Possible interactions within a methanotrophic-heterotrophic groundwater community able to transform linear alkylbenzenesulfonates

The relationships and interactions within a methanotrophic-heterotrophic groundwater community were studied in a closed system (shake culture) in the presence of methane as the primary carbon and energy source and with the addition of the pure linear alkylbenzenesulfonate (LAS) congener 2-[4-(sulfophenyl)]decan as a cometabolic substrate. When cultured under different conditions, this community was shown to be a stable association, consisting of one obligate type II methanotroph and four or five heterotrophs possessing different nutritional and physiological characteristics. The results of experiments examining growth kinetics and nutritional relationships suggested that a number of complex interactions existed in the community in which the methanotroph was the only member able to grow on methane and to cometabolically initiate LAS transformation. These growth and metabolic activities of the methanotroph ensured the supply of a carbon source and specific nutrients which sustained the growth of four or five heterotrophs. In addition to the obligatory nutritional relationships between the methanotroph and heterotrophs, other possible interactions resulted in the modification of basic growth parameters of individual populations and a concerted metabolic attack on the complex LAS molecule. Most of these relationships conferred beneficial effects on the interacting populations, making the community adaptable to various environmental conditions and more efficient in LAS transformation than any of the individual populations alone.     

Growth characteristics and metabolic activities of the methanotrophicheterotrophic groundwater community

In this work the growth characteristics and metabolic activities of the methanotrophicheterotrophic groundwater community (culture MM1) as well as of individual community members were studied. When growing in shake flasks, under various methane and oxygen tensions, culture MM1 revealed the capability of a stable association consisting of one obligate methanotroph with type II intracytoplasmic membranes as the dominant strain, and four or five heterotrophs of different morphological, physiological and metabolic characteristics. Coexistence of different populations and the stability of culture MM1 under various conditions suggested that complex relationships may exist between the community members. Most of these relationships seem to be beneficial for both the methanotroph and heterotrophs, making the community adaptable to a range of environmental conditions containing methane as the only carbon source. Furthermore, faster and more complete transformation of 2-[4- (sulphophenyl)]decane (2C₁₀LAS) by the community than by any of the community members alone, illustrates the role and importance of methanotrophicheterotrophic interactions in combined metabolic attack on complex linear alkylbenzenesulphonates molecules.     

Biodegradation of linear alkylbenzenesulphonates (LAS) by mixed methanotrophic-heterotrophic cultures

The biodegradation of undecylbenzenesulphonate (C₁₁LAS) was studied in shake flasks at 21°C using two mixed bacterial cultures. The first culture, MM1, contained a type II methanotroph and four heterotrophs, and was enriched from a groundwater aquifer. The second culture, MC, consisted of five heterotrophic strains, most of them belonging to the genus Pseudomonas , and was isolated from the wastewater of a detergent plant. Methane, carbon dioxide and oxygen concentrations were determined by gas chromatography. Concentrations of C₁₁LAS and the aromatic intermediates were determined by reversed-phase HPLC. In spite of faster transformation of the alkyl side-chain by the culture MC, the culture MM1 containing type II methanotroph was capable of further degradation of C₁₁LAS aromatic intermediates (sulphophenylalkanoates). The most probable mechanism for the degradation of the alkyl part of the C₁₁LAS molecule by both cultures was β-oxidation of the terminal methyl group followed by β-oxidation. Studies of methane utilization demonstrated an approximately three times higher second-order rate coefficient for methane consumption ( k _max/ K _s) in the absence of C₁₁LAS. This indicates a possible metabolic activity of methanotrophs in the transformation of the complex LAS molecule due to the methane monooxygenase enzyme system.     

