化能自养硫氧化细菌Halothiobacillus sp.LS2介导的以乙炔为电子受体的硫氧化反应

【目的】探究化能自养硫氧化细菌Halothiobacillus sp. LS2介导的以乙炔为电子受体的厌氧硫氧化反应。【方法】稀释涂布法测定细胞生长情况,离子色谱仪测试硫氧化动力学中SO_4~(2–)和S_2O_3~(2–)以及基于相对荧光定量法的基因表达分析。【结果】尽管菌株LS2在以氧气为电子受体时的最大反应速率V_(max)更高,但在厌氧条件下且以乙炔为电子受体时,菌株LS2的生长量是氧气为电子受体时的2倍,且硫氧化酶基因soxB的表达量显著高于氧气作为电子受体时。【结论】菌株LS2不仅可以以乙炔为电子受体完成厌氧硫氧化反应,且这一代谢过程的产能效率较有氧硫氧化过程更高。本研究首次发现了微生物介导的以乙炔为电子受体的厌氧硫氧化反应,对丰富硫的生物地球化学循环理论有积极意义。     

Effect of Reduced Sulfur Species on Chemolithoautotrophic Pyrite Oxidation with Nitrate

We compared the response at neutral pH of some denitrifiers to different electron donors such as reduced sulfur (pyrite, S(0), and marcasite) and reduced Fe. Chemolithoautotrophic oxidation of pyrite with nitrate as electron acceptor was not possible when the pyrite was in a pure crystalline form, whereas oxidation of synthesized FeS₂ of low crystallinity and of S(0) with nitrate as electron acceptor was possible. Neither nitrite nor sulfate was formed when Fe(II)-oxidizing strain Acidovorax sp. BoFeN1 was tested. Microbial reduction of nitrate appears to be induced via S oxidation but not via Fe oxidation.     

Nitrate-Controlled Anaerobic Oxidation of Pyrite by Thiobacillus Cultures

Nitrate-dependent pyrite oxidation is an important process as it may prevent pollution by nitrate from agriculture. Anaerobic oxidation of pyrite with nitrate as an electron acceptor was studied in cultures of Thiobacillus denitrificans and Thiobacillus thioparus. Both strains reduced nitrate, with pyrite added as sole electron donor, but T. thioparus reduced nitrate to nitrite only. Accumulation of nitrite, however, was prevented in co-cultures of T. denitrificans and T. thioparus. Furthermore, pyrite oxidation rates were dependent on pyrite pretreatment, which results in different specific surface areas of pyrite. Initial nitrate concentration or pyrite origin did not affect the pyrite oxidation rate.     

A model for the effects of primary substrates on the kinetics of reductive dehalogenation

