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.     

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.     

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.     

Optimization of diagnostic microarray for application in analysing landfill methanotroph communities under different plant covers

Landfill sites are responsible for 6-12% of global methane emission. Methanotrophs play a very important role in decreasing landfill site methane emissions. We investigated the methane oxidation capacity and methanotroph diversity in lysimeters simulating landfill sites with different plant vegetations. Methane oxidation rates were 35 g methane m-2 day-1 or higher for planted lysimeters and 18 g methane m-2 day-1 or less for bare soil controls. Best methane oxidation, as displayed by gas depth profiles, was found under a vegetation of grass and alfalfa. Methanotroph communities were analysed at high throughput and resolution using a microbial diagnostic microarray targeting the particulate methane monooxygenase (pmoA) gene of methanotrophs and functionally related bacteria. Members of the genera Methylocystis and Methylocaldum were found to be the dominant members in landfill site simulating lysimeters. Soil bacterial communities in biogas free control lysimeters, which were less abundant in methanotrophs, were dominated by Methylocaldum. Type Ia methanotrophs were found only in the top layers of bare soil lysimeters with relatively high oxygen and low methane concentrations. A competetive advantage of type II methanotrophs over type Ia methanotrophs was indicated under all plant covers investigated. Analysis of average and individual results from parallel samples was used to identify general trends and variations in methanotroph community structures in relation to depth, methane supply and plant cover. The applicability of the technology for the detection of environmental perturbations was proven by an erroneous result, where an unexpected community composition detected with the microarray indicated a potential gas leakage in the lysimeter being investigated.     

Syntrophic co-culture of a methanotroph and heterotroph for the efficient conversion of methane to mevalonate

As the bioconversion of methane becomes increasingly important for bio-industrial and environmental applications, methanotrophs have received much attention for their ability to convert methane under ambient conditions. This includes the extensive reporting of methanotroph engineering for the conversion of methane to biochemicals. To further increase methane usability, we demonstrated a highly flexible and efficient modular approach based on a synthetic consortium of methanotrophs and heterotrophs mimicking the natural methane ecosystem to produce mevalonate (MVA) from methane. In the methane-conversion module, we used Methylococcus capsulatus Bath as a highly efficient methane biocatalyst and optimized the culture conditions for the production of high amounts of organic acids. In the MVA-synthesis module, we used Escherichia coli SBA01, an evolved strain with high organic acid tolerance and utilization ability, to convert organic acids to MVA. Using recombinant E. coli SBA01 possessing genes for the MVA pathway, 61 mg/L (0.4 mM) of MVA was successfully produced in 48 h without any addition of nutrients except methane. Our platform exhibited high stability and reproducibility with regard to cell growth and MVA production. We believe that this versatile system can be easily extended to many other value-added processes and has a variety of potential applications.     

Aerobic methane oxidation and methanotroph community composition during seasonal stratification in Mono Lake, California (USA)

Patterns of aerobic methane (CH4) oxidation and associated methanotroph community composition were investigated during the development of seasonal stratification in Mono Lake, California (USA). CH4 oxidation rates were measured using a tritiated CH4 radiotracer technique. Fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) and sequence analysis were used to characterize methanotroph community composition. A temporally shifting zone of elevated CH4 oxidation (59-123 nM day(-1)) was consistently associated with a suboxycline, microaerophilic zone that migrated upwards in the water column as stratification progressed. FISH analysis revealed stable numbers of type I (4.1-9.3 x 10(5) cells ml(-1)) and type II (1.4-3.4 x 10(5) cells ml(-1)) methanotrophs over depth and over time. Denaturing gradient gel electrophoresis and sequence analysis indicated slight shifts in methanotroph community composition despite stable absolute cell numbers. Variable CH4 oxidation rates in the presence of a relatively stable methanotroph population suggested that zones of high CH4 oxidation resulted from an increase in activity of a subset of the existing methanotroph population. These results challenge existing paradigms suggesting that zones of elevated CH4 oxidation activity result from the accumulation of methanotrophic biomass and illustrate that type II methanotrophs may be an important component of the methanotroph population in saline and/or alkaline pelagic environments.     

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.     

