Adverse Effect of the Methanotroph Methylocystis sp. M6 on the Non-Methylotroph Microbacterium sp. NM2.

Several non-methylotrophic bacteria have been reported to improve the growth and activity of methanotrophs; however, their interactions remain to be elucidated. We investigated the interaction between Methylocystis sp. M6 and Microbacterium sp. NM2. A batch co-culture experiment showed that NM2 markedly increased the biomass and methane removal of M6. qPCR analysis revealed that NM2 enhanced both the growth and methane-monooxygenase gene expression of M6. A fed-batch experiment showed that co-culture was more efficient in removing methane than M6 alone (28.4 vs. 18.8 µmol·l-1·d-1), although the biomass levels were similar. A starvation experiment for 21 days showed that M6 population remained stable while NM2 population decreased by 66% in co-culture, but the results were opposite in pure cultures, indicating that M6 may cross-feed growth substrates from NM2. These results indicate that M6 apparently had no negative effect on NM2 when M6 actively proliferated with methane. Interestingly, a batch experiment involving a dialysis membrane indicates that physical proximity between NM2 and M6 is required for such biomass and methane removal enhancement. Collectively, the observed interaction is beneficial to the methanotroph but adversely affects the non-methylotroph; moreover, it requires physical proximity, suggesting a tight association between methanotrophs and non-methylotrophs in natural environments.

Co-cultivation of the strictly anaerobic methanogen Methanosarcina barkeri with aerobic methanotrophs in an oxygen-limited membrane bioreactor.

Wetlands contribute to 30% of global methane emissions due to an imbalance between microbial methane production and consumption. Methanogenesis and methanotrophy have mainly been studied separately, and little is known about their potential interactions in aquatic environments. To mimic the interaction between methane producers and oxidizers in the environment, we co-cultivated the methanogenic archaeon Methanosarcina barkeri with aerobic Methylocystaceae methanotrophs in an oxygen-limited bioreactor using acetate as methanogenic substrate. Methane, acetate, dissolved oxygen, available nitrogen, pH, temperature, and cell density were monitored to follow system stability and activity. Stable reactor operation was achieved for two consecutive periods of 2 months. Fluorescence in situ hybridization micrographs indicated close association between both groups of microorganisms. This association suggests that the methanotrophs profit from direct access to the methane that is produced from acetate, while methanogens are protected by the concomitant oxygen consumption of the methanotrophs. This proof of principle study can be used to set up systems to study their responses to environmental changes.

Colonization on Cucumber Root and Enhancement of Chlorimuron-ethyl Degradation in the Rhizosphere by Hansschlegelia zhihuaiae S113 and Root Exudates.

The colonization of Hansschlegelia zhihuaiae S113 and its degradation of the herbicide chlorimuron-ethyl in the cucumber rhizosphere was investigated. The results reveal that S113 colonized the cucumber roots (2.14 × 105cells per gram of roots) and were able to survive in the rhizosphere (maintained for 20 d). The root exudates promoted colonization on roots and increased the degradation of chlorimuron-ethyl by S113. Five organic acids in cucumber-root exudates were detected and identified by HPLC. Citric acid and fumaric acid significantly stimulated S113 colonization on cucumber roots, with 18.4 and 15.5% increases, respectively, compared with the control. After irrigation with an S113 solution for 10 days, chlorimuron-ethyl could not be detected in the roots, seedlings, or rhizosphere soil, which allowed for improved cucumber growth. Therefore, the degradation mechanism of chlorimuron-ethyl residues by S113 in the rhizosphere could be applied in situ for the bioremediation of chlorimuron-ethyl contaminated soil to ensure crop safety.

Living apart together-bacterial volatiles influence methanotrophic growth and activity.

Volatile organic compounds play an important role in microbial interactions. However, little is known about how volatile-mediated interactions modulate biogeochemical processes. In this study, we show the effect of volatile-mediated interaction on growth and functioning of aerobic methane-oxidizing bacteria, grown in co-culture with five different heterotrophs. Both growth and methane oxidation of Methylobacter luteus were stimulated by interaction with specific heterotrophs. In Methylocystis parvus, we observed significant growth promotion, while methane oxidation was inhibited. Volatolomics of the interaction of each of the methanotrophs with Pseudomonas mandelii, revealed presence of a complex blend of volatiles, including dimethylsulfide, dimethyldisulfide, and bicyclic sesquiterpenes. Although the ecological role of the detected compounds remains to be elucidated, our results provide unprecedented insights into interspecific relations and associated volatiles for stimulating methanotroph functioning, which is of substantial environmental and biotechnological significance.

