大豆根际土壤中氢氧化细菌的分离、筛选和基本特征

土壤中的氢氧化细菌能够利用土壤中的H2为能源并同化CO2,增加其种群数量并促进作物生长.采用气体循环培养体系(持续通氢气装置),通过电解水的方式产生H2,与通入的空气混合,形成流速为280ml.min-1、含H2量为41.6～125μmol.L-1的混合气体.采用矿质盐固体培养基,在适当的H2、O2和CO2下以H2作为唯一能源分离氢氧化细菌.采用此方法从大豆根际土壤样品中分离出40余株细菌,对其进行耗氢能力测定表明,有20株菌具有氧化氢功能和自养生长能力,初步判断这20株菌为氢氧化细菌,并测定了菌落形态及生理生化特征.     

紫花苜蓿根际氢氧化细菌的分离及其对小麦种子促生作用的研究

从大豆植物根际分离的氢氧化细菌对植物的生长有促进作用,但是关于其他的豆科植物根际分离的氢氧化细菌是否也有促生作用的研究甚少。从紫花苜蓿根际土壤分离氢氧化细菌,并进行其对小麦种子促生实验的研究,判断氢氧化细菌是否有促生作用,从而丰富具有促生作用的根际微生物资源。采用MSA培养基,从铜川新区紫花苜蓿根际土壤中分离得到氢氧化细菌疑似菌株,对其进行TTC法检测菌株氢化酶活性和自养能力的特性,以获得氢氧化细菌;通过小麦种子的萌发进行促生实验验证。结果表明,16株菌株处理过的小麦根长分别增加25%～128%,芽长增长27%～73%,鲜重增加48%～103%。从苜蓿根际土壤分离出的氢氧化细菌均具有较明显的促生作用。     

Bacterial sulfur cycling shapes microbial communities in surface sediments of an ultramafic hydrothermal vent field

The ultramafic-hosted Logatchev hydrothermal field (LHF) is characterized by vent fluids, which are enriched in dissolved hydrogen and methane compared with fluids from basalt-hosted systems. Thick sediment layers in LHF are partly covered by characteristic white mats. In this study, these sediments were investigated in order to determine biogeochemical processes and key organisms relevant for primary production. Temperature profiling at two mat-covered sites showed a conductive heating of the sediments. Elemental sulfur was detected in the overlying mat and metal-sulfides in the upper sediment layer. Microprofiles revealed an intensive hydrogen sulfide flux from deeper sediment layers. Fluorescence in situ hybridization showed that filamentous and vibrioid, Arcobacter-related Epsilonproteobacteria dominated the overlying mats. This is in contrast to sulfidic sediments in basalt-hosted fields where mats of similar appearance are composed of large sulfur-oxidizing Gammaproteobacteria. Epsilonproteobacteria (7-21%) and Deltaproteobacteria (20-21%) were highly abundant in the surface sediment layer. The physiology of the closest cultivated relatives, revealed by comparative 16S rRNA sequence analysis, was characterized by the capability to metabolize sulfur components. High sulfate reduction rates as well as sulfide depleted in (34)S further confirmed the importance of the biogeochemical sulfur cycle. In contrast, methane was found to be of minor relevance for microbial life in mat-covered surface sediments. Our data indicate that in conductively heated surface sediments microbial sulfur cycling is the driving force for bacterial biomass production although ultramafic-hosted systems are characterized by fluids with high levels of dissolved methane and hydrogen.     