Genomic Insights into Methanotrophy: The Complete Genome Sequence of Methylococcus capsulatus (Bath)

Methanotrophs are ubiquitous bacteria that can use the greenhouse gas methane as a sole carbon and energy source for growth, thus playing major roles in global carbon cycles, and in particular, substantially reducing emissions of biologically generated methane to the atmosphere. Despite their importance, and in contrast to organisms that play roles in other major parts of the carbon cycle such as photosynthesis, no genome-level studies have been published on the biology of methanotrophs. We report the first complete genome sequence to our knowledge from an obligate methanotroph, Methylococcus capsulatus (Bath), obtained by the shotgun sequencing approach. Analysis revealed a 3.3-Mb genome highly specialized for a methanotrophic lifestyle, including redundant pathways predicted to be involved in methanotrophy and duplicated genes for essential enzymes such as the methane monooxygenases. We used phylogenomic analysis, gene order information, and comparative analysis with the partially sequenced methylotroph Methylobacterium extorquens to detect genes of unknown function likely to be involved in methanotrophy and methylotrophy. Genome analysis suggests the ability of M. capsulatus to scavenge copper (including a previously unreported nonribosomal peptide synthetase) and to use copper in regulation of methanotrophy, but the exact regulatory mechanisms remain unclear. One of the most surprising outcomes of the project is evidence suggesting the existence of previously unsuspected metabolic flexibility in M. capsulatus, including an ability to grow on sugars, oxidize chemolithotrophic hydrogen and sulfur, and live under reduced oxygen tension, all of which have implications for methanotroph ecology. The availability of the complete genome of M. capsulatus (Bath) deepens our understanding of methanotroph biology and its relationship to global carbon cycles. We have gained evidence for greater metabolic flexibility than was previously known, and for genetic components that may have biotechnological potential.     

Enantiomeric Degradation of 2-(4-Sulfophenyl)Butyrate via 4-Sulfocatechol in Delftia acidovorans SPB1

Enrichment cultures with enantiomeric 2-(4-sulfophenyl)butyrate (SPB) as the sole added source(s) of carbon and energy for growth yielded a pure culture of a degradative bacterium, which was identified as Delftia acidovorans SPB1. The organism utilized the enantiomers sequentially. R-SPB was utilized first (specific growth rate [μ] = 0.28 h⁻¹), with transient excretion of an unknown intermediate, which was identified as 4-sulfocatechol (4SC). Utilization of S-SPB was slower (μ = 0.016 h⁻¹) and was initiated only after the first enantiomer was exhausted. Suspensions of cells grown in S-SPB excreted 4SC, so metabolism of the two enantiomers converged at 4SC. The latter was degraded by ortho cleavage via 3-sulfo-cis,cis-muconate. Strain SPB1 grew with 4SC and with 1-(4-sulfophenyl)octane (referred to herein as model LAS) but not with commercial linear alkylbenzenesulfonate (LAS) surfactant, which is subterminally substituted but nontoxic. It would appear that metabolism of the model LAS does not represent metabolism of commercial LAS.     

Genomic insights into methanotrophy: the complete genome sequence of Methylococcus capsulatus (Bath)

Methanotrophs are ubiquitous bacteria that can use the greenhouse gas methane as a sole carbon and energy source for growth, thus playing major roles in global carbon cycles, and in particular, substantially reducing emissions of biologically generated methane to the atmosphere. Despite their importance, and in contrast to organisms that play roles in other major parts of the carbon cycle such as photosynthesis, no genome-level studies have been published on the biology of methanotrophs. We report the first complete genome sequence to our knowledge from an obligate methanotroph, Methylococcus capsulatus (Bath), obtained by the shotgun sequencing approach. Analysis revealed a 3.3-Mb genome highly specialized for a methanotrophic lifestyle, including redundant pathways predicted to be involved in methanotrophy and duplicated genes for essential enzymes such as the methane monooxygenases. We used phylogenomic analysis, gene order information, and comparative analysis with the partially sequenced methylotroph Methylobacterium extorquens to detect genes of unknown function likely to be involved in methanotrophy and methylotrophy. Genome analysis suggests the ability of M. capsulatus to scavenge copper (including a previously unreported nonribosomal peptide synthetase) and to use copper in regulation of methanotrophy, but the exact regulatory mechanisms remain unclear. One of the most surprising outcomes of the project is evidence suggesting the existence of previously unsuspected metabolic flexibility in M. capsulatus, including an ability to grow on sugars, oxidize chemolithotrophic hydrogen and sulfur, and live under reduced oxygen tension, all of which have implications for methanotroph ecology. The availability of the complete genome of M. capsulatus (Bath) deepens our understanding of methanotroph biology and its relationship to global carbon cycles. We have gained evidence for greater metabolic flexibility than was previously known, and for genetic components that may have biotechnological potential.     