Effects of salinity on simultaneous reduction of perchlorate and nitrate in a methane-based membrane biofilm reactor.

This study builds upon prior work showing that methane (CH4) could be utilized as the sole electron donor and carbon source in a membrane biofilm reactor (MBfR) for complete perchlorate (ClO4-) and nitrate (NO3-) removal. Here, we further investigated the effects of salinity on the simultaneous removal of the two contaminants in the reactor. By testing ClO4- and NO3- at different salinities, we found that the reactor performance was very sensitive to salinity. While 0.2 % salinity did not significantly affect the hydrogen-based MBfR for ClO4- and NO3- removals, 1 % salinity completely inhibited ClO4- reduction and significantly lowered NO3- reduction in the CH4-based MBfR. In salinity-free conditions, NO3- and ClO4- removal fluxes were 0.171 g N/m2-day and 0.091 g/m2-day, respectively, but NO3- removal fluxes dropped to 0.0085 g N/m2-day and ClO4- reduction was completely inhibited when the medium changed to 1 % salinity. Scanning electron microscopy (SEM) showed that the salinity dramatically changed the microbial morphology, which led to the development of wire-like cell structures. Quantitative real-time PCR (qPCR) indicated that the total number of microorganisms and abundances of functional genes significantly declined in the presence of NaCl. The relative abundances of Methylomonas (methanogens) decreased from 31.3 to 5.9 % and Denitratisoma (denitrifiers) decreased from 10.6 to 4.4 % when 1 % salinity was introduced.

Biological Methanol Production by a Type II Methanotroph Methylocystis bryophila.

Methane (CH4) is the most abundant component in natural gas. To reduce its harmful environmental effect as a greenhouse gas, CH4 can be utilized as a low-cost feed for the synthesis of methanol by methanotrophs. In this study, several methanotrophs were examined for their ability to produce methanol from CH4; including Methylocella silvestris, Methylocystis bryophila, Methyloferula stellata, and Methylomonas methanica. Among these methanotrophs, M. bryophila exhibited the highest methanol production. The optimum process parameters aided in significant enhancement of methanol production up to 4.63 mM. Maximum methanol production was observed at pH 6.8, 30°C, 175 rpm, 100 mM phosphate buffer, 50 mM MgCl2 as a methanol dehydrogenase inhibitor, 50% CH4 concentration, 24 h of incubation, and 9 mg of dry cell mass ml(-1) inoculum load, respectively. Optimization of the process parameters, screening of methanol dehydrogenase inhibitors, and supplementation with formate resulted in significant improvements in methanol production using M. bryophila. This report suggests, for the first time, the potential of using M. bryophila for industrial methanol production from CH4.

Conversion of methane-derived carbon and microbial community in enrichment cultures in response to O2 availability.

Methanotrophs not only play an important role in mitigating CH4 emissions from the environment, but also provide a large quantity of CH4-derived carbon to their habitats. In this study, the distribution of CH4-derived carbon and microbial community was investigated in a consortium enriched at three O2 tensions, i.e., the initial O2 concentrations of 2.5 % (LO-2), 5 % (LO-1), and 21 % (v/v) (HO). The results showed that compared with the O2-limiting environments (2.5 and 5 %), more CH4-derived carbon was converted into CO2 and biomass under the O2 sufficient condition (21 %). Besides biomass and CO2, a high conversion efficiency of CH4-derived carbon to dissolved organic carbon was detected in the cultures, especially in LO-2. Quantitative PCR and Miseq sequencing both showed that the abundance of methanotroph increased with the increasing O2 concentrations. Type II methanotroph Methylocystis dominated in the enrichment cultures, accounting for 54.8, 48.1, and 36.9 % of the total bacterial 16S rRNA gene sequencing reads in HO, LO-1, and LO-2, respectively. Methylotrophs, mainly including Methylophilus, Methylovorus, Hyphomicrobium, and Methylobacillus, were also abundant in the cultures. Compared with the O2 sufficient condition (21 %), higher microbial biodiversity (i.e., higher Simpson and lower Shannon indexes) was detected in LO-2 enriched at the initial O2 concentration of 2.5 %. These findings indicated that compared with the O2 sufficient condition, more CH4-derived carbon was exuded into the environments and promoted the growth of non-methanotrophic microbes in O2-limiting environments.

