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Methanobactins (MBs) are ribosomally produced and post-translationally modified peptides (RiPPs) that are used by methanotrophs for copper acquisition. The signature post-translational modification of MBs is the formation of two heterocyclic groups, either an oxazolone, pyrazinedione or imidazolone group, with an associated thioamide from an X-Cys dipeptide. The precursor peptide (MbnA) for MB formation is found in a gene cluster of MB-associated genes. The exact biosynthetic pathway of MB formation is not yet fully understood, and there are still uncharacterized proteins in some MB gene clusters, particularly those that produce pyrazinedione or imidazolone rings. One such protein is MbnF, which is proposed to be a flavin monooxygenase (FMO) based on homology. To help to elucidate its possible function, MbnF from Methylocystis sp. strain SB2 was recombinantly produced in Escherichia coli and its X-ray crystal structure was resolved to 2.6 Å resolution. Based on its structural features, MbnF appears to be a type A FMO, most of which catalyze hydroxylation reactions. Preliminary functional characterization shows that MbnF preferentially oxidizes NADPH over NADH, supporting NAD(P)H-mediated flavin reduction, which is the initial step in the reaction cycle of several type A FMO enzymes. It is also shown that MbnF binds the precursor peptide for MB, with subsequent loss of the leader peptide sequence as well as the last three C-terminal amino acids, suggesting that MbnF might be needed for this process to occur. Finally, molecular-dynamics simulations revealed a channel in MbnF that is capable of accommodating the core MbnA fragment minus the three C-terminal amino acids.

Methylmercury is a potent neurotoxin that biomagnifies through food webs and which production depends on anaerobic microbial uptake of inorganic mercury (Hg) species. One outstanding knowledge gap in understanding Hg methylation is the nature of bioavailable Hg species. It has become increasingly obvious that Hg bioavailability is spatially diverse and temporally dynamic but current models are mostly built on single thiolated ligand systems, omitting ligand exchanges and interactions, or the inclusion of dissolved gaseous phases. In this study, we used a whole-cell anaerobic biosensor to determine the role of a mixture of thiolated ligands on Hg bioavailability. Serendipitously, we discovered how the diffusion of trace amounts of exogenous biogenic H

Aerobic methanotrophic activity is highly dependent on copper availability, and methanotrophs have developed multiple strategies to collect copper. Specifically, when copper is limiting (ambient concentrations less than 1 μM), some methanotrophs produce and secret a small modified peptide (less than 1,300 Da) termed methanobactin (MB) that binds copper with high affinity. As MB is secreted into the environment, other microbes that require copper for their metabolism may be inhibited as MB may make copper unavailable; e.g., inhibition of denitrifiers as complete conversion nitrate to dinitrogen involves multiple enzymes, some of which are copper-dependent. Of key concern is inhibition of the copper-dependent nitrous oxide reductase (NosZ), the only known enzyme capable of converting nitrous oxide (N(2)O) to dinitrogen. Herein, we show that different forms of MB differentially affect copper uptake and N(2)O reduction by Pseudomonas stutzeri strain DCP-Ps1 (that expresses clade I NosZ) and Dechloromonas aromatica strain RCB (that expresses clade II NosZ). Specifically, in the presence of MB from Methylocystis sp. strain SB2 (SB2-MB), copper uptake and nosZ expression were more significantly reduced than in the presence of MB from Methylosinus trichosporium OB3b (OB3b-MB). Further, N(2)O accumulation increased more significantly for both P. stutzeri strain DCP-Ps1 and D. aromatica strain RCB in the presence of SB2-MB versus OB3b-MB. These data illustrate that copper competition between methanotrophs and denitrifying bacteria can be significant and that the extent of such competition is dependent on the form of MB that methanotrophs produce. IMPORTANCE Herein, it was demonstrated that the different forms of methanobactin differentially enhance N(2)O emissions from Pseudomonas stutzeri strain DCP-Ps1 (harboring clade I nitrous oxide reductase) and Dechloromonas aromatica strain RCB (harboring clade II nitrous oxide reductase). This work contributes to our understanding of how aerobic methanotrophs compete with denitrifiers for the copper uptake and also suggests how MBs prevent copper collection by denitrifiers, thus downregulating expression of nitrous oxide reductase. This study provides critical information for enhanced understanding of microbe-microbe interactions that are important for the development of better predictive models of net greenhouse gas emissions (i.e., methane and nitrous oxide) that are significantly controlled by microbial activity.

Methanobactins (MBs) are ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by methanotrophs for copper uptake. The post-translational modification that define MBs is the formation of two heterocyclic groups with associated thioamines from X-Cys dipeptide sequences. Both heterocyclic groups in the MB from Methylosinus trichosporium OB3b (MB-OB3b) are oxazolone groups. The precursor gene for MB-OB3b, mbnA, which is part of a gene cluster that contains both annotated and unannotated genes. One of those unannotated genes, mbnC, is found in all MB operons, and in conjunction with mbnB, is reported to be involved in the formation of both heterocyclic groups in all MBs. To determine the function of mbnC, a deletion mutation was constructed in M. trichosporium OB3b, and the MB produced from the ΔmbnC mutant was purified and structurally characterized by UV-visible absorption spectroscopy, mass spectrometry and solution NMR spectroscopy. MB-OB3b from ΔmbnC was missing the C-terminal Met and also found to contain a Pro and a Cys in place of the pyrrolidiny-oxazolone-thioamide group. These results demonstrate MbnC is required for the formation of the C-terminal pyrrolidinyl-oxazolone-thioamide group from the Pro-Cys dipeptide, but not for the formation of the N-terminal 3-methylbutanol-oxazolone-thioamide group from the N-terminal dipeptide Leu-Cys. IMPORTANCE A number of environmental and medical applications have been proposed for MBs, including bioremediation of toxic metals, nanoparticle formation, as well as for the treatment of copper- and iron-related diseases. However, before MBs can be modified and optimized for any specific application, the biosynthetic pathway for MB production must be defined. The discovery that mbnC is involved in the formation of the C-terminal oxazolone group with associated thioamide but not for the formation of the N-terminal oxazolone group with associated thioamide in M. trichosporium OB3b suggests the enzymes responsible for post-translational modification(s) of the two oxazolone groups are not identical.