Culture dependent and independent analyses of 16S rRNA and ATP citrate lyase genes: a comparison of microbial communities from different black smoker chimneys on the Mid-Atlantic Ridge

The bacterial and archaeal communities of three deep-sea hydrothermal vent systems located on the Mid-Atlantic Ridge (MAR; Rainbow, Logatchev and Broken Spur) were investigated using an integrated culture-dependent and independent approach. Comparative molecular phylogenetic analyses, using the 16S rRNA gene and the deduced amino acid sequences of the alpha and beta subunits of the ATP citrate lyase encoding genes were carried out on natural microbial communities, on an enrichment culture obtained from the Broken Spur chimney, and on novel chemolithoautotrophic bacteria and reference strains originally isolated from several different deep-sea vents. Our data showed that the three MAR hydrothermal vent chimneys investigated in this study host very different microbial assemblages. The microbial community of the Rainbow chimney was dominated by thermophilic, autotrophic, hydrogen-oxidizing, sulfur- and nitrate-reducing Epsilonproteobacteria related to the genus Caminibacter. The detection of sequences related to sulfur-reducing bacteria and archaea (Archaeoglobus) indicated that thermophilic sulfate reduction might also be occurring at this site. The Logatchev bacterial community included several sequences related to mesophilic sulfur-oxidizing bacteria, while the archaeal component of this chimney was dominated by sequences related to the ANME-2 lineage, suggesting that anaerobic oxidation of methane may be occurring at this site. Comparative analyses of the ATP citrate lyase encoding genes from natural microbial communities suggested that Epsilonproteobacteria were the dominant primary producers using the reverse TCA cycle (rTCA) at Rainbow, while Aquificales of the genera Desulfurobacterium and Persephonella were prevalent in the Broken Spur chimney.     

The use of disposable gaas generating kits for the growth of hydrogen-oxidizing bacteria and the determination of hydrogen autotrophy

A simplified procedure for the determination of autotropic growth of hydrogen-oxidizing bacteria has been developed. The method uses commercially available disposable hydrogen and carbon dioxide kits, commonly used in anaerobic bacteriology, to produce a gaseous atmosphre containing by volume approximately 41% hydrogen, 6% carbon dioxide, 11% oxygen and 42% nitrogen. The atmosphere was suitable for the growth of strains assigned to the species Alcaligenes eutrophus, Alcaligenes paradoxus, Paracoccus denitrificans, Pseudomonas facilis, Pseudomonas flava, Pseudomonas palleronii, Pseudomonas saccahrophilia and Rhodococcus sp. (‘Nocardia opaca’). The method can also be used for the screening of hydrogen-oxidizing ability in bacterial isolates, thus eliminating the need for complex gas mixing devices or expensive gas mixtures.     

Growth and phylogenetic properties of novel bacteria belonging to the epsilon subdivision of the Proteobacteria enriched from Alvinella pompejana and deep-sea hydrothermal vents

Recent molecular characterizations of microbial communities from deep-sea hydrothermal sites indicate the predominance of bacteria belonging to the epsilon subdivision of Proteobacteria (epsilon Proteobacteria). Here, we report the first enrichments and characterizations of four epsilon Proteobacteria that are directly associated with Alvinella pompejana, a deep sea hydrothermal vent polychete, or with hydrothermal vent chimney samples. These novel bacteria were moderately thermophilic sulfur-reducing heterotrophs growing on formate as the energy and carbon source. In addition, two of them (Am-H and Ex-18.2) could grow on sulfur lithoautrotrophically using hydrogen as the electron donor. Optimal growth temperatures of the bacteria ranged from 41 to 45 degrees C. Phylogenetic analysis of the small-subunit ribosomal gene of the two heterotrophic bacteria demonstrated 95% similarity to Sulfurospirillum arcachonense, an epsilon Proteobacteria isolated from an oxidized marine surface sediment. The autotrophic bacteria grouped within a deeply branching clade of the epsilon Proteobacteria, to date composed only of uncultured bacteria detected in a sample from a hydrothermal vent along the mid-Atlantic ridge. A molecular survey of various hydrothermal vent environments demonstrated the presence of two of these bacteria (Am-N and Am-H) in more than one geographic location and habitat. These results suggest that certain epsilon Proteobacteria likely fill important niches in the environmental habitats of deep-sea hydrothermal vents, where they contribute to overall carbon and sulfur cycling at moderate thermophilic temperatures.     

