Bioremediation via Methanotrophy: Overview of Recent Findings and Suggestions for Future Research

Microbially mediated bioremediation of polluted sites has been a subject of much research over the past 30 years, with many different compounds shown to be degraded under both aerobic and anaerobic conditions. Aerobic-mediated bioremediation commonly examines the use of methanotrophs, microorganisms that consume methane as their sole source of carbon and energy. Given the diverse environments in which methanotrophs have been found, the range of substrates they can degrade and the fact that they can be easily stimulated with the provision of methane and oxygen, these microorganisms in particular have been examined for aerobic degradation of chlorinated hydrocarbons. The physiological and phylogenetic diversity of methanotrophy, however, has increased substantially in just the past 5 years. Here in this review, the current state of knowledge of methanotrophy, particularly as it applies to pollutant degradation is summarized, and suggestions for future research provided

Quantification of gene expression in methanotrophs by competitive reverse transcription-polymerase chain reaction

To improve the monitoring of methanotrophic activity, a competitive reverse transcription-polymerase chain reaction (RT-PCR) methodology was developed. Homologous internal RNA standards were created for mmoX and pmoA , genes encoding polypeptides of sMMO and pMMO, respectively. Using specific primer sets, expression of sMMO and pMMO could be quantified by means of competitive RT-PCR and capillary electrophoresis with uncoated bare-fused silica columns and UV detection. Using this technique, it was discovered that the amount of mRNA transcript for both mmoX and pmoA correlated well with whole-cell sMMO and pMMO activity respectively. A method for soil RNA extraction was also developed to utilize this RNA quantification technique for the monitoring of methanotrophic activity in situ . In a model soil slurry system with a background concentration of 2.9 µM copper, it was found that only pmoA was transcribed by cells capable of expressing both forms of MMO. As pMMO and sMMO have very different substrate ranges and kinetics, this methodology may prove useful for optimizing in situ bioremediation by methanotrophs. Provided sufficient sequence information is available to create specific primer sets, these techniques can be applied for monitoring and measuring the activity of other microbial communities in situ .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75134/1/j.1462-2920.2004.00572.x.pd

Methanotrophs and copper

Methanotrophs, cells that consume methane (CH 4 ) as their sole source of carbon and energy, play key roles in the global carbon cycle, including controlling anthropogenic and natural emissions of CH 4 , the second-most important greenhouse gas after carbon dioxide. These cells have also been widely used for bioremediation of chlorinated solvents, and help sustain diverse microbial communities as well as higher organisms through the conversion of CH 4 to complex organic compounds (e.g. in deep ocean and subterranean environments with substantial CH 4 fluxes). It has been well-known for over 30 years that copper (Cu) plays a key role in the physiology and activity of methanotrophs, but it is only recently that we have begun to understand how these cells collect Cu, the role Cu plays in CH 4 oxidation by the particulate CH 4 monooxygenase, the effect of Cu on the proteome, and how Cu affects the ability of methanotrophs to oxidize different substrates. Here we summarize the current state of knowledge of the phylogeny, environmental distribution, and potential applications of methanotrophs for regional and global issues, as well as the role of Cu in regulating gene expression and proteome in these cells, its effects on enzymatic and whole-cell activity, and the novel Cu uptake system used by methanotrophs.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79061/1/j.1574-6976.2010.00212.x.pd

Measurement and modeling of multiple substrate oxidation by methanotrophs at 20 °C

Earlier experiments have shown that when Methylosinus trichosporium OB3b was grown at 30 °C, greater growth and degradation of chlorinated ethenes was observed under particulate methane monooxygenase (pMMO)-expressing conditions than sMMO-expressing conditions. The effect of temperature on the growth and ability of methanotrophs to degrade chlorinated ethenes, however, has not been examined, particularly temperatures more representative of groundwater systems. Thus, experiments were performed at 20 °C to examine the effect of mixtures of trichloroethylene, trans -dichloroethylene and vinyl chloride in the presence of methane on the growth and ability of Methylosinus trichosporium OB3b cells to degrade these pollutants. Although the maximal rates of chlorinated ethane degradation were greater by M. trichosporium OB3b expressing sMMO as compared with the same cell expressing pMMO, the growth and ability of sMMO-expressing cells to degrade these cosubstrates was substantially inhibited in their presence as compared with the same cell expressing pMMO. The Δ model developed earlier was found to be useful for predicting the effect of chlorinated ethenes on the growth and ability of M. trichosporium OB3b to degrade these compounds at a growth temperature of 20 °C. Finally, it was also discovered that at 20 °C, cells expressing pMMO exhibited faster turnover of methane than sMMO-expressing cells, unlike that found earlier at 30 °C, suggesting that temperature may exert selective pressure on methanotrophic communities to express sMMO or pMMO.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75464/1/j.1574-6968.2008.01314.x.pd

Uptake and effect of rare earth elements on gene expression in Methylosinus trichosporium OB3b

It is well known that Methylosinus trichosporium OB3b has two forms of methane monooxygenase (MMO) responsible for the initial conversion of methane to methanol, a cytoplasmic (soluble) methane monooxygenase and a membrane-associated (particulate) methane monooxygenase, and that copper strongly regulates expression of these alternative forms of MMO. More recently, it has been discovered that M. trichosporium OB3b has multiple types of the methanol dehydrogenase (MeDH), i.e. the Mxa-type MeDH (Mxa-MeDH) and Xox-type MeDH (Xox-MeDH), and the expression of these two forms is regulated by the availability of the rare earth element (REE), cerium. Here, we extend these studies and show that lanthanum, praseodymium, neodymium and samarium also regulate expression of alternative forms of MeDH. The effect of these REEs on MeDH expression, however, was only observed in the absence of copper. Further, a mutant of M. trichosporium OB3b, where the Mxa-MeDH was knocked out, was able to grow in the presence of lanthanum, praseodymium and neodymium, but was not able to grow in the presence of samarium. Collectively, these data suggest that multiple levels of gene regulation by metals exist in M. trichosporium OB3b, but that copper overrides the effect of other metals by an as yet unknown mechanism