Copper is an important component of methanotrophic physiology as it controls the expression and activity of alternative forms of methane monooxygenase (MMO). To collect copper, some methanotrophs secrete a chalkophore or copper-binding compound called methanobactin (MB). MB is a ribosomally synthesized post-translationally modified polypeptide (RiPP) that, after binding copper, is collected by MbnT, a TonB-dependent transporter (TBDT). Structurally different forms of MB have been characterized, and here we show that different forms of MB are collected by specific TBDTs. Further, we report that in the model methanotroph, Methylosinus trichosporium OB3b, expression of the TBDT required for uptake of a different MB made by Methylocystis sp. strain SB2 (MB-SB2), is induced in the presence of MB-SB2, suggesting that methanotrophs have developed specific machinery and regulatory systems to actively take up MB from other methanotrophs for copper collection. Moreover, the canonical "copper-switch" in Ms. trichosporium OB3b that controls expression of alternative MMOs is apparent if one of the two TBDTs required for MB-OB3b and MB-SB2 uptake is knocked out, but is disrupted if both TBDTs are knocked out. These data indicate that MB uptake, including the uptake of exogenous MB, plays an important role in the copper switch in M. trichosporium OB3b and thus overall activity. Based on these data, we propose a revised model for the "copper-switch" in this methanotroph that involves MB uptake. IMPORTANCE In this study, we demonstrate that different TonB-dependent transporters (TBDTs) in the model methanotroph Methylosinus trichosporium OB3b are responsible for uptake of either endogenous MB or exogenous MB. Interestingly, the presence of exogenous MB induces expression of its specific TBDT in M. trichosporium OB3b, suggesting that this methanotroph is able to actively take up MB produced by others. This work contributes to our understanding of how microbes collect and compete for copper, and also helps inform how such uptake coordinates the expression of different forms of methane monooxygenase. Such studies are likely to be very important to develop a better understanding of methanotrophic interactions via synthesis and secretion of secondary metabolites such as methanobactin and thus provide additional means whereby these microbes can be manipulated for a variety of environmental and industrial purposes.

Aerobic methanotrophy is strongly controlled by copper, and methanotrophs are known to use different mechanisms for copper uptake. Some methanotrophs secrete a modified polypeptide-methanobactin-while others utilize a surface-bound protein (MopE) and a secreted form of it (MopE*) for copper collection. As different methanotrophs have different means of sequestering copper, competition for copper significantly impacts methanotrophic activity. Herein, we show that Methylomicrobium album BG8, Methylocystis sp. strain Rockwell, and Methylococcus capsulatus Bath, all lacking genes for methanobactin biosynthesis, are not limited for copper by multiple forms of methanobactin. Interestingly, Mm. album BG8 and Methylocystis sp. strain Rockwell were found to have genes similar to mbnT that encodes for a TonB-dependent transporter required for methanobactin uptake. Data indicate that these methanotrophs "steal" methanobactin and such "theft" enhances the ability of these strains to degrade methylmercury, a potent neurotoxin. Further, when mbnT was deleted in Mm. album BG8, methylmercury degradation in the presence of methanobactin was indistinguishable from when MB was not added. Mc. capsulatus Bath lacks anything similar to mbnT and was unable to degrade methylmercury either in the presence or absence of methanobactin. Rather, Mc. capsulatus Bath appears to rely on MopE/MopE* for copper collection. Finally, not only does Mm. album BG8 steal methanobactin, it synthesizes a novel chalkophore, suggesting that some methanotrophs utilize both competition and cheating strategies for copper collection. Through a better understanding of these strategies, methanotrophic communities may be more effectively manipulated to reduce methane emissions and also enhance mercury detoxification in situ.

Background & Aims In Wilson disease, ATP7B mutations impair copper excretion into bile. Hepatic copper accumulation may induce mild to moderate chronic liver damage or even acute liver failure. Etiologic factors for this heterogeneous phenotype remain enigmatic. Liver steatosis is a frequent finding in Wilson disease patients, suggesting that impaired copper homeostasis is linked with liver steatosis. Hepatic mitochondrial function is affected negatively both by copper overload and steatosis. Therefore, we addressed the question of whether a steatosis-promoting high-calorie diet aggravates liver damage in Wilson disease via amplified mitochondrial damage. Methods Control Atp7b+/- and Wilson disease Atp7b-/- rats were fed either a high-calorie diet (HCD) or a normal diet. Copper chelation using the high-affinity peptide methanobactin was used in HCD-fed Atp7b-/- rats to test for therapeutic reversal of mitochondrial copper damage. Results In comparison with a normal diet, HCD feeding of Atp7b-/- rats resulted in a markedly earlier onset of clinically apparent hepatic injury. Strongly increased mitochondrial copper accumulation was observed in HCD-fed Atp7b-/- rats, correlating with severe liver injury. Mitochondria presented with massive structural damage, increased H2O2 emergence, and dysfunctional adenosine triphosphate production. Hepatocellular injury presumably was augmented as a result of oxidative stress. Reduction of mitochondrial copper by methanobactin significantly reduced mitochondrial impairment and ameliorated liver damage. Conclusions A high-calorie diet severely aggravates hepatic mitochondrial and hepatocellular damage in Wilson disease rats, causing an earlier onset of the disease and enhanced disease progression., Graphical abstract

Aerobic methanotrophs are an intriguing group of microbes with the singular ability to consume methane as their sole source of carbon and energy. As such, methanotrophs are receiving increased attention to control methane emissions to limit future climate change. Methanotrophs have a wide range of other applications, including pollutant remediation and methane valorization (e.g. conversion of methane to protein, bioplastics, and biodiesel amongst other products). Methanotrophs also produce a novel copper-binding compound, methanobactin, that has significant potential for the treatment of copper-related human pathologies. Here we provide an overview of aerobic methanotrophy, describe current and future applications of these unique microbes, as well as discuss various strategies one can consider to better realize the opportunities these microbes present.

Methanotrophic bacteria catalyze the aerobic oxidation of methane to methanol using Cu-containing enzymes, thereby exerting a modulating influence on the global methane cycle. To facilitate the acquisition of Cu ions, some methanotrophic bacteria secrete small modified peptides known as “methanobactins,” which strongly bind Cu and function as an extracellular Cu recruitment relay, analogous to siderophores and Fe. In addition to Cu, methanobactins form complexes with other late transition metals, including the Group 12 transition metals Zn, Cd, and Hg, although the interplay among solution-phase configurations, metal interactions, and the spectroscopic signatures of methanobactin-metal complexes remains ambiguous. In this study, the complexation of Zn, Cd, and Hg by methanobactin from Methylocystis sp. strain SB2 was studied using a combination of absorbance, fluorescence, extended x-ray absorption fine structure (EXAFS) spectroscopy, and time-dependent density functional theory (TD-DFT) calculations. We report changes in sample absorbance and fluorescence spectral dynamics, which occur on a wide range of experimental timescales and characterize a clear stoichiometric complexation dependence. Mercury L3-edge EXAFS and TD-DFT calculations suggest a linear model for Hg S coordination, and TD-DFT suggests a tetrahedral model for Zn2+ and Cd2+. We observed an enhancement in the fluorescence of methanobactin upon interaction with transition metals and propose a mechanism of complexation-hindered isomerization drawing inspiration from the wild-type Green Fluorescent Protein active site. Collectively, our results represent the first combined computational and experimental spectroscopy study of methanobactins and shed new light on molecular interactions and dynamics that characterize complexes of methanobactins with Group 12 transition metals.