Isolation of hydrogenase regulatory mutants of hydrogen-oxidizing bacteria by a colony-screening method

Mutants derepressible for hydrogenases (Hox d) have been isolated from the wild type of Alcaligenes hydrogenophilus which is inducible for hydrogenases (Hox i). The mutants are able to form the hydrogenases during growth on gluconate under air while the wild type requires molecular hydrogen for hydrogenase systhesis.Mutant selection involved alternating growth under autotrophic and heterotrophic conditions. Mutants derepressed for hydrogenases after growth on gluconate were recognized by a new colony-screening method allowing differentiation between colonies of hydrogenase-containing and hydrogenase-free cells of aerobic hydrogen-oxidizing bacteria. The method is based on the ability of the colonies to reduce triphenyltetrazolium chloride in the presence of monoiodoacetate and gaseous hydrogen to its water-insoluble purple formazan. Endogenous dye reduction (under nitrogen) and the function of the cytoplasmic NAD-reducing hydrogenase were completely inhibited by monoiodoacetate. The applicability of the method has been demonstrated for wild type strains and mutants of various hydrogen-oxidizing bacteria. When mutants of A. hydrogenophilus and A. eutrophus H16 lacking the Hox-encoding plasmids pHG21-a and pHG1, respectively, were used as recipients and Hox d mutant M 201 of A. hydrogenophilus as a donor transconjugants appeared which had received the Hox d character and the megaplasmid pHG21-a.Abbreviations MIAc monoiodoacetate - TTC 2,3,5-triphenyl-2-tetrazolium chloride - Hox ability to oxidize hydrogen Dedicated to Gerhard Drews on the occasion of his 60th birthday, remembering the education and inspiration we received from our teacher Johannes Buder at the Martin-Luther University of Halle     

Growth and Phylogenetic Properties of Novel Bacteria Belonging to the Epsilon Subdivision of the Proteobacteria Enriched from Alvinella pompejana and Deep-Sea Hydrothermal Vents

Recent molecular characterizations of microbial communities from deep-sea hydrothermal sites indicate the predominance of bacteria belonging to the epsilon subdivision of Proteobacteria (epsilon Proteobacteria). Here, we report the first enrichments and characterizations of four epsilon Proteobacteria that are directly associated with Alvinella pompejana, a deep sea hydrothermal vent polychete, or with hydrothermal vent chimney samples. These novel bacteria were moderately thermophilic sulfur-reducing heterotrophs growing on formate as the energy and carbon source. In addition, two of them (Am-H and Ex-18.2) could grow on sulfur lithoautrotrophically using hydrogen as the electron donor. Optimal growth temperatures of the bacteria ranged from 41 to 45°C. Phylogenetic analysis of the small-subunit ribosomal gene of the two heterotrophic bacteria demonstrated 95% similarity to Sulfurospirillum arcachonense, an epsilon Proteobacteria isolated from an oxidized marine surface sediment. The autotrophic bacteria grouped within a deeply branching clade of the epsilon Proteobacteria, to date composed only of uncultured bacteria detected in a sample from a hydrothermal vent along the mid-Atlantic ridge. A molecular survey of various hydrothermal vent environments demonstrated the presence of two of these bacteria (Am-N and Am-H) in more than one geographic location and habitat. These results suggest that certain epsilon Proteobacteria likely fill important niches in the environmental habitats of deep-sea hydrothermal vents, where they contribute to overall carbon and sulfur cycling at moderate thermophilic temperatures.     

Complete genome sequence of Hydrogenobacter thermophilus type strain (TK-6)

Hydrogenobacter thermophilus Kawasumi et al. 1984 is the type species of the genus Hydrogenobacter. H. thermophilus was the first obligate autotrophic organism reported among aerobic hydrogen-oxidizing bacteria. Strain TK-6(T) is of interest because of the unusually efficient hydrogen-oxidizing ability of this strain, which results in a faster generation time compared to other autotrophs. It is also able to grow anaerobically using nitrate as an electron acceptor when molecular hydrogen is used as the energy source, and able to aerobically fix CO(2)via the reductive tricarboxylic acid cycle. This is the fifth completed genome sequence in the family Aquificaceae, and the second genome sequence determined from a strain derived from the original isolate. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,742,932 bp long genome with its 1,899 protein-coding and 49 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Cytochromes and hydrogen-oxidizing activity in the thermophilic hydrogen-oxidizing bacteria related to the genus Hydrogenobacter