丙烯经酶催化氧化制环氧丙烷——一株甲烷氧化细菌的分离筛选及生理特性的研究

环氧丙烷是聚氨酯、不饱和聚酯和优质洗涤剂的主要原料,还可用于油漆、化妆品等,是一种非常重要的精细化工原料。目前环氧丙烷主要用氯醇法和烷基过氧化氢法生产。1963年,Vender Lindent发现庚烷菌P.Seruginosa的休止细胞可使辛烯-1氧化成环氧辛烷,首次提出了烯烃经生物催化环氧化生成相应环氧化物的过程。1977年,Colby等报导了从Methylococcus capsu-latus(Bath)菌中提取了非专一性菌甲烷单加氧酶。1979年,C.T.Hou等分离出二十多种甲烷氧化细菌都能使C_2—C_4烯烃氧化成     

Diversity in methane enrichments from agricultural soil revealed by DGGE separation of PCR amplified 16S rDNA fragments

Denaturing gradient gel electrophoresis (DGGE) profiles of PCR amplified V3 regions of 16S rRNA genes were used to assess the diversity in enrichment cultures with methane as the only carbon and energy source. The enrichments originated from two agricultural soils. One was a sandy soil with low (10%) organic content, the other an organic soil with approximately 50% organic content. DGGE provided a fast evaluation of the distribution of amplifiable sequence types indicating that specific bacterial populations had been enriched from each soil. The DGGE profiles revealed a broader range of amplified V3 fragments in the community derived from organic soil than from sandy soil. Fragments from 19 individual DGGE bands were sequenced and compared with 27 previously published 16S rRNA gene sequences. The sequences confirmed the high diversity with the presence of different methylotrophic populations in each enrichment. No affiliation was found with type I methanotrophs, instead type II methanotroph sequences were found in the enrichments from both soil types. Some of the fragments from the organic soil enrichment were not affiliated with methylotrophs. Most of the sequences clustered distantly on a branch within the α-Proteobacteria. These facts suggested that previously undescribed methylotrophs are abundant in methane enrichments from agricultural soil.     

Termites Facilitate Methane Oxidation and Shape the Methanotrophic Community

Termite-derived methane contributes 3 to 4% to the total methane budget globally. Termites are not known to harbor methane-oxidizing microorganisms (methanotrophs). However, a considerable fraction of the methane produced can be consumed by methanotrophs that inhabit the mound material, yet the methanotroph ecology in these environments is virtually unknown. The potential for methane oxidation was determined using slurry incubations under conditions with high (12%) and in situ (∼0.004%) methane concentrations through a vertical profile of a termite (Macrotermes falciger) mound and a reference soil. Interestingly, the mound material showed higher methanotrophic activity. The methanotroph community structure was determined by means of a pmoA-based diagnostic microarray. Although the methanotrophs in the mound were derived from populations in the reference soil, it appears that termite activity selected for a distinct community. Applying an indicator species analysis revealed that putative atmospheric methane oxidizers (high-indicator-value probes specific for the JR3 cluster) were indicative of the active nest area, whereas methanotrophs belonging to both type I and type II were indicative of the reference soil. We conclude that termites modify their environment, resulting in higher methane oxidation and selecting and/or enriching for a distinct methanotroph population.     

Selection of associated heterotrophs by methane-oxidizing bacteria at different copper concentrations

Due to the increasing atmospheric concentration of the greenhouse gas methane, more knowledge is needed on the management of methanotrophic communities. While most studies have focused on the characteristics of the methane-oxidizing bacteria (MOB), less is known about their interactions with the associated heterotrophs. Interpretative tools based on denaturing gradient gel electrophoresis allowed to evaluate the influence of copper—an important enzymatic regulator for MOB—on the activity and composition of the bacterial community. Over 30 days, enrichments with 0.1, 1.0 and 10 μM Cu²⁺ respectively, showed comparable methane oxidation activities. The different copper concentrations did not create major shifts in the methanotrophic communities, as a Methylomonas sp. was able to establish dominance at all different copper concentrations by switching between both known methane monooxygenases. The associated heterotrophic communities showed continuous shifts, but over time all cultures evolved to a comparable composition, independent of the copper concentration. This indicates that the MOB selected for certain heterotrophs, possibly fulfilling vital processes such as removal of toxic compounds. The presence of a large heterotrophic food web indirectly depending on methane as sole carbon and energy source was confirmed by a clone library wherein MOB only formed a minority of the identified species.     