Methanobactins (MBs) are small (

Microbial production of the neurotoxin methylmercury (MeHg) is a significant health and environmental concern, as it can bioaccumulate and biomagnify in the food web. A chalkophore or a copper-binding compound, termed methanobactin (MB), has been shown to form strong complexes with mercury [as Hg(II)] and also enables some methanotrophs to degrade MeHg. It is unknown, however, if Hg(II) binding with MB can also impede Hg(II) methylation by other microbes. Contrary to expectations, MB produced by the methanotroph Methylosinus trichosporium OB3b (OB3b-MB) enhanced the rate and efficiency of Hg(II) methylation more than that observed with thiol compounds (such as cysteine) by the mercury-methylating bacteria Desulfovibrio desulfuricans ND132 and Geobacter sulfurreducens PCA. Compared to no-MB controls, OB3b-MB decreased the rates of Hg(II) sorption and internalization, but increased methylation by 5- to 7-fold, suggesting that Hg(II) complexation with OB3b-MB facilitated exchange and internal transfer of Hg(II) to the HgcAB proteins required for methylation. Conversely, addition of excess amounts of OB3b-MB or a different form of MB from Methylocystis strain SB2 (SB2-MB) inhibited Hg(II) methylation, likely due to greater binding of Hg(II). Collectively, our results underscore the complex roles of microbial exogenous metal-scavenging compounds in controlling net production and bioaccumulation of MeHg in the environment. IMPORTANCE Some anaerobic microorganisms convert inorganic mercury (Hg) into the neurotoxin methylmercury, which can bioaccumulate and biomagnify in the food web. While the genetic basis of microbial mercury methylation is known, factors that control net methylmercury production in the environment are still poorly understood. Here, it is shown that mercury methylation can be substantially enhanced by one form of an exogenous copper-binding compound (methanobactin) produced by some methanotrophs, but not by another. This novel finding illustrates that complex interactions exist between microbes and that these interactions can potentially affect the net production of methylmercury in situ.

Aerobic methane-oxidizing bacteria of the Alphaproteobacteria have been found to express a novel ribosomally synthesized post-translationally modified polypeptide (RiPP) termed methanobactin (MB). The primary function of MB in these microbes appears to be for copper uptake, but MB has been shown to have multiple capabilities, including oxidase, superoxide dismutase and hydrogen peroxide reductase activities, the ability to detoxify mercury species, as well as acting as an antimicrobial agent. Herein, we describe the diversity of known MBs as well as the genetics underlying MB biosynthesis. We further propose based on bioinformatics analyses that some methanotrophs may produce novel forms of MB that have yet to be characterized. We also discuss recent findings documenting that MBs play an important role in controlling copper availability to the broader microbial community, and as a result can strongly affect the activity of microbes that require copper for important enzymatic transformations, e.g. conversion of nitrous oxide to dinitrogen. Finally, we describe procedures for the detection/purification of MB, as well as potential medical and industrial applications of this intriguing RiPP.

Methanotrophs synthesize methanobactin, a secondary metabolite that binds copper with an unprecedentedly high affinity. Such a strategy may provide methanotrophs a "copper monopoly" that can inhibit the activity of copper-containing enzymes of other microbes, e.g., copper-dependent N2O reductases. Here, we show that methanobactin from Methylosinus trichosporium OB3b inhibited N2O reduction in denitrifiers. When Pseudomonas stutzeri DCP-Ps1 was incubated in cocultures with M. trichosporium OB3b or with purified methanobactin from M. trichosporium OB3b, stoichiometric N2O production was observed from NO3- reduction, whereas no significant N2O accumulation was observed in cocultures with a mutant defective in methanobactin production. Copper uptake by P. stutzeri DCP-Ps1 was inhibited by the presence of purified methanobactin, leading to a significant downregulation of nosZ transcription. Similar findings were observed with three other denitrifier strains. These results suggest that in situ stimulation of methanotrophs can inadvertently increase N2O emissions, with the potential for increasing net greenhouse gas emissions.

Aerobic methanotrophs are a novel group of microorganisms that play a critical role in the global carbon cycle. Expression and activity of a key enzyme in the metabolism in these microbes, the methane monooxygenase, is controlled by the availability of copper. To sequester copper, some methanotrophs produce a ribosomally synthesized post-translationally modified polypeptide called methanobactin. This peptide, or chalkophore, has unique features, with all known forms being small (

In Wilson disease (WD), functional loss of ATPase copper-transporting β (ATP7B) impairs biliary copper excretion, leading to excessive copper accumulation in the liver and fulminant hepatitis. Current US Food and Drug Administration- and European Medicines Agency-approved pharmacological treatments usually fail to restore copper homeostasis in patients with WD who have progressed to acute liver failure, leaving liver transplantation as the only viable treatment option. Here, we investigated the therapeutic utility of methanobactin (MB), a peptide produced by Methylosinus trichosporium OB3b, which has an exceptionally high affinity for copper. We demonstrated that ATP7B-deficient rats recapitulate WD-associated phenotypes, including hepatic copper accumulation, liver damage, and mitochondrial impairment. Short-term treatment of these rats with MB efficiently reversed mitochondrial impairment and liver damage in the acute stages of liver copper accumulation compared with that seen in untreated ATP7B-deficient rats. This beneficial effect was associated with depletion of copper from hepatocyte mitochondria. Moreover, MB treatment prevented hepatocyte death, subsequent liver failure, and death in the rodent model. These results suggest that MB has potential as a therapeutic agent for the treatment of acute WD.

SUMMARY Methanobactins (mbs) are low-molecular-mass (2+ to Cu 1+ . In addition to binding copper, mbs will bind most transition metals and near-transition metals and protect the host methanotroph as well as other bacteria from toxic metals. Several other physiological functions have been assigned to mbs, based primarily on their redox and metal-binding properties. In this review, we examine the current state of knowledge of this novel type of metal-binding peptide. We also explore its potential applications, how mbs may alter the bioavailability of multiple metals, and the many roles mbs may play in the physiology of methanotrophs.