Effect of CH4/O2 ratio on fatty acid profile and polyhydroxybutyrate content in a heterotrophic-methanotrophic consortium.

Understanding the role of heterotrophic-methanotrophic (H-Meth) communities is important for improvement of methane (CH4) oxidation capacities (MOC) particularly in conjunction with bio-product development in industrial bio-filters. Initially, a H-Meth consortium was established and enriched from marine sediments and characterized by next generation sequencing of the 16s rDNA gene. The enriched consortium was subjected to 10-50% CH4 (i.e., 0.20-1.6 CH4/O2 ratios) to study the effects on MOCs, biomass growth, fatty acid profiles and biopolymer (e.g. polyhydroxybutyrate; PHB) content. Methylocystis, Methylophaga and Pseudoxanthomonas dominated the H-Meth consortium. Culture enrichment of the H-Meth consortium resulted in 15-20-folds higher MOC compared to seed sediments. Increasing CH4 concentration (and decreased O2 levels) yielded higher MOCs, but did not improve total fatty acid contents. PHB contents varied between 2.5% and 8.5% independently of CH4/O2 ratios. The results suggest that H-Meth consortia could potentially be used in industrial bio-filters for production of biopolymer/biofuel precursors from CH4.

Optimization of Methanotrophic Growth and Production of Poly(3-Hydroxybutyrate) in a High-Throughput Microbioreactor System.

Production of poly(3-hydroxybutyrate) (P3HB) from methane has economic and environmental advantages over production by agricultural feedstock. Identification of high-productivity strains and optimal growth conditions is critical to efficient conversion of methane to polymer. Current culture conditions, including serum bottles, shake flasks, and agar plates, are labor-intensive and therefore insufficient for systematic screening and isolation. Gas chromatography, the standard method for analysis of P3HB content in bacterial biomass, is also incompatible with high-throughput screening. Growth in aerated microtiter plates coupled with a 96-well Nile red flow-cytometric assay creates an integrated microbioreactor system for high-throughput growth and analysis of P3HB-producing methanotrophic cultures, eliminating the need for individual manipulation of experimental replicates. This system was tested in practice to conduct medium optimization for P3HB production in pure cultures of Methylocystis parvus OBBP. Optimization gave insight into unexpected interactions: for example, low calcium concentrations significantly enhanced P3HB production under nitrogen-limited conditions. Optimization of calcium and copper concentrations in the growth medium increased final P3HB content from 18.1% to 49.4% and P3HB concentration from 0.69 g/liter to 3.43 g/liter while reducing doubling time from 10.6 h to 8.6 h. The ability to culture and analyze thousands of replicates with high mass transfer in completely mixed culture promises to streamline medium optimization and allow the detection and isolation of highly productive strains. Applications for this system are numerous, encompassing analysis of biofuels and other lipid inclusions, as well as analysis of heterotrophic and photosynthetic systems.

Mercury binding by methanobactin from Methylocystis strain SB2.

Methanobactin (mb) is a post-translationally modified copper-binding compound, or chalkophore, secreted by many methane-oxidizing bacteria or methanotrophs in response to copper limitation. In addition to copper, methanobactin from Methylosinus trichosporium OB3b (mb-OB3b) has been shown to bind a variety of metals including Hg(2+). In this report, Hg binding by the structurally unique methanobactin from Methylocystis strain SB2 (mb-SB2) was examined and compared to mb-OB3b. Mb-SB2 is shown to bind the common forms of Hg found in aqueous environments, Hg(2+), Hg(CN)2 and CH3Hg(+). The spectral and thermodynamic properties of binding for each form of mercury differed. UV-visible absorption spectra suggested that Hg(2+) binds to both the oxazolone and imidazolone rings of mb-SB2, whereas CH3Hg(+) appeared to only bind to the oxazolone ring. Hg(CN)2 showed spectral properties between Hg(2+) and CH3Hg(+). Isothermal titration calorimetry (ITC) showed both Hg(CN)2 and CH3Hg(+) fit into two-site binding models. For Hg(CN)2 the first site was exothermic and the second endothermic. Both binding sites in CH3Hg(+) were exothermic, but at equilibrium the reaction never moved back to the baseline, suggesting a slow residual reaction. ITC results for Hg(2+) were more complex and suggested a 3- or 4-site model. The spectral, kinetic and thermodynamic changes following Hg binding by mb-SB2 also differed from the changes associated with mb-OB3b. Like mb-OB3b, copper did not displace Hg bound to mb-SB2. In contrast to mb-OB3b Hg(2+) could displace Cu from Cu-containing mb-SB2 and preferentially bound Hg(2+) over Cu(2+) at metal to mb-SB2 molar ratios above 1.0.