Degradation of chlorinated and brominated hydrocarbons by Methylomicrobium album BG8

The degradation kinetics of ten halogenated hydrocarbons by Methylomicrobium album BG8 expressing particulate methane monooxygenase (pMMO) and the inhibitory effects of these compounds on microbial growth and whole-cell pMMO activity were measured. When M. album BG8 was grown with methane, growth was completely inhibited by dichloromethane (DCM), bromoform (BF), chloroform (CF), vinyl chloride (VC), 1,1-dichloroethylene (1,1-DCE), and cis -dichloroethylene ( cis -DCE). Trichloroethylene (TCE) partially inhibited growth on methane, while dibromomethane (DBM), trans -dichloroethylene ( trans -DCE), and 1,1,1-trichloroethane (1,1,1-TCA) had no effect. If the cells were grown with methanol, DCM, BF, CF, and 1,1-DCE completely inhibited growth, while VC, trans -DCE, TCE, and 1,1,1-TCA partially inhibited growth. Both DBM and cis -DCE had no effect on growth with methanol. Whole-cell pMMO activity was also affected by these compounds, with all but 1,1,1-TCA, DCM, and DBM reducing activity by more than 25%. DCM, DBM, VC, trans -DCE, cis -DCE, 1,1-DCE, and TCE were degraded and followed Michaelis-Menten kinetics. CF, BF, and 1,1,1-TCA were not measurably degraded. These results suggested that the products of DCM, TCE, VC, and 1,1-DCE inactivated multiple enzymatic processes, while trans -DCE oxidation products were also toxic but to a lesser extent. cis -DCE toxicity, however, appeared to be localized to pMMO. Finally, DBM and 1,1,1-TCA were not inhibitory, and CF and BF were themselves toxic to M. album BG8. Based on these results, the compounds could be separated into four general categories, namely (1) biodegradable with minimal inactivation, (2) biodegradable with substantial inactivation, (3) not biodegradable with minimal inactivation, and (4) not biodegradable but substantial inactivation of cell activity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41918/1/203-172-6-393_91720393.pd

Dichloromethane and trichloroethylene inhibition of methane oxidation by the membrane-associated methane monooxygenase of Methylosinus trichosporium OB3b

Whole-cell assays were used to measure the effect of dichloromethane and trichloroethylene on methane oxidation by Methylosinus trichosporium OB3b synthesizing the membrane-associated or particulate methane monooxygenase (pMMO). For M. trichosporium OB3b grown with 20 μM copper, no inhibition of methane oxidation was observed in the presence of either dichloromethane or trichloroethylene. If 20 mM formate was added to the reaction vials, however, methane oxidation rates increased and inhibition of methane oxidation was observed in the presence of dichloromethane and trichloroethylene. In the presence of formate, dichloromethane acted as a competitive inhibitor, while trichloroethylene acted as a noncompetitive inhibitor. The finding of noncompetitive inhibition by trichloroethylene was further examined by measuring the inhibition constants K iE and K iES . These constants suggest that trichloroethylene competes with methane at some sites, although it can bind to others if methane is already bound. Whole-cell oxygen uptake experiments for active and acetylene-treated cells also showed that provision of formate could stimulate both methane and trichloroethylene oxidation and that trichloroethylene did not affect formate dehydrogenase activity. The finding that different chlorinated hydrocarbons caused different inhibition patterns can be explained by either multiple substrate binding sites existing in pMMO or multiple forms of pMMO with different activities. The whole-cell analysis performed here cannot distinguish between these models, and further work should be done on obtaining active preparations of the purified pMMO.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41917/1/203-171-5-301_91710301.pd

Transformation of ortho-substituted biphenyls by Methylosinus trichosporium OB3b: substituent effects on oxidation kinetics and product formation

The ability of Methylosinus trichosporium OB3b, expressing soluble methane monooxygenase, to oxidize a range of ortho- substituted biphenyls was examined to better understand how substituents affect both the rate and products of oxidation in comparison to biphenyl. Inhibition of oxidation was observed over the tested substrate range for both biphenyl and ortho- halogenated biphenyls (2-chloro-, 2-bromo-, and 2-iodobiphenyl). No inhibition was observed during the oxidation of 2-hydroxybiphenyl and 2-methylbiphenyl. Analysis of the products of oxidation showed that, depending on the substituent, ring hydroxylation, substituent oxidation, and elimination pathways could occur. The type and abundance of products formed along with the relatively high kinetic isotope effect observed for deuterated vs. nondeuterated biphenyl ( k h / k d = 3.4±0.02) are consistent with mechanisms that include both hydrogen abstraction and NIH-shift pathways. Knowledge of these substituent-dependent reaction rates and mechanisms enhances our understanding of the methanotrophic aryl transformation potential and allows for better prediction of the formation of oxidized intermediates by methanotrophic bacteria.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41919/1/203-174-1-2-35_s002030000170.pd

Microbial Fouling of a Reverse Osmosis Municipal Water Treatment System

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146838/1/wer0703.pd

Draft genome sequences of gammaproteobacterial methanotrophs isolated from marine ecosystems

The genome sequences of Methylobacter marinus A45, Methylobacter sp. strain BBA5.1, and Methylomarinum vadi IT-4 were obtained. These aerobic methanotrophs are typical members of coastal and hydrothermal vent marine ecosystems