Bacterial [methyl-3H]thymidine incorporation in substrate-amended estuarine sediment slurries

Abstract Five different bacterial communities were enriched in substrate-amended slurries of sediment from the Tay Estuary, Scotland. During incubation of the slurries, concentrations of volatile fatty acids, sulphate, sulphide and methane were monitored to clearly define the activity of the stimulated populations. An aerobic population, a ‘microaerophilic’ population and three anaerobic populations (fermentative heterotrophs, sulphate-reducing bacteria and methanogens plus acetogens) were established to reflect community growth and metabolism both in surface oxic and deeper anoxic layers. Similar numbers of cells involved in division were observed in all five slurries, demonstrating the potential for bacterial production. Thymidine incorporation rates in glucose-stimulated slurries under both aerobic and fully anaerobic conditions were similar, confirming the ability of fermentative anaerobic heterotrophs to incorporate [ methyl -³H]thymidine into DNA during growth. Although anaerobic communities of sulphate-reducing, acetogenic plus methanogenic bacteria were stimulated and actively growing, they did not incorporate [ methyl -³H]thymidine into DNA. Since the thymidine technique does not measure the growth of these important groups, calculated productivity values based upon thymidine incorporation within anoxic sediment systems will be substantially underestimated, even if growth substrates are not limiting.     

Exploring methanotroph diversity in acidic northern wetlands: Molecular and cultivation-based studies

Acidic wetlands of the northern hemisphere are an important source of methane, a major greenhouse gas. The taxonomic identity of the aerobic methanotrophic bacteria, which colonize these environments and reduce the potential flux of methane to the atmosphere, has remained elusive for a long time. Both cultivation-independent molecular approaches and cultivation-based studies have been used to identify methanotrophs in this acidic habitat. It was shown that acidic peat is colonized mainly by methanotrophic representatives of the Alphaproteobacteria: Methylocystis spp., Methylocella spp. and Methylocapsa spp. Novel methanotrophic isolates from acidic wetlands display a number of unique characteristics and metabolic traits including unusual cell ultrastructure and fatty acid composition, ability to utilize some multicarbon compounds as growth substrates, and new regulatory mechanisms of methane oxidation. Several other methanotroph populations, which have been detected in acidic peat by molecular approaches but have so far eluded isolation, represent a challenge for further cultivation studies.     

Population dynamics of type I and II methanotrophic bacteria in rice soils

Methane-oxidizing bacteria (methanotrophs) consume a significant but variable fraction of greenhouse-active methane gas produced in wetlands and rice paddies before it can be emitted to the atmosphere. Temporal and spatial dynamics of methanotroph populations in California rice paddies were quantified using phospholipid biomarker analyses in order to evaluate the relative importance of type I and type II methanotrophs with depth and in relation to rice roots. Methanotroph population fluctuations occurred primarily within the top 0-2 cm of soil, where methanotroph cells increased by a factor of 3-5 over the flooded rice-growing season. The results indicate that rice roots and rhizospheres were less important than the soil-water interface in supporting methanotroph growth. Both type I and type II methanotrophs were abundant throughout the year. However, only type II populations were strongly correlated with soil porewater methane concentrations and rice growth.     