The highly thermophilic, hydrogen-oxidizing aerobic bacteria related to Hydrogenobacter possess a respiratory chain comprising a quinone and b-type (alpha band at 556 nm and 562 nm) and c-type (alpha band at 552 nm) cytochromes. They have no aa₃-type cytochromes and their terminal oxidase is an o-type cytochrome. A polarographic method with an oxygen electrode was used for the measurement of the hydrogen-oxidizing activity. This activity was strongly inhibited by HQNO (2-N-heptyl-4-hydroxyquinoline N-oxide), an inhibitor of the respiratory chain in the quinone-cytochrome b region, and by KCN, an inhibitor of the terminal cytochrome oxidase. This study shows that the electrons released from hydrogen oxidation by the membrane-bound hydrogenase probably enter the respiratory chain at the level of the quinone-cytochrome b region.Abbreviations HQNO 2-N-heptyl-4-hydroxyquinoline N-oxide - TMPD N,N,N',N'-tetramethyl-p-phenylenediamine - DW dry weight     

Hydrogen Oxidation by the Host-Controlled Uptake Hydrogenase Phenotype of Bradyrhizobium japonicum in Symbiosis with Soybean Host Plants

Symbioses between uptake hydrogenase host-regulated (Hup-hr) phenotypes of Bradyrhizobium japonicum and exotic, agronomically unadapted soybean germ plasm were examined for expression of uptake hydrogenase activity. Determinations for hydrogen evolution and uptake hydrogenase activity identified five plant introduction (PI) lines which formed hydrogen-oxidizing symbioses with strains USDA 61 and PA3 6c. Hup-hr strains belonging to serogroup 94 expressed uptake hydrogenase activity in symbioses with PI 181696 and PI 219655 at rates sufficient to prevent hydrogen from escaping the nodules. The identification of soybean germ plasm forming hydrogen-oxidizing symbioses with Hup-hr bradyrhizobia potentially has implications for enhancing nitrogen fixation efficiency in soybean production.     

Fermentation of primary alcohols and diols and pure culture of syntrophically alcohol-oxidizing anaerobes

Ethanol, propanol, ethylene glycol, 1,2-propanediol, 1,2-butanediol, acetoin, diacetyl, and 2,3-pentanedione were used as substrates for enrichment and isolation of alcohol-oxidizing fermentative bacteria. Diacetyl and 2,3-pentanedione proved to be highly toxic. With the other substrates, various kinds of bacteria could be isolated which were assigned to three different metabolic groups: (i) homoacetogenic bacteria, and (ii) bacteria forming propionate as reduced end product were isolated from freshwater sources; (iii) bacteria disproportionating acetoin and 1,2-diols to acids and primary alcohols were isolated from marine sediments. The latter oxidized primary alcohols to fatty acids in the presence of hydrogen-oxidizing partners. Syntrophically ethanol-oxidizing cocultures enriched with primary alcohols could be separated with 1,2-diols as substrates into an alcohol-oxidizing organism and a hydrogen-oxidizing homoacetogen. The pathways of alcohol conversion in the disproportionating isolates were studied in detail. Growth experiments as well as enzymological studies demonstrated that acetoin and 1,2-diols were degraded via acetaldehyde which was also an intermediate in syntrophic oxidation of primary alcohols. The environmental importance of the various metabolic types isolated was assessed by most-probable-number enumerations.     