A kinetic model that describes substrate interactions during reductive dehalogenation reactions is developed. This model describes how the concentrations of primary electron-donor and -acceptor substrates affect the rates of reductive dehalogenation reactions. A basic model, which considers only exogenous electron-donor and -acceptor substrates, illustrates the fundamental interactions that affect reductive dehalogenation reaction kinetics. Because this basic model cannot accurately describe important phenomena, such as reductive dehalogenation that occurs in the absence of exogenous electron donors, it is expanded to include an endogenous electron donor and additional electron acceptor reactions. This general model more accurately reflects the behavior that has been observed for reductive dehalogenation reactions. Under most conditions, primary electron-donor substrates stimulate the reductive dehalogenation rate, while primary electron acceptors reduce the reaction rate. The effects of primary substrates are incorporated into the kinetic parameters for a Monod-like rate expression. The apparent maximum rate of reductive dehalogenation (q _{m, ap} ) and the apparent half-saturation concentration (K _ap ) increase as the electron donor concentration increases. The electron-acceptor concentration does not affect q _{m, ap} , but K _ap is directly proportional to its concentration.Definitions for model parameters RX halogenated aliphatic substrate - E-M ⁿ reduced dehalogenase - E-M ⁿ⁺² oxidized dehalogenase - [E-M ⁿ ] steady-state concentration of the reduced dehalogenase (moles of reduced dehalogenase per unit volume) - [E-M ⁿ⁺²] steady-state concentration of the oxidized dehalogenase (moles of reduced dehalogenase per unit volume) - DH₂ primary exogenous electron-donor substrate - A primary exogenous electron-acceptor substrate - A2 second primary exogenous electron-acceptor substrate - X biomass concentration (biomass per unit volume) - f fraction of biomass that is comprised of the dehalogenase (moles of dehalogenase per unit biomass) - stoichiometric coefficient for the reductive dehalogenation reaction (moles of dehalogenase oxidized per mole of halogenated substrate reduced) - stoichiometric coefficient for oxidation of the primary electron donor (moles of dehalogenase reduced per mole of donor oxidized) - stoichiometric coefficient for oxidation of the endogenous electron donor (moles of dehalogenase reduced per unit biomass oxidized) - stoichiometric coefficient for reduction of the primary electron acceptor (moles of dehalogenase oxidized per mole of acceptor reduced) - stoichiometric coefficient for reduction of the second electron acceptor (moles of dehalogenase oxidized per mole of acceptor reduced) - r _RX rate of the reductive dehalogenation reaction (moles of halogenated substrate reduced per unit volume per unit time) - r _d1 rate of oxidation of the primary exogenous electron donor (moles of donor oxidized per unit volume per unit time) - r _d2 rate of oxidation of the endogenous electron donor (biomass oxidized per unit volume per unit time) - r _a1 rate of reduction of the primary exogenous electron acceptor (moles of acceptor reduced per unit volume per unit time) - r _a2 rate of reduction of the second primary electron acceptor (moles of acceptor reduced per unit volume per unit time) - k _RX mixed second-order rate coefficient for the reductive dehalogenation reaction (volume per mole dehalogenase per unit time) - k _d1 mixed-second-order rate coefficient for oxidation of the primary electron donor (volume per mole dehalogenase per unit time) - k _d2 mixed-second-order rate coefficient for oxidation of the endogenous electron donor (volume per mole dehalogenase per unit time) - b first-order biomass decay coefficient (biomass oxidized per unit biomass per unit time) - k _a1 mixed-second-order rate coefficient for reduction of the primary electron acceptor (volume per mole dehalogenase per unit time) - k _a2 mixed-second-order rate coefficient for reduction of the second primary electron acceptor (volume per mole dehalogenase per unit time) - q _m,ap apparent maximum specific rate of reductive dehalogenation (moles of RX per unit biomass per unit time) - K _ap apparent half-saturation concentration for the halogenated aliphatic substrate (moles of RX per unit volume) - k _ap apparent pseudo-first-order rate coefficient for reductive dehalogenation (volume per unit biomass per unit time)     

Changes of Photosystem Ⅱ Electron Transport in the ChlorophyⅡ-deficient Oilseed Rape Mutant Studied by ChlorophyⅡ Fluorescence and Thermoluminescence

The photosystem Ⅱ （PSII） complex of photosynthetic membranes comprises a number of chlorophyll-binding proteins that are important to the electron flow. Here we report that the chlorophyll b-deficient mutant has decreased the amount of light-harvesting complexes with an increased amount of some core polypeptldes of PSII, including CP43 and CP47. By means of chlorophyll fluorescence and thermolumlnescence, we found that the ratio of Fv/Fm, qP and electron transport rate in the chlorophyll b-deficient mutant was higher compared to the wild type. In the chlorophyll lPdeflclent mutant, the decay of the primary electron acceptor quinones （QA-） reoxidation was decreased, measured by the fluorescence. Furthermore, the thermoluminescence studies in the chlorophyll bdeficient mutant showed that the B band （S2/S3QB-） decreased slightly and shifted up towards higher temperatures. In the presence of dlchlorophenyl-dlmethylurea, which is inhibited in the electron flow to the second electron acceptor quinines （QB） at the PSll acceptor side, the maximum of the Q band （S2QA-） was decreased slightly and shifted down to lower temperatures, compared to the wild type. Thus, the electron flow within PSll of the chlorophyⅡ b-deficient mutant was down-regulated and characterized by faster oxidation of the primary electron acceptor quinine QA-via forward electron flow and slower reduction of the oxidation S states.     

An assessment of growth yields and energy coupling inDesulfovibrio

The yield coefficients forDesulfovibrio vulgaris andD. gigas varied with the electron donoracceptor combinations and with the bacterial strain. The only evidence for electron transport coupled formation of adenosine triphosphate (ATP) was with sulfate as the electron acceptor. WithD. vulgaris the ATP formation coupling to electron flow with pyruvate oxidation was 1:4 electrons and with lactate oxidation was 1:8 electrons. WithD. gigas these ratios were 1:8 electrons and 1:16 electrons for the oxidation of pyruvate and lactate. The clearest resolution of energy coupling was withD. vulgaris growing on formatesulfate medium where 2 ATP appear to be formed with the transfer of electrons from formate to adenosine phosphosulfate and one ATP with the transfer of electrons from formate to sulfite.     