含有甲烷氧化菌的混合菌群特性研究

为获得高效甲烷氧化微生物体系,从农业土壤中采样,以甲烷作为唯一碳源进行好氧选择性传代培养,得到生长性能稳定、生长优于Methylosinus trichosporium OB3b纯培养的具有甲烷单加氧酶(Methane Monooxygenase,MMO)活性甲烷氧化混合菌。利用MMO的共代谢特性,分别以苯酚和环氧丙烷作为目标对象,考察该混合菌对有机污染物的降解及用于生产有用化学物质的催化特性。结果表明,所得混合菌具有高效降解苯酚能力,对初始浓度为600mg/L的苯酚,经过11h培养,苯酚降解率可达99%。另外,以该混合菌为催化剂可以实现丙烯氧化生产环氧丙烷。通过降低磷酸盐浓度可以有效提高环氧丙烷的积累浓度,最大可至5mmol/L。此外,采用纯种分离方法结合PCR扩增、16SrRNA和MMO功能基因分析技术对混合菌群结构进行解析。结果表明,该混合菌群由Ⅱ型甲烷氧化菌及其它至少4种非甲烷氧化菌组成,它们分别属于Methylosinus trichosporium和Acinetobacter junii、Cupriavidusme tallidurans、Comamonas testosteroni和Stenotrophomonas maltophilia。采用PCR方法从混合菌及纯化菌株M.trichosporiums Y9总DNA中都能扩增得到mmoB、mmoX和pmoA基因片段,表明该甲烷氧化菌同时具有sMMO和pMMO两种形式的MMO。通过对从甲烷氧化混合菌中分离纯化得到的甲烷氧化菌进行PCR产物测序,结果发现其与Methylosinus trichosporium的同源性为99.9%。     

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     

Effect of mineral media on trichloroethylene oxidation by aquifer methanotrophs

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

Methanotroph and methanogen coupling in granular biofilm under O2-limited conditions

Summary The co-culture between Methylosinus sporium, a strictly aerobic methanotroph, and strictly anaerobic methanogens was studied in 5 L aerobic/anaerobic coupled granular sludge reactors under O₂-limited conditions. The methanogenic bacteria maintained very good metabolic activities and were able to produce sufficient methane which serviced as substrate for methanotrophic growth. Although other strictly aerobic population proliferated by two orders of magnitude after the granular sludge had been operated under O₂-limited conditions for one month, only a limited amount of the added methanotroph remained in the sludge. This result may indicate that M. sporium lacks sufficient O₂ affinity to compete with facultative bacteria for the dissolved O₂ for their growth.     

Inhibition of trichloroethylene oxidation by the transformation intermediate carbon monoxide

Inhibition of trichloroethylene (TCE) oxidation by the transformation intermediate carbon monoxide (CO) was evaluated with the aquifer methanotroph Methylomonas sp. strain MM2. CO was a TCE transformation intermediate. During TCE oxidation, approximately 9 mol% of the TCE was transformed to CO. CO was oxidized by Methylomonas sp. strain MM2, and when formate was provided as an electron donor, the CO oxidation rate doubled. The rate of CO oxidation without formate was 4.6 liter mg (dry weight)-1 day-1, and the rate with formate was 10.2 liter mg (dry weight)-1 day-1. CO inhibited TCE oxidation, both by exerting a demand for reductant and through competitive inhibition. The Ki for CO inhibition of TCE oxidation, 4.2 microM, was much less than the Ki for methane inhibition of TCE oxidation, 116 microM. CO also inhibited methane oxidation, and the degree of inhibition increased with increasing CO concentration. When CO was present, formate amendment was necessary for methane oxidation to occur and both substrates were simultaneously oxidized. CO at a concentration greater than that used in the inhibition studies was not toxic to Methylomonas sp. strain MM2.     

Inhibition of trichloroethylene oxidation by the transformation intermediate carbon monoxide.

Inhibition of trichloroethylene (TCE) oxidation by the transformation intermediate carbon monoxide (CO) was evaluated with the aquifer methanotroph Methylomonas sp. strain MM2. CO was a TCE transformation intermediate. During TCE oxidation, approximately 9 mol% of the TCE was transformed to CO. CO was oxidized by Methylomonas sp. strain MM2, and when formate was provided as an electron donor, the CO oxidation rate doubled. The rate of CO oxidation without formate was 4.6 liter mg (dry weight)-1 day-1, and the rate with formate was 10.2 liter mg (dry weight)-1 day-1. CO inhibited TCE oxidation, both by exerting a demand for reductant and through competitive inhibition. The Ki for CO inhibition of TCE oxidation, 4.2 microM, was much less than the Ki for methane inhibition of TCE oxidation, 116 microM. CO also inhibited methane oxidation, and the degree of inhibition increased with increasing CO concentration. When CO was present, formate amendment was necessary for methane oxidation to occur and both substrates were simultaneously oxidized. CO at a concentration greater than that used in the inhibition studies was not toxic to Methylomonas sp. strain MM2.     