Methanotrophs have remarkable redundancy in multiple steps of the central pathway of methane oxidation to carbon dioxide. For example, it has been known for over 30 years that two forms of methane monooxygenase, responsible for oxidizing methane to methanol, exist in methanotrophs, i.e., soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), and that expression of these two forms is controlled by the availability of copper. Specifically, sMMO expression occurs in the absence of copper, while pMMO expression increases with increasing copper concentrations. More recently, it was discovered that multiple forms of methanol dehydrogenase (MeDH), Mxa MeDH and Xox MeDH, also exist in methanotrophs and that the expression of these alternative forms is regulated by the availability of cerium. That is, expression of Xox MeDH increases in the presence of cerium, while Mxa MeDH expression decreases in the presence of cerium. As it had been earlier concluded that pMMO and Mxa MeDH form a supercomplex in which electrons from Mxa MeDH are back donated to pMMO to drive the initial oxidation of methane, we speculated that Mxa MeDH could be rendered inactive through marker-exchange mutagenesis but growth on methane could still be possible if cerium was added to increase the expression of Xox MeDH under sMMO-expressing conditions. Here we report that mxaF , encoding the large subunit of Mxa MeDH, could indeed be knocked out in Methylosinus trichosporium OB3b, yet growth on methane was still possible, so long as cerium was added. Interestingly, growth of this mutant occurred in both the presence and the absence of copper, suggesting that Xox MeDH can replace Mxa MeDH regardless of the form of MMO expressed.

Methanotrophs have multiple methane monooxygenases that are well known to be regulated by copper, i.e., a “copper switch.” At low copper/biomass ratios the soluble methane monooxygenase (sMMO) is expressed while expression and activity of the particulate methane monooxygenase (pMMO) increases with increasing availability of copper. In many methanotrophs there are also multiple methanol dehydrogenases (MeDHs), one based on Mxa and another based on Xox. Mxa-MeDH is known to have calcium in its active site, while Xox-MeDHs have been shown to have rare earth elements in their active site. We show here that the expression levels of Mxa-MeDH and Xox-MeDH in Methylosinus trichosporium OB3b significantly decreased and increased, respectively, when grown in the presence of cerium but the absence of copper compared to the absence of both metals. Expression of sMMO and pMMO was not affected. In the presence of copper, the effect of cerium on gene expression was less significant, i.e., expression of Mxa-MeDH in the presence of copper and cerium was slightly lower than in the presence of copper alone, but Xox-MeDH was again found to increase significantly. As expected, the addition of copper caused sMMO and pMMO expression levels to significantly decrease and increase, respectively, but the simultaneous addition of cerium had no discernible effect on MMO expression. As a result, it appears Mxa-MeDH can be uncoupled from methane oxidation by sMMO in M. trichosporium OB3b but not from pMMO.

Nitrate and nitrite reduction are of paramount importance for nitrogen assimilation and anaerobic metabolism, and understanding the specific roles of each participating reductase is necessary to describe the biochemical balance that dictates cellular responses to their environments. The soluble, cytoplasmic siroheme NADH-nitrite reductase (Nir) in Escherichia coli is necessary for nitrate/nitrite assimilation but has also been reported to either “detoxify” nitrite, or to carry out fermentative ammonification in support of anaerobic catabolism. Theoretically, nitrite detoxification would be important for anaerobic growth on nitrate, during which excess nitrite would be reduced to ammonium. Fermentative ammonification by Nir would be important for maximization of non-respiratory ATP production during anaerobic growth in the presence of nitrite. Experiments reported here were designed to test the potential role of Nir in fermentative ammonification directly by growing E. coli along with mutant strains lacking Nir or the respiratory nitrite reductase (Nrf) under anaerobic conditions in defined media while monitoring nitrogen utilization and fermentation metabolites. To focus on the role of Nir in fermentative ammonification, pH control was used in most experiments to eliminate nitrite toxicity due to nitric acid formation. Our results demonstrate that Nir confers a significant benefit during fermentative growth that reflects fermentative ammonification rather than detoxification. We conclude that fermentative ammonification by Nir allows for the energetically favorable fermentation of glucose to formate and acetate. These results and conclusions are discussed in light of the roles of Nir in other bacteria and in plants.

Nonendospore-forming pathogenic bacteria in the long-term survival (LTS) phase can remain viable for months or years and may show reduced susceptibility to various antimicrobial interventions. In the present study, we investigated the response of LTS phase Salmonella enterica serovar Typhimurium (ATCC 14028) to ultraviolet (UV) radiation in 0.85% (w/v) saline and apple juice and the extent of sublethal injury in LTS phase survivors. The LTS-phase Salmonella Typhimurium cells were cultured at 35°C for 14 days in tryptic soy broth with 0.6% (w/v) yeast extract (TSBYE). Exponential- and stationary-phase cells, cultured in TSBYE (35°C) for 2.5 and 18 h, respectively, served as control samples. Cells (10

Aerobic methanotrophs have long been known to play a critical role in the global carbon cycle, being capable of converting methane to biomass and carbon dioxide. Interestingly, these microbes exhibit great sensitivity to copper and rare-earth elements, with the expression of key genes involved in the central pathway of methane oxidation controlled by the availability of these metals. That is, these microbes have a “copper switch” that controls the expression of alternative methane monooxygenases and a “rare-earth element switch” that controls the expression of alternative methanol dehydrogenases. Further, it has been recently shown that some methanotrophs can detoxify inorganic mercury and demethylate methylmercury; this finding is remarkable, as the canonical organomercurial lyase does not exist in these methanotrophs, indicating that a novel mechanism is involved in methylmercury demethylation. Here, we review recent findings on methanotrophic interactions with metals, with a particular focus on these metal switches and the mechanisms used by methanotrophs to bind and sequester metals.

Methanotrophs can express a cytoplasmic (soluble) methane monooxygenase (sMMO) or membrane-bound (particulate) methane monooxygenase (pMMO). Expression of these MMOs is strongly regulated by the availability of copper. Many methanotrophs have been found to synthesize a novel compound, methanobactin (Mb), that is responsible for the uptake of copper, and methanobactin produced by Methylosinus trichosporium OB3b plays a key role in controlling expression of MMO genes in this strain. As all known forms of methanobactin are structurally similar, it was hypothesized that methanobactin from one methanotroph may alter gene expression in another. When Methylosinus trichosporium OB3b was grown in the presence of 1 μM CuCl 2 , expression of mmoX , encoding a subunit of the hydroxylase component of sMMO, was very low. mmoX expression increased, however, when methanobactin from Methylocystis sp. strain SB2 (SB2-Mb) was added, as did whole-cell sMMO activity, but there was no significant change in the amount of copper associated with M. trichosporium OB3b. If M. trichosporium OB3b was grown in the absence of CuCl 2 , the mmoX expression level was high but decreased by several orders of magnitude if copper prebound to SB2-Mb (Cu-SB2-Mb) was added, and biomass-associated copper was increased. Exposure of Methylosinus trichosporium OB3b to SB2-Mb had no effect on expression of mbnA , encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl 2 . mbnA expression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl 2 . These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin “piracy” may be commonplace.