Oxidation of organic compounds to CO2 with sulfur or thiosulfate as electron acceptor in the anaerobic hyperthermophilic archaea Thermoproteus tenax and Pyrobaculum islandicum proceeds via the citric acid cycle

The oxidation of organic compounds with elemental sulfur or thiosulfate as electron acceptor was studied in the anaerobic hyperthermophilic archaea Thermoproteus tenax and Pyrobaculum islandicum. T. tenax was grown on either glucose or casamino acids and sulfur; P. islandicum on peptone and either elemental sulfur or thiosulfate as electron acceptor. During exponential growth only CO₂ and H₂S rather than acetate, alanine, lactate, and succinate were detected as fermentation products of both organisms; the ratio of CO₂/H₂S formed was 1:2 with elemental sulfur and 1:1 with thiosulfate as electron acceptor. Cell extracts of T. tenax and P. islandicum contained all enzymes of the citric acid cycle in catabolic activities: citrate synthase, aconitase, isocitrate dehydrogenase (NADP⁺-reducing), oxoglutarate: benzylviologen oxidoreductase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase and malate dehydrogenase (NAD⁺-reducing). Carbon monoxide dehydrogenase activity was not detected. We conclude that in T. tenax and P. islandicum organic compounds are completely oxidized to CO₂ with sulfur or thiosulfate as electron acceptor and that acetyl-CoA oxidation to CO₂ proceeds via the citric acid cycle.     

Energetic aspects of the metabolism of reduced sulphur compounds in Thiobacillus denitrificans

Yields of Thiobacillus denitrificans on different substrates were compared. The organism was grown in a chemostat at a dilution rate of 0.03 h^-1. From the difference in the cell yields with (1) oxygen (6.40 g carbon per mole thiosulphate) and (2) nitrate (4.51 g carbon per mole thiosulphate) as an electron acceptor the experimental value for Y_ATP was estimated to be 1.75. The efficiency of the biosynthetic system would be 42% if 1 ATP should be needed in reversed electron transport, and 57% if this was 2 ATP per electron pair.It could be calculated that during anaerobic oxidation of thiosulphate with nitrate 1.41 or 1.16 ATP per 2 electrons are generated if 1 or 2 ATP respectively per thiosulphate is formed in substrate-level phosphorylation. For aerobic oxidation these figures are 2.40 and 2.16, respectively     

Role of formate and hydrogen in the degradation of propionate and butyrate by defined suspended cocultures of acetogenic and methanogenic bacteria

The butyrate-degradingSyntrophospora bryantii degrades butyrate and a propionate-degrading strain (MPOB) degrades propionate in coculture with the hydrogen- and formate-utilizingMethanospirillum hungatii orMethanobacterium formicicum. However, the substrates are not degraded in constructed cocultures with twoMethanobrevibacter arboriphilus strains which are only able to consume hydrogen. Pure cultures of the acetogenic bacteria form both hydrogen and formate during butyrate oxidation with pentenoate as electron acceptor and during propionate oxidation with fumarate as electron acceptor. Using the highest hydrogen and formate levels which can be reached by the acetogens and the lowest hydrogen and formate levels which can be maintained by the methanogens it appeared that the calculated formate diffusion rates are about 100 times higher than the calculated hydrogen diffusion rates.     

pH dependent changes in the reactivity of the primary electron acceptor of System II in spinach chloroplasts to external oxidant and reductant

Changes in the rates of dark oxidation and reduction of the primary electron acceptor of System II by added oxidant and reductant were investigated by measuring the induction of chlorophyll fluorescence under moderate actinic light in 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea-inhibited chloroplasts at pH values between 3.6 and 9.5. It was found that:

1. (1) The rate of dark oxidation of photoreduced primary acceptor was very slow at all the pH values tested without added electron acceptor.

2. (2) The rate was accelerated by the addition of ferricyanide in the whole pH range. It was dependent approximately on the 0.8th power of the ferricyanide concentration.

3. (3) The rate constant for the oxidation of the primary acceptor by ferricyanide was pH-dependent and became high at low pH. The value at pH 3.6 was more than 100 times that at pH 7.8.

4. (4) The pH-dependent change in the rate constant was almost reversible when the chloroplasts were suspended at the original pH after a large pH change (acid treatment).

5. (5) An addition of carbonylcyanide m-chlorophenylhydrazone or heavy metal chelators had little effect on the rate of dark oxidation of the primary acceptor by ferricyanide.

6. (6) The dark reduction of the primary acceptor by sodium dithionite also became faster at low pH.

From these results it is concluded that at low pH the primary acceptor of System II becomes accessible to the added hydrophilic reagents even in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea.     