Formate oxidation-driven calcium carbonate precipitation by Methylocystis parvus OBBP.

Microbially induced carbonate precipitation (MICP) applied in the construction industry poses several disadvantages such asammonia release to the air and nitric acid production. An alternative MICP from calcium formate by Methylocystis parvus OBBP is presented here to overcome these disadvantages. To induce calcium carbonate precipitation, M. parvus was incubated at different calcium formate concentrations and starting culture densities. Up to 91.4% ± 1.6% of the initial calcium was precipitated in the methane-amended cultures compared to 35.1% ± 11.9% when methane was not added. Because the bacteria could only utilize methane for growth, higher culture densities and subsequently calcium removals were exhibited in the cultures when methane was added. A higher calcium carbonate precipitate yield was obtained when higher culture densities were used but not necessarily when more calcium formate was added. This was mainly due to salt inhibition of the bacterial activity at a high calcium formate concentration. A maximum 0.67 ± 0.03 g of CaCO3 g of Ca(CHOOH)2(-1) calcium carbonate precipitate yield was obtained when a culture of 10(9) cells ml(-1) and 5 g of calcium formate liter(-)1 were used. Compared to the current strategy employing biogenic urea degradation as the basis for MICP, our approach presents significant improvements in the environmental sustainability of the application in the construction industry.

Effects of ammonium on the activity and community of methanotrophs in landfill biocover soils.

The influence of NH4(+) on microbial CH4 oxidation is still poorly understood in landfill cover soils. In this study, effects of NH4(+) addition on the activity and community structure of methanotrophs were investigated in waste biocover soil (WBS) treated by a series of NH4(+)-N contents (0, 100, 300, 600 and 1200mgkg(-1)). The results showed that the addition of NH4(+)-N ranging from 100 to 300mgkg(-1) could stimulate CH4 oxidation in the WBS samples at the first stage of activity, while the addition of an NH4(+)-N content of 600mgkg(-1) had an inhibitory effect on CH4 oxidation in the first 4 days. The decrease of CH4 oxidation rate observed in the last stage of activity could be caused by nitrogen limitation and/or exopolymeric substance accumulation. Type I methanotrophs Methylocaldum and Methylobacter, and type II methanotrophs (Methylocystis and Methylosinus) were abundant in the WBS samples. Of these, Methylocaldum was the main methanotroph in the original WBS. With incubation, a higher abundance of Methylobacter was observed in the treatments with NH4(+)-N contents greater than 300mgkg(-1), which suggested that NH4(+)-N addition might lead to the dominance of Methylobacter in the WBS samples. Compared to type I methanotrophs, the abundance of type II methanotrophs Methylocystis and/or Methylosinus was lower in the original WBS sample. An increase in the abundance of Methylocystis and/or Methylosinus occurred in the last stage of activity, and was likely due to a nitrogen limitation condition. Redundancy analysis showed that NH4(+)-N and the C/N ratio had a significant influence on the methanotrophic community in the WBS sample.

Stoichiometry and kinetics of the PHB-producing Type II methanotrophs Methylosinus trichosporium OB3b and Methylocystis parvus OBBP.

In this study, modeling is used to describe how oxygen and nitrogen source affect the stoichiometry and kinetics of growth and PHB production in the Type II methanotrophs Methylosinus trichosporium OB3b and Methylocystis parvus OBBP. Significant differences were observed, with major implications for the use of these species in biotechnology applications. Such analyses can better inform bioreactor design, scale-up models, and life cycle assessments (LCAs).

The relative contribution of methanotrophs to microbial communities and carbon cycling in soil overlying a coal-bed methane seep.