It is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that in Methylosinus trichosporium OB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced by M. trichosporium OB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and active in situ . Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin.

Methanotrophs synthesize methanobactin, a secondary metabolite that binds copper with an unprecedentedly high affinity. Such a strategy may provide methanotrophs a “copper monopoly” that can inhibit the activity of copper-containing enzymes of other microbes, e.g., copper-dependent N(2)O reductases. Here, we show that methanobactin from Methylosinus trichosporium OB3b inhibited N(2)O reduction in denitrifiers. When Pseudomonas stutzeri DCP-Ps1 was incubated in cocultures with M. trichosporium OB3b or with purified methanobactin from M. trichosporium OB3b, stoichiometric N(2)O production was observed from NO(3)(−) reduction, whereas no significant N(2)O accumulation was observed in cocultures with a mutant defective in methanobactin production. Copper uptake by P. stutzeri DCP-Ps1 was inhibited by the presence of purified methanobactin, leading to a significant downregulation of nosZ transcription. Similar findings were observed with three other denitrifier strains. These results suggest that in situ stimulation of methanotrophs can inadvertently increase N(2)O emissions, with the potential for increasing net greenhouse gas emissions.

Certain methanotrophs can take up and degrade methylmercury, signifying a potentially important demethylation pathway in the environment., Methylmercury (CH3Hg+) is a potent neurotoxin produced by certain anaerobic microorganisms in natural environments. Although numerous studies have characterized the basis of mercury (Hg) methylation, no studies have examined CH3Hg+ degradation by methanotrophs, despite their ubiquitous presence in the environment. We report that some methanotrophs, such as Methylosinus trichosporium OB3b, can take up and degrade CH3Hg+ rapidly, whereas others, such as Methylococcus capsulatus Bath, can take up but not degrade CH3Hg+. Demethylation by M. trichosporium OB3b increases with increasing CH3Hg+ concentrations but was abolished in mutants deficient in the synthesis of methanobactin, a metal-binding compound used by some methanotrophs, such as M. trichosporium OB3b. Furthermore, addition of methanol (>5 mM) as a competing one-carbon (C1) substrate inhibits demethylation, suggesting that CH3Hg+ degradation by methanotrophs may involve an initial bonding of CH3Hg+ by methanobactin followed by cleavage of the C–Hg bond in CH3Hg+ by the methanol dehydrogenase. This new demethylation pathway by methanotrophs indicates possible broader involvement of C1-metabolizing aerobes in the degradation and cycling of toxic CH3Hg+ in the environment.

Gene expression in methanotrophs has been shown to be affected by the availability of a variety of metals, most notably copper-regulating expression of alternative forms of methane monooxygenase. A copper-binding compound, or chalkophore, called methanobactin plays a key role in copper uptake in methanotrophs. Methanobactin is a ribosomally synthesized and posttranslationally modified peptide (RiPP) with two heterocyclic rings with an associated thioamide for each ring, formed from X-Cys dipeptide sequences that bind copper. The gene coding for the precursor polypeptide of methanobactin, mbnA , is part of a gene cluster, but the role of other genes in methanobactin biosynthesis is unclear. To begin to elucidate the function of these genes, we constructed an unmarked deletion of mbnABCMN in Methylosinus trichosporium OB3b and then homologously expressed mbnABCM using a broad-host-range cloning vector to determine the function of mbnN , annotated as coding for an aminotransferase. Methanobactin produced by this strain was found to be substantially different from wild-type methanobactin in that the C-terminal methionine was missing and only one of the two oxazolone rings was formed. Rather, in place of the N-terminal 3-methylbutanoyl-oxazolone-thioamide group, a leucine and a thioamide-containing glycine (Gly-Ψ) were found, indicating that MbnN is used for deamination of the N-terminal leucine of methanobactin and that this posttranslational modification is critical for closure of the N-terminal oxazolone ring in M. trichosporium OB3b. These studies provide new insights into methanobactin biosynthesis and also provide a platform for understanding the function of other genes in the methanobactin gene cluster. IMPORTANCE Methanotrophs, microbes that play a critical role in the carbon cycle, are influenced by copper, with gene expression and enzyme activity changing as copper levels change. Methanotrophs produce a copper-binding compound, or chalkophore, called methanobactin for copper uptake, and methanobactin plays a key role in controlling methanotrophic activity. Methanobactin has also been shown to be effective in the treatment of Wilson disease, an autosomal recessive disorder where the human body cannot correctly assimilate copper. It is important to characterize the methanobactin biosynthesis pathway to understand how methanotrophs respond to their environment as well as to optimize the use of methanobactin for the treatment of copper-related diseases such as Wilson disease. Here we show that mbnN , encoding an aminotransferase, is involved in the deamination of the N-terminal leucine and necessary for the formation of one but not both of the heterocyclic rings in methanobactin that are responsible for copper binding.

The genomes of the aerobic methanotrophs “ Methyloterricola oryzae ” strain 73a T and Methylomagnum ishizawai strain 175 were sequenced. Both strains were isolated from rice plants. Methyloterricola oryzae strain 73a T represents the first isolate of rice paddy cluster I, and strain 175 is the second representative of the recently described genus Methylomagnum .

Many methanotrophs have been shown to synthesize methanobactin, a novel biogenic copper-chelating agent or chalkophore. Methanobactin binds copper via two heterocyclic rings with associated enethiol groups. The structure of methanobactin suggests that it can bind other metals, including mercury. Here we report that methanobactin from Methylosinus trichosporium OB3b does indeed bind mercury when added as HgCl 2 and, in doing so, reduced toxicity associated with Hg(II) for both Alphaproteobacteria methanotrophs, including M. trichosporium OB3b, M. trichosporium OB3b Δ mbnA (a mutant defective in methanobactin production), and Methylocystis sp. strain SB2, and a Gammaproteobacteria methanotroph, Methylomicrobium album BG8. Mercury binding by methanobactin was evident in both the presence and absence of copper, despite the fact that methanobactin had a much higher affinity for copper due to the rapid and irreversible binding of mercury by methanobactin. The formation of a gray precipitate suggested that Hg(II), after being bound by methanobactin, was reduced to Hg(0) but was not volatilized. Rather, mercury remained associated with methanobactin and was also found associated with methanotrophic biomass. It thus appears that although the mercury-methanobactin complex was cell associated, mercury was not removed from methanobactin. The amount of biomass-associated mercury in the presence of methanobactin from M. trichosporium OB3b was greatest for M. trichosporium wild-type strain OB3b and the Δ mbnA mutant and least for M. album BG8, suggesting that methanotrophs may have selective methanobactin uptake systems that may be based on TonB-dependent transporters but that such uptake systems exhibit a degree of infidelity.