Utilization of nitrate byBeggiatoa alba

Beggiatoa alba B18LD utilizes both nitrate and nitrite as sole nitrogen sources, although nitrite was toxic above 1 mM.B. alba coupledin vivo acetate oxidation, but not sulfide oxidation, with nitrate and nitrite reduction.B. alba could not, however, grow anaerobically with nitrate as the sole electron acceptor. Furthermore, the incorporation of acetate into macromolecules under anaerobic conditions with nitrate as the sole electron acceptor was less 10% of the incorporation with oxygen as the electron acceptor. The product of nitrate reduction byB. alba was ammonia; N₂ or N₂O were not produced. The nitrate reductase activity inB. alba was soluble and it utilized reduced flavins or methyl viologen and dithionite as electron donors. Pyrimidine nucleotides were not used as in vitro electron donors, either alone or with flavins in coupled assays. TheB. alba nitrate reductase activity was competitively inhibited with chlorate and was only mildly inhibited by azide and cyanide. Nitrate was not required for induction of theB. alba nitrate reductase, and neither oxygen nor ammonia repressed its activity. Thus,B. alba nitrate reductase appears to be an assimilatory nitrate reductase with unusual regulatory properties.Non-standard abbreviations MV Methyl viologen - DT dithionite - GS glutamine synthetase - GOGAT glutamine 2-oxoglutarate aminotransferase - PPO 2-diphenyloxazole - POPOP 1,4-(bis)-[2-(5-phenyloxazolyl)] benzene - TCA trichloroacetic acid - CCCP carbonylcyanidem-chlorophenylhydrazone - FCCP carbonylcyanidep-trifluoromethoxyphenylhydrazone - TTFA thenoyltrifluoroacetone - PHEN 1,10-phenanthroline - HOQNO 2-heptyl 4-hydroxyquinoline-n-oxide - 8HQ 8-hydroxyquinoline     

Phorphorylative electron transport chains lacking a cytochromebc 1 complex

Electron transport-coupled phosphorylation with fumarate as terminal acceptor inWolinella succinogenes yields less than 1 ATP/2 electrons. The generated by the electron transport is 0.18V and the H⁺/electron ratio is 1. The electron transport chain is made up of two dehydrogenases (hydrogenase and formate dehydrogenase) that catalyze the reduction of menaquinone, and fumarate reductase which catalyzes the oxidation of menaquinol.C-type cytochromes are not involved. The phosphorylative electron transport with sulfur as terminal acceptor inW. succinogenes orDesulfuromonas acetoxidans does not involve known quinones. The ATP yields should be even smaller than those with fumarate. Succinate oxidation by sulfur, which is a catabolic reaction inD. acetoxidans, is accomplished by reversed electron transport.     

Enzymatic Improvement of Food Flavor IV. Oxidation of Aldehydes in Soybean Extracts by Aldehyde Oxidase

Aldehyde oxidase (aldehyde: oxygen oxidoreductase, EC 1.2.3.1) was partially purified from bovine liver. The enzyme irreversibly oxidized various aldehydes to the corresponding acids by using dissolved oxygen as an electron acceptor. Although the Km value for n-hexanal was low (6 µm), that for acetaldehyde was high (20 mm).

Medium-chain aldehydes such as hexanal and pentanal appear to be mainly responsible for green beany odor of soybean products. A great reduction in the beany odor was observed after the soybean extract was incubated with aldehyde oxidase under aerobic conditions. Dissolved oxygen was utilized as the electron acceptor throughout the enzyme-catalyzed oxidation of aldehydes and none of other cofactors were found to be required.

It has been shown that bovine liver mitochondrial aldehyde dehydrogenase oxidizes the soybean protein-bound aldehyde with a rate comparable to that for free n-hexanal (Agric. Biol. Chem., 43, in press). Comparative studies of aldehyde oxidase and aldehyde dehydrogenase with respect to oxidation-rates of free aldehydes and the soybean protein-bound aldehydes indicated that aldehyde oxidase acted on the bound aldehyde with a much slower rate.     

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。     

NADH-dependent polyvanadate reduction by microsomes

NADH-dependent reduction of polyvanadate was observed by using rat liver microsomes as the enzyme source. The reduced vanadate form obtained was blue in color with a broad absorption maximum in the red region around 650 nm. Microsomes and phosphate anions were found to be essential for polyvanadate reduction. The rate and the extent of formation of blue color compound was dependent on the amount of vanadate present. Cytochrome b ₅ was found to be involved in this SOD-insensitive reaction. The rate of disappearance of the blue-colored compound was dependent on concentration of NADH and was found to be sensitive to SOD. Catalase and Mn²⁺. which inhibit oxygen consumption accompanying NADH oxidation, increased both the rate and extent of the blue color compound formed. The results suggest that vanadate acts as an electron acceptor.     