Atmospheric constraints on the evolution of metabolism

Earth's early history may have been characterized by coevolution of microbial metabolism and atmospheric composition. Metabolic developments affected the composition of the atmosphere and the resultant changes in the atmosphere stimulated the evolution of new metabolic capabilities.The first organisms were presumably fermenting heterotrophs, exploiting organic molecules abiotically synthesized. These organisms multiplied, developing new biosynthetic capabilities to overcome deficiencies in the abiotic supply of particular compounds, until their growth was limited by the energy source provided by abiotic synthesis of fermentable organic compounds. Further growth required a new energy source, which may have been the chemical energy represented by the mixture of carbon dioxide and hydrogen in the primitive atmosphere. Chemotrophic organisms resembling methane bacteria may have evolved to exploit this source. They would have flourished, along with the heterotrophs that fed on them, until they had decreased the level of atmospheric hydrogen to the point where further extraction of chemical energy from the atmosphere was not possible. Once again, the expansion of life was limited by the availability of energy.The origin of bacterial photosynthesis overcame the second energy crisis. Photosynthetic bacteria could exploit the abundant energy of sunlight while using atmospheric hydrogen and reduced compounds derived from it only as electron donors. Life flourished again, drawing atmospheric hydrogen (replenished only by volcanoes) down to levels so low as to limit even bacterial photosynthesis. Before the full potential of photosynthesis could be exploited the evolution of the metabolic apparatus to process an electron donor of unlimited abundance was necessary. This donor, of course, was water, and the new metabolic process was algal photosynthesis. The oxygen released changed the world from anaerobic to aerobic and made possible the last great advance in energy-yielding metabolism, aerobic respiration.Proceedings of the Fourth College Park Colloquium on Chemical Evolution:Limits of Life, University of Maryland, College Park, 18–20 October 1978.     

甲烷氧化菌研究进展

甲烷氧化菌以甲烷为其唯一的碳源和能源 ,在全球大气甲烷平衡中起着重要的作用 ,它还可以降解卤代化合物 ,在污染治理方面具有潜在价值。本文从甲烷氧化菌的分类出发 ,对甲烷氧化菌氧化甲烷的机理及影响因素、甲烷氧化菌的生理、生态分布及检测方法、甲烷氧化菌降解有机污染物的潜在应用等作一综述 ,分析目前研究中存在的问题 ,并指出今后应加强研究的方面。     

Toxic Effects of Linear Alkylbenzene Sulfonate on Metabolic Activity, Growth Rate, and Microcolony Formation of Nitrosomonas and Nitrosospira Strains

Strong inhibitory effects of the anionic surfactant linear alkylbenzene sulfonate (LAS) on four strains of autotrophic ammonia-oxidizing bacteria (AOB) are reported. Two Nitrosospira strains were considerably more sensitive to LAS than two Nitrosomonas strains were. Interestingly, the two Nitrosospira strains showed a weak capacity to remove LAS from the medium. This could not be attributed to adsorption or any other known physical or chemical process, suggesting that biodegradation of LAS took place. In each strain, the metabolic activity (50% effective concentration [EC₅₀], 6 to 38 mg liter⁻¹) was affected much less by LAS than the growth rate and viability (EC₅₀, 3 to 14 mg liter⁻¹) were. However, at LAS levels that inhibited growth, metabolic activity took place only for 1 to 5 days, after which metabolic activity also ceased. The potential for adaptation to LAS exposure was investigated with Nitrosomonas europaea grown at a sublethal LAS level (10 mg liter⁻¹); compared to control cells, preexposed cells showed severely affected cell functions (cessation of growth, loss of viability, and reduced NH₄⁺ oxidation activity), demonstrating that long-term incubation at sublethal LAS levels was also detrimental. Our data strongly suggest that AOB are more sensitive to LAS than most heterotrophic bacteria are, and we hypothesize that thermodynamic constraints make AOB more susceptible to surfactant-induced stress than heterotrophic bacteria are. We further suggest that AOB may comprise a sensitive indicator group which can be used to determine the impact of LAS on microbial communities.     

Effects of hydrocarbon enrichment on trichloroethylene biodegradation and microbial populations in finished compost

This study focused on the capacity of finished compost, often used as packing material in biofiltration units, to support microbial biodegradation of trichloroethylene (TCE). Finished compost was enriched with methane or propane (10% head space) to stimulate cometabolic biodegradation of gaseous TCE. Successful hydrocarbon enrichment, as indicated by rapid depletion of hydrocarbon gas and measurable growth of hydrocarbon-utilizing micro-organisms, occurred within a week. Within batch reactor flasks, approximately 75% of head space TCE (1–40 ppmv) was rapidly sorbed onto compost material. Up to 99% of the remaining head space TCE was removed via biodegradation in compost enriched with either hydrocarbon. Hydrocarbon enrichment with methane or propane corresponded to 10-fold increases in methanotrophic or propanotrophic populations, respectively. Based on growth assessment under different nutritional regimes, there appeared to be complex metabolic interactions within the microbial community in enriched compost. Five separate bacterial cultures were derived from the hydrocarbon-enriched compost and assayed for the ability to degrade TCE.     

