terminal methyl group of the alkane but at a subterminal position. The detection of two equally abundant trimethylsilyl-esterified metabolites in culture extracts suggests that the analysis is resolving diastereomers which have the succinyl moiety located at the same subterminal carbon in two different absolute configurations. Alternatively, there may be more than one methylene group in the alkane that undergoes the proposed fumarate addition reaction, giving at least two structural isomers in equal amounts.     

Photoproduction of ammonium ion from N2 inRhodospirillum rubrum

NH ₄ ⁺ excretion was undetectable in N₂-fixing cultures ofRhodospirillum rubrum (S-1) and nitrogenase activity in these cultures was repressed by the addition of 10 mM NH ₄ ⁺ to the medium. The glutamate analog,l-methionine-dl-sulfoximine (MSX), derepressed N₂ fixation even in the presence of 10 mM extracellular NH ₄ ⁺ . When 10 mg MSX/ml was added to cultures just prior to nitrogenase induction they developed nitrogenase activity (20% of the control activities) and excreted most of their fixed N₂ as NH ₄ ⁺ . Nitrogenase activities and NH ₄ ⁺ production from fixed N₂ were increased considerably when a combined nitrogen source, NH ₄ ⁺ (>40 moles NH ₄ ⁺ /mg cell protein in 6 days) orl-glutamate (>60 moles NH ₄ ⁺ /mg cell protein in 6 days) was added to the cultures together with MSX.Biochemical analysis revealed thatR. rubrum produced glutamine synthetase and glutamate synthase (NADP-dependent) but no detectable NADP-dependent glutamate dehydrogenase. The specific activity of glutamine synthetase was observed to be maximal when nitrogenase activity was also maximal. Nitrogenase and glutamine synthetase activities were repressed by NH ₄ ⁺ as well as by glutamate.The results demonstrate that utilization of solar energy to photoproduce large quantities of NH ₄ ⁺ from N₂ is possible with photosynthetic bacteria by interfering with their regulatory control of N₂ fixation.     

Degradation of Cyanophycin by Sedimentibacter hongkongensis Strain KI and Citrobacter amalonaticus Strain G Isolated from an Anaerobic Bacterial Consortium

Using a combination of various enrichment techniques, the strictly anaerobic, gram-positive, endospore-forming bacterium Sedimentibacter hongkongensis strain KI as revealed by 16S rRNA analysis and the gram-negative enterobacterium Citrobacter amalonaticus strain G as revealed by physiological tests were isolated from an anaerobic cyanophycin (CGP)-degrading bacterial consortium. S. hongkongensis strain KI is the first anaerobic bacterium with the ability to hydrolyze CGP to β-Asp-Arg and β-Asp-Lys dipeptides, as revealed by electrospray ionization-mass spectrometry and reversed-phase high-performance liquid chromatography analysis. However, these primary accumulated hydrolysis products were only partially used by S. hongkongensis strain KI, and significant growth on CGP did not occur. On the other hand, C. amalonaticus strain G did not degrade CGP but grew on the β-linked iso-dipeptides formed in vitro by enzymatic CGP degradation or in vivo by metabolic activity of S. hongkongensis strain KI. Dipeptide utilization occurred at the highest rate if both strains were used in cocultivation experiments with CGP, indicating that cooperation between different bacteria occurs in anaerobic natural environments for complete CGP turnover. The amino acids obtained from the cleavage of dipeptides were fermented to ethanol, acetic acid, and succinic acid, as revealed by gas chromatographic analysis and by spectrophotometric enzyme assays.     