Seepage of coal-bed methane (CBM) through soils is a potential source of atmospheric CH4 and also a likely source of ancient (i.e. (14) C-dead) carbon to soil microbial communities. Natural abundance (13) C and (14) C compositions of bacterial membrane phospholipid fatty acids (PLFAs) and soil gas CO2 and CH4 were used to assess the incorporation of CBM-derived carbon into methanotrophs and other members of the soil microbial community. Concentrations of type I and type II methanotroph PLFA biomarkers (16:1ω8c and 18:1ω8c, respectively) were elevated in CBM-impacted soils compared with a control site. Comparison of PLFA and 16s rDNA data suggested type I and II methanotroph populations were well estimated and overestimated by their PLFA biomarkers, respectively. The δ(13) C values of PLFAs common in type I and II methanotrophs were as negative as -67‰ and consistent with the assimilation of CBM. PLFAs more indicative of nonmethanotrophic bacteria had δ(13) C values that were intermediate indicating assimilation of both plant- and CBM-derived carbon. Δ(14) C values of select PLFAs (-351 to -936‰) indicated similar patterns of CBM assimilation by methanotrophs and nonmethanotrophs and were used to estimate that 35-91% of carbon assimilated by nonmethanotrophs was derived from CBM depending on time of sampling and soil depth.

Priority pollutant degradation by the facultative methanotroph, Methylocystis strain SB2.

Methylocystis strain SB2, a facultative methanotroph capable of growth on multi-carbon compounds, was screened for its ability to degrade the priority pollutants 1,2-dichloroethane (1,2-DCA), 1,1,2-trichloroethane (1,1,2-TCA), and 1,1-dichloroethylene (1,1-DCE), as well as cis-dichloroethylene (cis-DCE) when grown on methane or ethanol. Methylocystis strain SB2 degraded 1,2-DCA and 1,1,2-TCA when grown on either substrate and cis-DCE when grown on methane. Growth of Methylocystis strain SB2 on methane was inhibited in the presence of all compounds, while only 1,1-DCE and cis-DCE inhibited growth on ethanol. No degradation of any chlorinated hydrocarbon was observed in ethanol-grown cultures when particulate methane monooxygenase (pMMO) activity was inhibited with the addition of acetylene, indicating that competition for binding to the pMMO between the chlorinated hydrocarbons and methane limited both methanotrophic growth and pollutant degradation when this strain was grown on methane. Characterization of Methylocystis strain SB2 found no evidence of a high-affinity form of pMMO for methane, nor could this strain utilize 1,2-DCA or its putative oxidative products 2-chloroethanol or chloroactetic acid as sole growth substrates, suggesting that this strain lacks appropriate dehydrogenases for the conversion of 1,2-DCA to glyoxylate. As ethanol: (1) can be used as an alternative growth substrate for promoting pollutant degradation by Methylocystis strain SB2 as the pMMO is not required for its growth on ethanol and (2) has been used to enhance the mobility of chlorinated hydrocarbons in situ, it is proposed that ethanol can be used to enhance both pollutant transport and biodegradation by Methylocystis strain SB2.

[Methanotrophs and their applications in environment treatment: a review].

Methanotrophs can oxidize methane, playing an important role in regulating methane emission, and gaining increasing attention by the researchers around the world. Two biological pathways are involved in methane oxidation, i.e., anaerobic oxidation and aerobic oxidation, which are governed by anaerobic and aerobic methanotrophs, respectively. In this paper, the research advances about methanotrophs were summarized, with the focus on the phylogeny and taxonomy of methanotrophs, the key enzymes responsible for the aerobic oxidation of methane, the microorganisms involved in the anaerobic oxidation of methane, and the mechanisms of microbial methane consumption. The application prospects of the two methane oxidizers in greenhouse gases removal, pollutants degradation, biological denitrification, and recovery of metals and sulfur compounds were also analyzed.

Composition of methane-oxidizing bacterial communities as a function of nutrient loading in the Florida everglades.

Agricultural runoff of phosphorus (P) in the northern Florida Everglades has resulted in several ecosystem level changes, including shifts in the microbial ecology of carbon cycling, with significantly higher methane being produced in the nutrient-enriched soils. Little is, however, known of the structure and activities of methane-oxidizing bacteria (MOB) in these environments. To address this, 0 to 10 cm plant-associated soil cores were collected from nutrient-impacted (F1), transition (F4), and unimpacted (U3) areas, sectioned in 2-cm increments, and methane oxidation rates were measured. F1 soils consumed approximately two-fold higher methane than U3 soils; additionally, most probable numbers of methanotrophs were 4-log higher in F1 than U3 soils. Metabolically active MOB containing pmoA sequences were characterized by stable-isotope probing using 10 % (v/v) (13)CH(4). pmoA sequences, encoding the alpha subunit of methane monooxygenase and related to type I methanotrophs, were identified from both impacted and unimpacted soils. Additionally, impacted soils also harbored type II methanotrophs, which have been shown to exhibit preferences for high methane concentrations. Additionally, across all soils, novel pmoA-type sequences were also detected, indicating presence of MOB specific to the Everglades. Multivariate statistical analyses confirmed that eutrophic soils consisted of metabolically distinct MOB community that is likely driven by nutrient enrichment. This study enhances our understanding on the biological fate of methane being produced in productive wetland soils of the Florida Everglades and how nutrient-enrichment affects the composition of methanotroph bacterial communities.