Characteristics of proteins synthesized by hydrogen-oxidizing microorganisms

The study was conducted to determine the biological value of proteins synthesized by hydrogen-oxidizing microorganisms—the hydrogen bacteria Alcaligenes eutrophus Z1 and Ralstonia eutropha B5786 and the CO-resistant strain of carboxydobacterium Seliberia carboxydohydrogena Z1062. Based on a number of significant parameters characterizing the biological value of a product, the proteins of hydrogen-oxidizing microorganisms have been found to occupy an intermediate position between traditional animal and plant proteins. The high total protein in biomass of these microorganisms, their complete amino acid content, and availability to proteolytic enzymes allow for us to consider these microorganisms as potential protein producers.     

Kinetics of pure cultures of hydrogen-oxidizing denitrifying bacteria and modeling of the interactions among them in mixed cultures

In this study we report the isolation of four denitrifying bacteria from a batch reactor, where the progress of hydrogenotrophic denitrification was examined. Only three of the strains had the ability to use hydrogen as electron donor. In the present work, kinetic batch experiments were carried out in order to study the dynamic characteristics of pure and defined mixed cultures of hydrogen-oxidizing denitrifying bacteria, under anoxic conditions, in a defined synthetic medium, in the presence of nitrates. Kinetic models were developed and the kinetic parameters were determined from the batch experiments for each bacterium separately. The behavior of mixed cultures and the interactions between the bacteria were described using kinetic models based on the kinetic models developed for each bacterium separately and their predictions were compared with the results from mixed culture experiments. The mathematical models that were developed and validated in the present work are capable of describing the behavior of the bacteria in pure and mixed cultures, and in particular, the kinetics of nitrate and nitrite reduction and cell growth.     

Postprandial changes in methanogenic and acidogenic bacteria in the rumens of steers fed high- or low-forage diets once daily.

Four ruminally fistulated Hereford steers (400 kg) were fed two isocaloric diets at 1.5 x maintenance once daily in a repeated measurement crossover experiment. Postprandial changes in hydrogen-oxidizing, carbon dioxide-reducing bacterial groups were monitored. The methanogenic bacterial populations were present at densities of 4 x 10(8) to 8 x 10(8)/g of ruminal contents on either the high- or low-forage diet. Numbers remained constant postprandially on the high-forage diet but showed a distinct rise and fall with the once-daily feeding of the low-forage diet. Presumed hydrogen- and carbon dioxide-utilizing, acid-producing (acidogenic) bacteria were present between 2 x 10(8) and 12 x 10(8)/g of ruminal contents, with the density of the low-forage population being twofold higher than that of the high-forage population. Acidogenic bacteria exhibited similar postprandial changes on both diets, with the predominant shift being associated with the feeding event. This is the first study which documents the postfeeding trends in ruminal methanogenic bacteria on specified, production-level diets. It is also the first study to suggest that other hydrogen-oxidizing, carbon dioxide-reducing bacteria which produce acid instead of methane are present at high population densities in the normally fed adult ruminant.     

Effect of hydrogen on soil bacterial community structure in two soils as determined by terminal restriction fragment length polymorphism

The metabolism of hydrogen evolved from HUP^? legume nodules can alter bacterial community structures in the rhizosphere. Our earlier experiments demonstrated increased hydrogen uptake and appearance of white spots within bacterial colonies in H₂-treated soil. We were also able to isolate hydrogen-oxidizing bacteria from soil samples exposed to hydrogen, but not from samples exposed to air. To further understand the effect of hydrogen metabolism on soil microbial communities, in this study 16S rRNA terminal restriction fragment (TRF) profiles of different soil samples exposed to hydrogen gas under laboratory, greenhouse, and field conditions were analyzed. Relationships between soil bacterial community structures from hydrogen-treated soil samples and controls, illustrated by UPGMA (unpaired group mathematical averages) dendrograms, indicated a significant contribution of hydrogen metabolism to the variation in bacterial community. The intensity variation of TRF peaks includes both hydrogen-utilizing bacteria, whose growth were stimulated by hydrogen exposure, and other bacterial species whose growth was inhibited. Comparison of TRF profiles between laboratory and greenhouse samples showed that T-RFLP is a useful technique in the detection of root-related effects on soil bacterial community structure.     