Analysis of methane monooxygenase genes in mono lake suggests that increased methane oxidation activity may correlate with a change in methanotroph community structure

Mono Lake is an alkaline hypersaline lake that supports high methane oxidation rates. Retrieved pmoA sequences showed a broad diversity of aerobic methane oxidizers including the type I methanotrophs Methylobacter (the dominant genus), Methylomicrobium, and Methylothermus, and the type II methanotroph Methylocystis. Stratification of Mono Lake resulted in variation of aerobic methane oxidation rates with depth. Methanotroph diversity as determined by analysis of pmoA using new denaturing gradient gel electrophoresis primers suggested that variations in methane oxidation activity may correlate with changes in methanotroph community composition.     

Stable coexistence of five bacterial strains as a cellulose-degrading community

A cellulose-degrading defined mixed culture (designated SF356) consisting of five bacterial strains (Clostridium straminisolvens CSK1, Clostridium sp. strain FG4, Pseudoxanthomonas sp. strain M1-3, Brevibacillus sp. strain M1-5, and Bordetella sp. strain M1-6) exhibited both functional and structural stability; namely, no change in cellulose-degrading efficiency was observed, and all members stably coexisted through 20 subcultures. In order to investigate the mechanisms responsible for the observed stability, "knockout communities" in which one of the members was eliminated from SF356 were constructed. The dynamics of the community structure and the cellulose degradation profiles of these mixed cultures were determined in order to evaluate the roles played by each eliminated member in situ and its impact on the other members of the community. Integration of each result gave the following estimates of the bacterial relationships. Synergistic relationships between an anaerobic cellulolytic bacterium (C. straminisolvens CSK1) and two strains of aerobic bacteria (Pseudoxanthomonas sp. strain M1-3 and Brevibacillus sp. strain M1-5) were observed; the aerobes introduced anaerobic conditions, and C. straminisolvens CSK1 supplied metabolites (acetate and glucose). In addition, there were negative relationships, such as the inhibition of cellulose degradation by producing excess amounts of acetic acid by Clostridium sp. strain FG4, and growth suppression of Bordetella sp. strain M1-6 by Brevibacillus sp. strain M1-5. The balance of the various types of relationships (both positive and negative) is thus considered to be essential for the stable coexistence of the members of this mixed culture.     

Amsterdam urban canals contain novel niches for methane-cycling microorganisms

Urbanised environments have been identified as hotspots of anthropogenic methane emissions. Especially urban aquatic ecosystems are increasingly recognised as important sources of methane. However, the microbiology behind these emissions remains unexplored. Here, we applied microcosm incubations and molecular analyses to investigate the methane-cycling community of the Amsterdam canal system in the Netherlands. The sediment methanogenic communities were dominated by Methanoregulaceae and Methanosaetaceae, with co-occurring methanotrophic Methanoperedenaceae and Methylomirabilaceae indicating the potential for anaerobic methane oxidation. Methane was readily produced after substrate amendment, suggesting an active but substrate-limited methanogenic community. Bacterial 16S rRNA gene amplicon sequencing of the sediment revealed a high relative abundance of Thermodesulfovibrionia. Canal wall biofilms showed the highest initial methanotrophic potential under oxic conditions compared to the sediment. During prolonged incubations the maximum methanotrophic rate increased to 8.08 mmol g_DW⁻¹ d⁻¹ that was concomitant with an enrichment of Methylomonadaceae bacteria. Metagenomic analysis of the canal wall biofilm lead to the recovery of a single methanotroph metagenome-assembled genome. Taxonomic analysis showed that this methanotroph belongs to the genus Methyloglobulus. Our results underline the importance of previously unidentified and specialised environmental niches at the nexus of the natural and human-impacted carbon cycle.     

The more,the merrier: heterotroph richness stimulates methanotrophic activity

Although microorganisms coexist in the same environment, it is still unclear how their interaction regulates ecosystem functioning. Using a methanotroph as a model microorganism, we determined how methane oxidation responds to heterotroph diversity. Artificial communities comprising of a methanotroph and increasing heterotroph richness, while holding equal starting cell numbers were assembled. We considered methane oxidation rate as a functional response variable. Our results showed a significant increase of methane oxidation with increasing heterotroph richness, suggesting a complex interaction in the cocultures leading to a stimulation of methanotrophic activity. Therefore, not only is the methanotroph diversity directly correlated to methanotrophic activity for some methanotroph groups as shown before, but also the richness of heterotroph interacting partners is relevant to enhance methane oxidation too. In this unprecedented study, we provide direct evidence showing how heterotroph richness exerts a response in methanotroph–heterotroph interaction, resulting in increased methanotrophic activity. Our study has broad implications in how methanotroph and heterotroph interact to regulate methane oxidation, and is particularly relevant in methane-driven ecosystems.     

甲烷利用细菌降解三氯乙烯的研究

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