Anaerobic degradation of α-resorcylate by Thauera aromatica strain AR-1 proceeds via oxidation and decarboxylation to hydroxyhydroquinone

Anaerobic degradation of α-resorcylate (3,5-dihydroxybenzoate) was studied with the denitrifying strain AR-1, which was assigned to the described species Thauera aromatica. α-Resorcylate degradation does not proceed via the benzoyl-CoA, the resorcinol, or the phloroglucinol pathway. Instead, α-resorcylate is converted to hydroxyhydroquinone (1,2,4-trihydroxybenzene) by dehydrogenative oxidation and decarboxylation. Nitrate, K₃[Fe(CN)₆], dichlorophenol indophenol, and the NAD⁺ analogue 3-acetylpyridine adeninedinucleotide were suitable electron acceptors for the oxidation reaction; NAD⁺ did not function as an electron acceptor. The oxidation reaction was strongly accelerated by the additional presence of the redox carrier phenazine methosulfate, which could also be used as sole electron acceptor. Oxidation of α-resorcylate with molecular oxygen in cell suspensions or in cell-free extracts of α-resorcylate- and nitrate-grown cells was also detected although this bacterium did not grow with α-resorcylate under an air atmosphere. α-Resorcylate degradation to hydroxyhydroquinone proceeded in two steps. The α-resorcylate-oxidizing enzyme activity was membrane-associated and exhibited maximal activity at pH 8.0. The primary oxidation product was not hydroxyhydroquinone. Rather, formation of hydroxyhydroquinone by decarboxylation of the unknown intermediate in addition required the cytoplasmic fraction and needed lower pH values since hydroxyhydroquinone was not stable at alkaline pH. Received: 8 July 1997 / Accepted: 20 October 1997     

Effects of the medium on the rate of electron transfer in a reconstituted system of mitochondrial hydroxylation]

Effects of ionic strength, pH, viscosity, concentrations of components and nature of acceptor on the rate of NADPH oxidation and acceptor reduction were studied in a hydroxylation system containing adrenodoxin reductase, adrenodoxin and cytochrome P450 or cytochrome c. The maximal rate was observed with 0.05--0.10 M phosphate buffer, pH 6.0--6.5 and at the adrenoxin/flavoprotein/cytochrome ratio of 1 : 1 : 1. The electron transfer rate was decreased with an increase in viscosity. Cytochrome P450 is more efficient as a terminal acceptor as compared to cytochrome c or indigodisulphonate.     

Oxaloacetate inhibition of succinate oxidation in tightly coupled liver mitochondria with ferricyanide as an electron acceptor

When ferricyanide is used as an artificial electron acceptor, succinate oxidation by tightly coupled liver mitochondria becomes inhibited after 1–3 min. No inhibition occurs in the presence of rotenone or glutamate establishing that oxaloacetate causes the inhibtion. Oxygen consumption by mitochondria oxidizing succinate does not become inhibited in the absence of rotenone suggesting that oxaloacetate accumulates to a greater extent when ferricyanide is added than when oxygen is the terminal acceptor. Higher levels of oxaloacetate in the ferricyanide reaction are apparently due to an increased rate of synthesis rather than a decreased rate of removal. Thus it appears that when succinate is the substrate and oxygen the terminal acceptor a control mechanism exists which blocks oxidation of malate. When ferricyanide is added as an artificial electron acceptor this control is lost and oxaloacetate accumulates to inhibit succinate oxidation.     

Methanofuran-b is required for CO2 formation from formaldehyde by Methanosarcina barkeri

Abstract Extracts of Methanosarcina barkeri strain Fasaro oxidized formaldehyde to CO₂ with methyl-coenzyme M as the natural terminal electron acceptor resulting in methanogenesis. A combination of the artificial electron acceptors methylviologen and metronidazole could substitute for methyl-coenzyme M. The rate of formaldehyde oxidation was thereby increased. Taking advantage of this artificial electron acceptor system the role of cofactors in formaldehyde oxidation was investigated. Cofactor-free extract of M. barkeri did not catalyze the oxidation of formaldehyde. CO₂ formation could be restored by the addition of tetrahydromethanopterin-b (H₄MPT-b) and methanofuran-b (MFR-b) from M. barkeri . Other low molecular weight or heat-resistant compounds stimulating formaldehyde oxidation were not found. Formaldehyde oxidation seems, therefore, to proceed via H₄ MPT-b and MFR-b-derivatives already shown to be involved in methanogenesis from H₂+ CO₂.