Utilization of cathodic hydrogen by hydrogen-oxidizing bacteria

Summary Hydrogen is consumed by methanogenic, sulphate-reducing, and homoacetogenic bacteria and members of these bacterial groups are able to grow chemolithotrophically with hydrogen as sole energy source. Cathodic hydrogen consumption by sulphate-reducing bacteria has been proposed as one of the factors in the anaerobic corrosion of metals. Desulfovibrio spp. were able to utilize cathodic hydrogen from mild steel as the only source of energy for growth with sulphate or nitrate as terminal electron acceptor. Other hydrogen-oxidizing bacteria such as Methanospirillum hungatei, Acetobacterium woodii and Wolinella succinogenes were also able to utilize cathodic hydrogen from mild steel for energy generation and growth. Weight loss studies of mild steel coupons under different growth conditions of Desulfovibrio spp. indicated that hydrogen removal alone is not the cause of corrosion and the depolarization phenomenon probably plays a role only in the initiation of the anaerobic microbial corrosion process.     

Postprandial changes in methanogenic and acidogenic bacteria in the rumens of steers fed high- or low-forage diets once daily

Four ruminally fistulated Hereford steers (400 kg) were fed two isocaloric diets at 1.5 x maintenance once daily in a repeated measurement crossover experiment. Postprandial changes in hydrogen-oxidizing, carbon dioxide-reducing bacterial groups were monitored. The methanogenic bacterial populations were present at densities of 4 x 10(8) to 8 x 10(8)/g of ruminal contents on either the high- or low-forage diet. Numbers remained constant postprandially on the high-forage diet but showed a distinct rise and fall with the once-daily feeding of the low-forage diet. Presumed hydrogen- and carbon dioxide-utilizing, acid-producing (acidogenic) bacteria were present between 2 x 10(8) and 12 x 10(8)/g of ruminal contents, with the density of the low-forage population being twofold higher than that of the high-forage population. Acidogenic bacteria exhibited similar postprandial changes on both diets, with the predominant shift being associated with the feeding event. This is the first study which documents the postfeeding trends in ruminal methanogenic bacteria on specified, production-level diets. It is also the first study to suggest that other hydrogen-oxidizing, carbon dioxide-reducing bacteria which produce acid instead of methane are present at high population densities in the normally fed adult ruminant.     

Hydrogen-oxidizing Methane Bacteria I. Cultivation and Methanogenesis

A method for the mass culture of hydrogen-oxidizing methane bacteria has been developed; yields of 50 to 60 g (wet weight) of cells per 12 to 14 liters of culture medium were obtained. The methanogenic organism from the culture of Methanobacillus omelianskii was grown in a complex medium which was aerated with a gas mixture of hydrogen and carbon dioxide. Extracts prepared from hydrogen-grown cells formed methane from methyl cobalamin, 5-methyl tetrahydrofolate, serine, pyruvate, and carbon dioxide; these substrates have been shown to be precursors of methane in extracts of the ethyl alcohol-grown culture of M. omelianskii.     

Anaerobic degradation of isovalerate by a defined methanogenic coculture

Isovalerate-oxidizing strictly aneerobic bacteria were isolated from marine sediment and sewage sludge in coculture with Desulfovibrio sp. Cells stained Gram positive and behaved Gram positive also in Gram classification with KOH. Isovalerate degradation depended on interspecies hydrogen transfer to syntrophic hydrogen-oxidizing sulfate reducers or methanogens. Isovalerate was the only substrate utilized and was fermented to 3 mol acetate and 1 mol hydrogen per mol substrate. The degradation pathway was studied by enzyme assays in crude cell extracts, and included acetyl-CoA dependent activation of isovalerate, oxidation to methylcrotonyl-CoA and carboxylation to methylgluta-conyl-CoA which is hydrated and cleaved to acetoacetate and acetyl-CoA. Studies with inhibitors and ionophores suggest that energy conservation with this organism depends on either acetate efflux-driven proton symport or on an ion-gradient driven carboxylation mechanism.