Anaerobic Degradation of Propionate by a Mesophilic Acetogenic Bacterium in Coculture and Triculture with Different Methanogens

A mesophilic acetogenic bacterium (MPOB) oxidized propionate to acetate and CO₂ in cocultures with the formate- and hydrogen-utilizing methanogens Methanospirillum hungatei and Methanobacterium formicicum. Propionate oxidation did not occur in cocultures with two Methanobrevibacter strains, which grew only with hydrogen. Tricultures consisting of MPOB, one of the Methanobrevibacter strains, and organisms which are able to convert formate into H₂ plus CO₂ (Desulfovibrio strain G11 or the homoacetogenic bacterium EE121) also degraded propionate. The MPOB, in the absence of methanogens, was able to couple propionate conversion to fumarate reduction. This propionate conversion was inhibited by hydrogen and by formate. Formate and hydrogen blocked the energetically unfavorable succinate oxidation to fumarate involved in propionate catabolism. Low formate and hydrogen concentrations are required for the syntrophic degradation of propionate by MPOB. In triculture with Methanospirillum hungatei and the aceticlastic Methanothrix soehngenii, propionate was degraded faster than in biculture with Methanospirillum hungatei, indicating that low acetate concentrations are favorable for propionate oxidation as well.     

Anaerobic Biotransformation of High Concentrations of Chloroform by an Enrichment Culture and Two Bacterial Isolates

A fermentative enrichment culture (designated DHM-1) was developed that is capable of cometabolically biotransforming high concentrations of chloroform (CF) to nontoxic end products. Two Pantoea spp. were isolated from DHM-1 that also possess this dechlorination capability. Following acclimation to increasing levels of CF, corn syrup-grown DHM-1 was able to transform over 500 mg/liter CF in the presence of vitamin B₁₂ (approximately 3% of CF on a molar basis) at a rate as high as 22 mg/liter/day in a mineral salts medium. CO, CO₂, and organic acids were the predominant biodegradation products, suggesting that hydrolytic reactions predominate during CF transformation. DHM-1 was capable of growing on corn syrup in the presence of high concentrations of CF (as may be present near contaminant source zones in groundwater), which makes it a promising culture for bioaugmentation. Strains DHM-1B and DHM-1T transform CF at rates similar to that of the DHM-1 enrichment culture. The ability of these strains to grow in the presence of high concentrations of CF appears to be related to alteration of membrane fluidity or homeoviscous and homeophasic adaptation.Chloroform (CF) is a toxic organic compound that is frequently detected in groundwater. In the 2007 “CERCLA Priority List of Hazardous Substances,” CF ranks eleventh overall and is the third highest among chlorinated organics after vinyl chloride and polychlorinated biphenyls (4). When present at hazardous waste sites, CF is often a focal point for evaluating the feasibility of bioremediation, since it is toxic to many obligate anaerobic prokaryotes (44). For example, 1 mg/liter of CF completely inhibited dechlorination of tetrachloroethene (PCE) by a chlororespiring anaerobic isolate (32). Inhibition of reductive dechlorination of chloroethenes by CF is a general problem for sites cocontaminated with CF, which can only be overcome by first removing the CF (6).In spite of major recent advances in bioremediation of chlorinated organic compounds, treatment of CF, especially at high concentrations (e.g., >100 mg/liter), remains challenging. Although aerobic biotransformation of CF is possible (e.g., cometabolism by a butane-grown strain) (14), CF is more difficult to cometabolize than trichloroethene (42). Biotransformation of CF by mixed or pure cultures under methanogenic (5, 21) and sulfate-reducing (20) conditions has been reported, however, only at low-mg/liter CF concentrations.Corrinoids such as vitamin B₁₂ (i.e., cyanocobalamin) are effective catalysts for increasing the rate of halomethane biotransformation under anaerobic conditions. Addition of vitamin B₁₂ also shifts the pathway away from reductive dechlorination and toward hydrolytic and substitutive reactions, forming CO, CO₂, and organic acids as the major products (8, 23, 24). With low levels of B₁₂ added (3 to 5% molar ratios of CF), an enrichment culture grown on dichloromethane (DCM) as the sole substrate (8) and a lactate-grown sulfate-reducing enrichment culture (18) were able to biotransform up to 260 mg/liter and 270 mg/liter CF, respectively. However, use of a DCM-grown culture is not feasible for in situ bioremediation since DCM is also a regulated contaminant and the culture grows well only at 35°C. A major drawback with using a lactate-grown culture is the accumulation of DCM during CF transformation. Organohalide respiration of CF by a Dehalobacter population was recently reported (19), although DCM is the end product. Further treatment of the DCM would be necessary for bioremediation to be successful, although bioaugmentation cultures for this purpose are not yet available. It is apparent that a strategy for bioremediation of high concentrations of chloroform is still lacking.In a recent microcosm study using soil and groundwater from an industrial site, we demonstrated that bioaugmentation is a technically feasible remediation strategy for high concentrations of chloroform as well as carbon tetrachloride (CT) and trichlorofluoromethane (35). High rates of transformation were achieved with a fermentative enrichment culture that grows on corn syrup, supplemented with B₁₂ (1.3 to 1.4 mol%). The objectives of this study were to characterize the fermentative culture in terms of its maximum rate of CF transformation, its dependence on B₁₂ for transformation of CF, and its ability to grow on corn syrup in the presence of CF; to identify the transformation products from CF; to isolate and identify the microbes in the enrichment culture that are responsible for CF biotransformation; and to investigate the adaptation mechanisms used by the isolates to tolerate the toxicity of high concentrations of CF.     