Successional Changes in an Evolving Anaerobic Chlorophenol-Degrading Community Used To Infer Relationships between Population Structure and System-Level Processes

The response of a complex methanogenic sediment community to 2-chlorophenol (2-CP) was evaluated by monitoring the concentrations of this model contaminant and important metabolic intermediates and products and by using rRNA-targeted probes to track several microbial populations. Key relationships between the evolving population structure, formation of metabolic intermediates, and contaminant mineralization were identified. The nature of these relationships was intrinsically linked to the metabolism of benzoate, an intermediate that transiently accumulated during the mineralization of 2-CP. Before the onset of benzoate fermentation, reductive dehalogenation of 2-CP competed with methanogenesis for endogenous reducing equivalents. This suppressed H₂ levels, methane production, and archaeal small-subunit (SSU)-rRNA concentrations in the sediment community. The concentrations of bacterial SSU rRNA, including SSU rRNA derived from “Desulfovibrionaceae” populations, tracked with 2-CP levels, presumably reflecting changes in the activity of dehalogenating organisms. After the onset of benzoate fermentation, the abundance of Syntrophus-like SSU rRNA increased, presumably because these syntrophic organisms fermented benzoate to methanogenic substrates. Consequently, although the parent substrate 2-CP served as an electron acceptor, cleavage of its aromatic nucleus also influenced the sediment community by releasing the electron donors H₂ and acetate. Increased methane production and archaeal SSU-rRNA levels, which tracked with the Syntrophus-like SSU-rRNA concentrations, revealed that methanogenic populations in particular benefited from the input of reducing equivalents derived from 2-CP.     

Towards elucidation of microbial community metabolic pathways: unravelling the network of carbon sharing in a pollutant-degrading bacterial consortium by immunocapture and isotopic ratio mass spectrometry

Although much information on metabolic pathways within individual organisms is available, little is known about the pathways operating in natural communities in which extensive sharing of nutritional resources is the rule. In order to analyse such a consortium pathway, we have investigated the flow of 4-chlorosalicylate as carbon substrate within a simple chemostat microbial community using ¹³C-labelled metabolites and isotopic ratio mass spectrometric analysis of label enrichment in immunocaptured member populations of the community. A complex pathway network of carbon sharing was thereby revealed, involving two different metabolic routes, one of which is completely novel and involves the toxic metabolite protoanemonin. The high stability of the community results, at least in part, from interdependencies based on carbon sharing and the rapid removal of toxic metabolites.     

Revealing the community and metabolic potential of active methanotrophs by targeted metagenomics in the Zoige wetland of the Tibetan Plateau

The Zoige wetland of the Tibetan Plateau is one of the largest alpine wetlands in the world and a major emission source of methane. Methane oxidation by methanotrophs can counteract the global warming effect of methane released in the wetlands. Understanding methanotroph activity, diversity and metabolism at the molecular level can guide the isolation of the uncultured microorganisms and inform strategy-making decisions and policies to counteract global warming in this unique ecosystem. Here we applied DNA stable isotope probing using ¹³C-labelled methane to label the genomes of active methanotrophs, examine the methane oxidation potential and recover metagenome-assembled genomes (MAGs) of active methanotrophs. We found that gammaproteobacteria of type I methanotrophs are responsible for methane oxidation in the wetland. We recovered two phylogenetically novel methanotroph MAGs distantly related to extant Methylobacter and Methylovulum. They belong to type I methanotrophs of gammaproteobacteria, contain both mxaF and xoxF types of methanol dehydrogenase coding genes, and participate in methane oxidation via H₄MPT and RuMP pathways. Overall, the community structure of active methanotrophs and their methanotrophic pathways revealed by DNA-SIP metagenomics and retrieved methanotroph MAGs highlight the importance of methanotrophs in suppressing methane emission in the wetland under the scenario of global warming.