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.

Methanobactin, a small modified polypeptide synthesized by methanotrophs for copper uptake, has been found to be chromosomally encoded. The gene encoding the polypeptide precursor of methanobactin, mbnA , is part of a gene cluster that also includes several genes encoding proteins of unknown function (but speculated to be involved in methanobactin formation) as well as mbnT , which encodes a TonB-dependent transporter hypothesized to be responsible for methanobactin uptake. To determine if mbnT is truly responsible for methanobactin uptake, a knockout was constructed in Methylosinus trichosporium OB3b using marker exchange mutagenesis. The resulting M. trichosporium mbnT ::Gm r mutant was found to be able to produce methanobactin but was unable to internalize it. Further, if this mutant was grown in the presence of copper and exogenous methanobactin, copper uptake was significantly reduced. Expression of mmoX and pmoA , encoding polypeptides of the soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), respectively, also changed significantly when methanobactin was added, which indicates that the mutant was unable to collect copper under these conditions. Copper uptake and gene expression, however, were not affected in wild-type M. trichosporium OB3b, indicating that the TonB-dependent transporter encoded by mbnT is responsible for methanobactin uptake and that methanobactin is a key mechanism used by methanotrophs for copper uptake. When the mbnT ::Gm r mutant was grown under a range of copper concentrations in the absence of methanobactin, however, the phenotype of the mutant was indistinguishable from that of wild-type M. trichosporium OB3b, indicating that this methanotroph has multiple mechanisms for copper uptake.

Methanotrophs are a group of phylogenetically diverse microorganisms characterized by their ability to utilize methane as their sole source of carbon and energy. Early studies suggested that growth on methane could be stimulated with the addition of some small organic acids, but initial efforts to find facultative methanotrophs, i.e., methanotrophs able to utilize compounds with carbon-carbon bonds as sole growth substrates were inconclusive. Recently, however, facultative methanotrophs in the genera Methylocella, Methylocapsa, and Methylocystis have been reported that can grow on acetate, as well as on larger organic acids or ethanol for some species. All identified facultative methanotrophs group within the Alphaproteobacteria and utilize the serine cycle for carbon assimilation from formaldehyde. It is possible that facultative methanotrophs are able to convert acetate into intermediates of the serine cycle (e.g. malate and glyoxylate), because a variety of acetate assimilation pathways convert acetate into these compounds (e.g. the glyoxylate shunt of the tricarboxylic acid cycle, the ethylmalonyl-CoA pathway, the citramalate cycle, and the methylaspartate cycle). In this review, we summarize the history of facultative methanotrophy, describe scenarios for the basis of facultative methanotrophy, and pose several topics for future research in this area.

The ability of ferrous hemoglobins to reduce nitrite to form nitric oxide has been demonstrated for hemoglobins from animals, including myoglobin, blood cell hemoglobin, neuroglobin, and cytoglobin. In all cases, the rate constants for the bimolecular reactions with nitrite are relatively slow, with maximal values of ~5 M(-1) s(-1) at pH 7. Combined with the relatively low concentrations of nitrite found in animal blood plasma (normally no greater than 13 μM), these slow reaction rates are unlikely to contribute significantly to hemoglobin oxidation, nitrite reduction, or NO production. Plants and cyanobacteria, however, must contend with much higher (millimolar) nitrite concentrations necessitated by assimilatory nitrogen metabolism during hypoxic growth, such as the conditions commonly found during flooding or in waterlogged soil. Here we report rate constants for nitrite reduction by a ferrous plant hemoglobin (rice nonsymbiotic hemoglobin 1) and a ferrous cyanobacterial hemoglobin from Synechocystis that are more than 10 times faster than those observed for animal hemoglobins. These rate constants, along with the relatively high concentrations of nitrite present during hypoxia, suggest that plant and cyanobacterial hemoglobins could serve as anaerobic nitrite reductases in vivo.

International audience; Methanobactins (mb) are low-molecular mass, copper-binding molecules secreted by most methanotrophic bacteria. These molecules have been identified for a number of methanotrophs, but only the one produced by Methylosinus trichosporium OB3b (mb-OB3b) has to date been chemically characterized. Here we report the chemical characterization and copper binding properties of a second methanobactin, which is produced by Methylocystis strain SB2 (mb-SB2). mb-SB2 shows some significant similarities to mb-OB3b, including its spectral and metal binding properties, and its ability to bind and reduce Cu(II) to Cu(I). Like mb-OB3b, mb-SB2 contains two five-member heterocyclic rings with associated enethiol groups, which together form the copper ion binding site. mb-SB2 also displays some significant differences compared to mb-OB3b, including the number and types of amino acids used to complete the structure of the molecule, the presence of an imidazolone ring in place of one of the oxazolone rings found in mb-OB3b, and the presence of a sulfate group not found in mb-OB3b. The sulfate is bonded to a threonine-like side chain that is associated with one of the heterocyclic rings and may represent the first example of this type of sulfate group found in a bacterially derived peptide. Acid-catalyzed hydrolysis and decarboxylation of the oxazolone rings found in mb-OB3b and mb-SB2 produce pairs of amino acid residues and suggest that both mb-OB3b and mb-SB2 are derived from peptides. In support of this, the gene for a ribosomally produced peptide precursor for mb-OB3b has been identified in the genome of M. trichosporium OB3b. The gene sequence indicates that the oxazolone rings in mb-OB3b are derived from the combination of a cysteine residue and the carbonyl from the preceding residue in the peptide sequence. Taken together, the results suggest methanobactins make up a structurally diverse group of ribosomally produced, peptide-derived molecules, which share a common pair of five-member rings with associated enethiol groups that are able to bind, reduce, and stabilize copper ions in an aqueous environment.