Community structure, abundance, and activity of methanotrophs in the Zoige wetland of the Tibetan Plateau.

The Zoige wetland of the Tibetan Plateau is a high-altitude tundra wetland and one of the biggest methane emission centers in China. In this study, methanotrophs with respect to community structure, abundance, and activity were investigated in peat soils collected in the vicinity of different marshland plants that dominate different regions of the wetland, including Polygonum amphibium, Carex muliensis, and Eleocharis valleculosa (EV). 16S rRNA gene and particulate methane monooxygenase gene (pmoA) clone library sequence data indicated the presence of methanotrophs with two genera, Methylobacter and Methylocystis. Methylococcus, like pmoA gene sequences, were also retrieved and showed low similarity to those from Methylococcus spp. and thus indicates the existence of novel methanotrophs in the Zoige wetland. Quantitative polymerase chain reaction (qPCR) assays were used to measure the abundance of methantrophs and detected 10(7) to 10(8) of total pmoA gene copies per gram dry weight of soil in the three marshes. Group-specific qPCR and reverse transcriptase qPCR results found that the Methylobacter genus dominates the wetland, and Methylocystis methanotrophs were less abundant, although this group of methanotrophs was estimated to be more active according to mRNA/DNA ratio. Furthermore, EV marsh demonstrated the highest methanotrophs abundance and activity among the three marshes investigated. Our study suggests that both type I and type II methanotrophs contribute to the methane oxidation in the Zoige wetland.

Selection of Type I and Type II methanotrophic proteobacteria in a fluidized bed reactor under non-sterile conditions.

Type II methanotrophs produce polyhydroxybutyrate (PHB), while Type I methanotrophs do not. A laboratory-scale fluidized bed reactor was initially inoculated with a Type II Methylocystis-like dominated culture. At elevated levels of dissolved oxygen (DO, 9 mg/L), pH of 6.2-6.5 with nitrate as the N-source, a Methylobacter-like Type I methanotroph became dominant within the biofilms which did not produce PHB. A shift to biofilms capable of PHB production was achieved by re-inoculating with Type II Methylosinus culture, providing dissolved N(2) as the N-source, and maintaining a low influent DO (2.0mg/L). The resulting biofilms contained both Types I and II methanotrophs. Batch tests indicated that biofilm samples grown with N(2) became dominated by Type II methanotrophs and produced PHB. Enrichments with nitrate or ammonium were dominated by Type I methanotrophs without PHB production capability. The key selection factors favoring Type II were N(2) as N-source and low DO.

Pollutant degradation by a Methylocystis strain SB2 grown on ethanol: bioremediation via facultative methanotrophy.

A facultative methanotroph, Methylocystis strain SB2, was examined for its ability to degrade chlorinated hydrocarbons when grown on methane or ethanol. Strain SB2 grown on methane degraded vinyl chloride (VC), trans-dichloroethylene (t-DCE), trichloroethylene (TCE), 1,1,1-trichloroethane (1,1,1-TCA), and chloroform (CF), but not dichloromethane (DCM). Growth on methane was reduced in the presence of any chlorinated hydrocarbon. Strain SB2 grown on ethanol degraded VC, t-DCE, and TCE, and 1,1,1-TCA, but not DCM or CF. With the exception of 1,1,1-TCA, the growth of strain SB2 on ethanol was not affected by any individual chlorinated hydrocarbon. No degradation of any chlorinated hydrocarbon was observed when acetylene was added to ethanol-grown cultures, indicating that this degradation was due to particulate methane monooxygenase (pMMO) activity. When mixtures of chlorinated alkanes or alkenes were added to cultures growing on methane or ethanol, chlorinated alkene degradation occurred, but chlorinated alkanes were not, and growth was reduced on both methane and ethanol. Collectively, these data indicate that competitive inhibition of pMMO activity limits methanotrophic growth and pollutant degradation. Facultative methanotrophy may thus be useful to extend the utility of methanotrophs for bioremediation as the use of alternative growth substrates allows for pMMO activity to be focused on pollutant degradation.