[3H]Thymidine Incorporation To Estimate Growth Rates of Anaerobic Bacterial Strains

The incorporation of [³H]thymidine by axenic cultures of anaerobic bacteria was investigated as a means to measure growth. The three fermentative strains and one of the methanogenic strains tested incorporated [³H]thymidine, whereas the sulfate-reducing bacterium and two of the methanogenic bacteria were unable to incorporate [³H]thymidine during growth. It is concluded that the [³H]thymidine incorporation method underestimates bacterial growth in anaerobic environments.     

细菌纤维素的研究进展

细菌纤维素是一种新型微生物合成材料,在食品、医药、纺织、化工等方面有着巨大的应用潜力。简要介绍了细菌纤维素的性质和结构特点,系统阐述了细菌纤维素的生物合成途径及影响细菌纤维素产量的因素。     

Sustained Photoproduction of Ammonia from Nitrate by Anacystis nidulans

Exposure of crude cell lysates of Staphylococcus aureus MF-31 to 5 or 10 mM hydrogen peroxide resulted in a linear decrease in superoxide dismutase activity. Approximately 13% of the superoxide dismutase activity was lost after 16 min. Thermally stressed and nonstressed cells were exposed to a photochemically generated exogenous flux of superoxide radicals (O2.-). The death of thermally stressed cells was linear with time. Addition of superoxide dismutase or catalase to the O2.- generating system resulted in protection of thermally stressed and nonstressed cells, with the protective effect being greater for thermally stressed cells. Incorporation of O2-, hydroxyl radical, or singlet oxygen scavengers or antioxidants to tryptic soy agar containing 7.5% NaCl did not increase the enumeration of thermally stressed cells.     

H(2)-CO(2)-Dependent Anaerobic O-Demethylation Activity in Subsurface Sediments and by an Isolated Bacterium

The ability of microorganisms in sediments from the Atlantic Coastal Plain to biodegrade methoxylated aromatic compounds was examined. O-demethylation activity was detected in deep (121- and 406-m) sediments, as well as in the surface soil. A syringate-demethylating consortium, containing at least three types of bacteria, was enriched from a deep-sediment sample in a medium containing syringate as the sole organic carbon source and with a N(2)-CO(2) atmosphere. An isolate which demethylated syringate was obtained from the enrichment on an agar medium incubated under a H(2)-CO(2) but not a N(2)-CO(2) or N(2) atmosphere. O demethylation of syringate of this isolate was dependent on the presence of both H(2) and CO(2) in the gas phase. The metabolism of syringate occurred in a sequential manner: methylgallate accumulated transiently before it was converted to gallate. Mass balance analysis suggests that the stoichiometry of the reaction in this isolate proceeds in accordance with the following generalized equation: C(7)H(3)O(3)(OCH(3))(n) + nHCO(3) + nH(2) --> C(7)H(3)O(3)(OH)(n) + nCH(3)COO + nH(2)O.