Summary The particulate methane monooxygenase (pMMO) in Methylocystis strain SB2 was found to be constitu- tively expressed in the absence of methane when the strain was grown on either acetate or ethanol. Real- timequantitativepolymerasechainreaction(PCR)and reversetranscription-PCRshowedthattheexpression of pmoA decreased by one to two orders of magnitude when grown on acetate as compared with growth of strain SB2 on methane. The capability of strain SB2 to degrade a mixture of chlorinated ethenes in the absence of methane was examined to verify the pres- ence and activity of pMMO under acetate-growth con- ditions as well determine the effectiveness of such conditions for bioremediation. It was found that when strain SB2 was grown on acetate and exposed to 40 mM each of trichloroethylene (TCE), trans- dichloroethylene (t-DCE) and vinyl chloride (VC), approximately 30% of VC and t-DCE was degraded but no appreciable TCE removal was measured after 216 h of incubation. The ability to degrade VC andt-DCE was lostwhenacetylenewasadded,confirmingthatpMMO was responsible for the degradation of these chlori- nated ethenes by Methylocystis strain SB2 when the strain was grown on acetate.

Summary A non-motile strain of Methylocystis, strain SB2, isolated from a spring bog in southeast Michigan, had a curved rod morphology with a typical type II intracytoplasmic membrane system. This organism expressed the membrane-bound or particulate methane monooxygenase (pMMO) as well as a chalkophore with high affinity for copper and did not express the cytoplasmic or soluble methane monooxygenase (sMMO). Strain SB2 was found to grow within the pH range of 6–9, with optimal growth at 6.8. Growth was observed at temperatures ranging between 10°C and 30°C, with no growth at 37°C. The DNA G+C content was 62.9 mol%. Predominant fatty acids were 18:1ω7c (72.7%) and 18:1ω9c (24%) when grown on methane. Phylogenetic comparisons based on both pmoA and 16S rRNA sequences indicated that this organism belonged to the Methylocystis genus, and was closely related to Methylocystis rosea SV97T and Methylocystis echinoides IMET10491T (98% 16S rRNA gene sequence similarity to both strains). DNA : DNA hybridizations indicated that strain SB2 had 70% similarity with M. rosea SV97T. Unlike M. rosea SV97T, strain SB2 was able to utilize not only methane for growth, but also ethanol and acetate. Furthermore, the predominant fatty acids in strain SB2 were different from those found in M. rosea SV97T, i.e. 54.2% and 39.7% of fatty acids are 18:1ω8 and 18:1ω7 in M. rosea SV97T, while 18:1ω8 is completely absent in strain SB2.

Methanotrophs, bacteria that utilize methane as their sole carbon and energy source, are known to have high requirements for copper. These bacteria have recently been found to synthesize a copper-chelating agent, or chalkophore, termed methanobactin. To aid in screening methanobactin production by methanotrophs, a plate assay developed from the chrome azurol S (CAS) assay for siderophore production, was modified. In the typical CAS assay, a colour change from blue to orange in iron-CAS plates is observed as iron (III) ion weakly bound to CAS is sequestered by siderophores with higher affinities. In our modified assay, iron (III) chloride of the original CAS solution was substituted with copper (II) chloride, and removal of copper from CAS caused a colour change from blue to yellow. Assay results indicated that of the four tested methanotrophs (Methylosinus trichosporium OB3b, Methylococcus capsulatus Bath, Methylomicrobium album BG8 and Methylocystis parvus OBBP), only M. trichosporium OB3b, M. capsulatus Bath and M. album BG8 produced chalkophores capable of competing with CAS for copper, while M. parvus OBBP did not or did not export sufficient concentrations of methanobactin for detection by this assay. It was also found using Fe-CAS plates that at least M. trichosporium OB3b and M. album BG8 produce siderophores. These results may be expanded for the detection of chalkophores in other microorganisms as well as for screening of putative mutants of chalkophore synthesis.

Aerobic methane-oxidizing bacteria (methanotrophs) have a key role in the global carbon cycle, converting methane to biomass and carbon dioxide. Although these bacteria have been isolated from many environments, until recently, it was not known if they survived, much less thrived in thermoacidic environments, that is, locations with pH values of approximately 1 and temperatures greater than 50 degrees C. Recently, three independent studies have isolated unusual methanotrophs from such extreme environments, expanding the known functional and phylogenetic diversity of methanotrophs.

Improvements in the purification of methanobactin (mb) from eitherMethylosinus trichosporiumOB3bTorMethylococcus capsulatusBath resulted in preparations that stimulated methane-oxidation activity in both whole-cell and cell-free fractions ofMethylococcus capsulatusBath expressing the membrane-associated methane monooxygenase (pMMO). By using washed membrane factions with pMMO activities in the 290 nmol propylene oxidized min−1(mg protein)−1range, activities approaching 400 nmol propylene oxidized min−1(mg protein)−1were commonly observed following addition of copper-containing mb (Cu–mb), which represented 50–75 % of the total whole-cell activity. The stimulation of methane-oxidation activity by Cu–mb was similar to or greater than that observed with equimolar concentrations of Cu(II), without the inhibitory effects observed with high copper concentrations. Stimulation of pMMO activity was not observed with copper-free mb, nor was it observed when the copper-to-mb ratio was 4and/or O2). The results indicated that Cu–mb increased electron flow to the pMMO, increased the free radical formed following the addition of O2and decreased the residual free radical following the addition of O2plus CH4. The increase in pMMO activity and EPR spectral changes to the pMMO following Cu–mb addition represent the first positive evidence of interactions between the pMMO and Cu–mb.

Cells of the magnetotactic marine vibrio, strain MV-1, produce magnetite-containing magnetosomes when grown anaerobically or microaerobically. Stable, spontaneous, non-magnetotactic mutants were regularly observed when cells of MV-1 were cultured on solid media incubated under anaerobic or microaerobic conditions. Randomly amplified polymorphic DNA analysis showed that these mutants are not all genetically identical. Cellular iron content of one non-magnetotactic mutant strain, designated MV-1nm1, grown anaerobically, was ∼20- to 80-fold less than the iron content of wild-type (wt) MV-1 for the same iron concentrations, indicating that MV-1nm1 is deficient in some form of iron uptake. Comparative protein profiles of the two strains showed that MV-1nm1 did not produce several proteins produced by wt MV-1. To understand the potential roles of these proteins in iron transport better, one of these proteins was purified and characterized. This protein, a homodimer with an apparent subunit mass of about 19 kDa, was an iron-regulated, periplasmic protein (p19). Two potential ‘copper-handling’ motifs (MXM/MX2M) are present in the amino acid sequence of p19, and the native protein binds copper in a 1 : 1 ratio. The structural gene for p19,chpA(copperhandlingprotein) and two other putative genes upstream ofchpAwere cloned and sequenced. These putative genes encode a protein similar to the iron permease, Ftr1, from the yeastSaccharomyces cerevisiae, and a ferredoxin-like protein of unknown function. A periplasmic, copper-containing, iron(II) oxidase was also purified from wt MV-1 and MV-1nm1. This enzyme, like p19, was regulated by media iron concentration and contained four copper atoms per molecule of enzyme. It is hypothesized that ChpA, the iron permease and the iron(II) oxidase might have analogous functions for the three components of theS. cerevisiaecopper-dependent high-affinity iron uptake system (Ctr1, Ftr1 and Fet3, respectively), and that strain MV-1 may have a similar iron uptake system. However, iron(II) oxidase purified from both wt MV-1 and MV-1nm1 displayed comparable iron oxidase activities using O2as the electron acceptor, indicating that ChpA does not supply the multi-copper iron(II) oxidase with copper.

Temporal pigmentation changes resulting from the development of a purple color in anaerobic swine waste lagoons were investigated during a 4-year period. The major purple photosynthetic bacterium responsible for these color changes and the corresponding reductions in odor was isolated from nine photosynthetic lagoons. By using morphological, physiological, and phylogenetic characterization methods we identified the predominant photosynthetic bacterium as a new strain of Rhodobacter , designated Rhodobacter sp. strain PS9. Rhodobacter sp. strain PS9 is capable of photoorganotrophic growth on a variety of organic compounds, including all of the characteristic volatile organic compounds (VOC) responsible for the odor associated with swine production facilities (J. A. Zahn, A. A. DiSpirito, Y. S. Do, B. E. Brooks, E. E. Copper, and J. L. Hatfield, J. Environ. Qual. 30: 624-634, 2001). The seasonal variations in airborne VOC emitted from waste lagoons showed that there was a 80 to 93% decrease in the concentration of VOC during a photosynthetic bloom. During the height of a bloom, the Rhodobacter sp. strain PS9 population accounted for 10% of the total community and up to 27% of the eubacterial community based on 16S ribosomal DNA signals. Additional observations based on seasonal variations in meteorological, biological, and chemical parameters suggested that the photosynthetic blooms of Rhodobacter sp. strain PS9 were correlated with lagoon water temperature and with the concentrations of sulfate and phosphate. In addition, the photosynthetic blooms of Rhodobacter sp. strain PS9 were inversely correlated with the concentrations of protein and fluoride.

Succinic acid production medium for Actinobacillus succinogenes was simplified. Vitamins and fatty acids were removed from the medium and industrial quality yeast extract was substituted to yield revised succinic acid medium. Customized plastic composite support (PCS) blends were screened for biofilm and succinic acid production. Twenty different PCS blends with and without mineral salt additions were evaluated in twenty simulated repeated-batch fermentations using MgCO3 for pH control and CO2 supply. Succinic acid concentrations, percentage yield succinic acid, and biofilm formation for each PCS blend were determined. No correlation was observed for biofilm formation and succinic acid production. The selected customized PCS blend for A. succinogenes succinic acid production consisted of 50% (wt/wt) polypropylene, 35% (wt/wt) dried ground soybean hulls, 5% (wt/wt) dried bovine albumin, 2.5% (wt/wt) soybean flour, 2.5% (wt/wt) yeast extract, 2.5% (wt/wt) dried red blood cells, and 2.5% (wt/wt) peptone, whi...

Gaseous emissions from swine (Sus scrofa) manure storage systems represent a concern to air quality due to the potential effects of hydrogen sulfide, ammonia, methane, and volatile organic compounds on environmental quality and human health. The lack of knowledge concerning functional aspects of swine manure management systems has been a major obstacle in the development and optimization of emission abatement technologies for these point sources. In this study, a classification system based on gas emission characteristics and effluent concentrations of total phosphorus (P) and total sulfur (S) was devised and tested on 29 swine manure management systems in Iowa, Oklahoma, and North Carolina in an effort to elucidate functional characteristics of these systems. Four swine manure management system classes were identified that differed in effluent concentrations of P and S, methane (CH 4 ) emission rate, odor intensity, and air concentration of volatile organic compounds (VOCs). Odor intensity and the concentration of VOCs in air emitted from swine manure management systems were strongly correlated (r 2 = 0.88). The concentration of VOC in air samples was highest with outdoor swine manure management systems that received a high input of volatile solids (Type 2). These systems were also shown to have the highest odor intensity levels. The emission rate for VOCs and the odor intensity associated with swine manure management systems were inversely correlated with CH 4 and ammonia (NH 3 ) emission rates. The emission rates of CH 4 , NH 3 , and VOCs were found to be dependent upon manure loading rate and were indirectly influenced by animal numbers.

Gaseous emissions from swine manure storage systems represent a concern to air quality due to the potential impacts of hydrogen sulfide, ammonia, methane, and volatile organic compounds on environmental quality, human health, and wellbeing. The lack of knowledge concerning functional aspects of swine manure management systems has been a major obstacle in the development and optimization of emission abatement technologies for these point sources. In this study, a classification system based on emission characteristics and solution-phase concentrations of phosphorus (P) and sulfur (S) was devised and tested on 29 swine manure management systems in Iowa, Oklahoma, and North Carolina in an effort to elucidate functional characteristics of swine manure management systems. Four swine manure management system classes were identified that differed in solution-phase concentrations of P and S, methane emission rate, odor intensity, and air concentration of volatile organic compounds (VOCs). Odor intensity and the air concentration of VOCs emitted from swine manure management systems were shown to be strongly correlated (r = 0.88). The concentration of VOC in air samples from the 29 swine manure management systems was highest with high-load, outdoor manure management systems (Type 2). These swine manure management systems were also observed to have the highest odor intensity levels. Volatile organic compound (VOC) emission rate and odor intensity for swine manure management systems were found to be inversely correlated with methane and ammonia emission rates. Emission rate of methane, ammonia, and volatile organic compounds were found to be dependent upon manure loading rate and indirectly influenced by animal numbers. Data show that current swine production activities have the potential to produce up to 386 tons of air pollutants • site • year.

Whole-cell assays were used to measure the ef- fect of dichloromethane and trichloroethylene on methane oxidation by Methylosinus trichosporiumOB3b synthesiz- ing the membrane-associated or particulate methane mono- oxygenase (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 re- action 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 competi- tive inhibitor, while trichloroethylene acted as a noncom- petitive inhibitor. The finding of noncompetitive inhibi- tion by trichloroethylene was further examined by mea- suring the inhibition constants KiE and KiES. These constants suggest that trichloroethylene competes with methane at some sites, although it can bind to others if methane is al- ready bound. Whole-cell oxygen uptake experiments for ac- tive and acetylene-treated cells also showed that provision of formate could stimulate both methane and trichloroeth- ylene oxidation and that trichloroethylene did not affect formate dehydrogenase activity. The finding that different chlorinated hydrocarbons caused different inhibition pat- terns 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 pu- rified pMMO.