Methanobactin from Methylocystis sp. Strain SB2 Affects Gene Expression and Methane Monooxygenase Activity in Methylosinus trichosporium OB3b

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 CuCl₂, 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 CuCl₂, 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 CuCl₂. mbnA expression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl₂. These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin “piracy” may be commonplace.     

A TonB-Dependent Transporter Is Responsible for Methanobactin Uptake by Methylosinus trichosporium OB3b

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.     

Spectral and thermodynamic properties of methanobactin from γ-proteobacterial methane oxidizing bacteria: A case for copper competition on a molecular level

Methanobactin (mb) is a low molecular mass copper-binding molecule analogous to iron-binding siderophores. The molecule is produced by many methanotrophic or methane oxidizing bacteria (MOB), but has only been characterized to date in one MOB, Methylosinus trichosporium OB3b. To explore the potential molecular diversity in this novel class of metal binding compound, the spectral (UV-visible, fluorescent, and electron paramagnetic resonance) and thermodynamic properties of mb from two γ-proteobacterial MOB, Methylococcus capsulatus Bath and Methylomicrobium album BG8, were determined and compared to the mb from the α-proteobacterial MOB, M. trichosporium OB3b. The mb from both γ-proteobacterial MOB differed from the mb from M. trichosporium OB3b in molecular mass and spectral properties. Compared to mb from M. trichosporium OB3b, the extracellular concentrations were low, as were copper-binding constants of mb from both γ-proteobacterial MOB. In addition, the mb from M. trichosporium OB3b removed Cu(I) from the mb of both γ-proteobacterial MOB. Taken together the results suggest mb may be a factor in regulating methanotrophic community structure in copper-limited environments.     

Oxidase, superoxide dismutase, and hydrogen peroxide reductase activities of methanobactin from types I and II methanotrophs

Methanobactin (mb) is a copper-binding chromopeptide that appears to be involved in oxidation of methane by the membrane-associated or particulate methane monooxygenase (pMMO). To examine this potential physiological role, the redox and catalytic properties of mb from three different methanotrophs were examined in the absence and presence of O₂. Metal free mb from the type II methanotroph Methylosinus trichosporium OB3b, but not from the type I methanotrophs Methylococcus capsulatus Bath or Methylomicrobium album BG8, were reduced by a variety of reductants, including NADH and duroquinol, and catalyzed the reduction of O₂ to . Copper-containing mb (Cu-mb) from all three methanotrophs showed several interesting properties, including reductase dependent oxidase activity, dismutation of to H₂O₂, and the reductant dependent reduction of H₂O₂ to H₂O. The superoxide dismutase-like and hydrogen peroxide reductase activities of Cu-mb were 4 and 1 order(s) of magnitude higher, respectively, than the observed oxidase activity. The results demonstrate that Cu-mb from all three methanotrophs are redox-active molecules and oxygen radical scavengers, with the capacity to detoxify both superoxide and hydrogen peroxide without the formation of the hydroxyl radicals associated with Fenton reactions. As previously observed with Cu-mb from Ms. trichosporium OB3b, Cu-mb from both type I methanotrophs stimulated pMMO activity. However, in contrast to previous studies using mb from Ms. trichosporium OB3b, pMMO activity was not inhibited by mb from the two type I methanotrophs at low copper to mb ratios.     

Factors affecting competition between type I and type II methanotrophs in two-organism,continuous-flow reactors

Competition experiments were performed in a continuous-flow reactor using Methylosinus trichosporium OB3b, a type II methanotroph, and Methylomonas albus BG8, a type I methanotroph. The experiments were designed to establish conditions under which type II methanotrophs, which have significant cometabolic potential, prevail over type I strains. The primary determinants of species selection were dissolved methane, copper, and nitrate concentrations. Dissolved oxygen and methanol concentrations played secondary roles. M. trichosporium OB3b proved dominant under copper and nitratelimited conditions. The ratio of M. trichosporium to M. albus in the reactor increased ten-fold in less than 100 hours following the removal of copper from the reactor feed. Numbers of M. albus declined to levels that were below detection limits (<106/ml) under nitrogen-limited conditions. In the latter experiment, the competitive success of M. trichosporiumdepended on the maintenance of an ambient dissolved oxygen level below about 7.5 × 10^–5 M, or 30% of saturation with air. The ability of M. trichosporium to express soluble methane monooxygenase under copper limitation and nitrogenase under nitrate limitation was very significant. M. albus predominated under methane-limited conditions, especially when low levels of methanol were simultaneously added with methane to the reactor. The results imply that nitrogen limitation can be used to select for type II strains such as M. trichosporium OB3b. Offprint requests to: Pierre Servais     

Methane and Trichloroethylene Degradation by Methylosinus trichosporium OB3b Expressing Particulate Methane Monooxygenase

Whole-cell assays of methane and trichloroethylene (TCE) consumption have been performed on Methylosinus trichosporium OB3b expressing particulate methane monooxygenase (pMMO). From these assays it is apparent that varying the growth concentration of copper causes a change in the kinetics of methane and TCE degradation. For M. trichosporium OB3b, increasing the copper growth concentration from 2.5 to 20 μM caused the maximal degradation rate of methane (V_max) to decrease from 300 to 82 nmol of methane/min/mg of protein. The methane concentration at half the maximal degradation rate (K_s) also decreased from 62 to 8.3 μM. The pseudo-first-order rate constant for methane, V_max/K_s, doubled from 4.9 × 10⁻³ to 9.9 × 10⁻³ liters/min/mg of protein, however, as the growth concentration of copper increased from 2.5 to 20 μM. TCE degradation by M. trichosporium OB3b was also examined with varying copper and formate concentrations. M. trichosporium OB3b grown with 2.5 μM copper was unable to degrade TCE in both the absence and presence of an exogenous source of reducing equivalents in the form of formate. Cells grown with 20 μM copper, however, were able to degrade TCE regardless of whether formate was provided. Without formate the V_max for TCE was 2.5 nmol/min/mg of protein, while providing formate increased the V_max to 4.1 nmol/min/mg of protein. The affinity for TCE also increased with increasing copper, as seen by a change in K_s from 36 to 7.9 μM. V_max/K_s for TCE degradation by pMMO also increased from 6.9 × 10⁻⁵ to 5.2 × 10⁻⁴ liters/min/mg of protein with the addition of formate. From these whole-cell studies it is apparent that the amount of copper available is critical in determining the oxidation of substrates in methanotrophs that are expressing only pMMO.     

Methylosinus trichosporium OB3b Mutants Having Constitutive Expression of Soluble Methane Monooxygenase in the Presence of High Levels of Copper

The methanotrophic bacterium Methylosinus trichosporium OB3b is unusually active in degrading recalcitrant haloalkanes such as trichloroethylene (TCE). The first and rate-limiting step in the degradation of TCE is catalyzed by a soluble methane monooxygenase (sMMO). This enzyme is not expressed when the cells are grown in the presence of copper at concentrations typically found in polluted groundwater. Under these conditions, M. trichosporium OB3b expresses a particulate form of the enzyme (pMMO), which has a narrow substrate specificity and does not degrade TCE at any significant rate. We have isolated M. trichosporium OB3b mutants that are deficient in pMMO and express sMMO constitutively in the presence of elevated concentrations of copper. One mutant (PP358) exhibited a TCE degradation rate which was almost twice as high as that of the wild-type strain grown under optimal conditions (without copper). All of the mutants lost the ability to express pMMO activity and to form stacked intracellular membranes characteristic of wild-type cells expressing pMMO.     

Ammonium and Nitrite Inhibition of Methane Oxidation by Methylobacter albus BG8 and Methylosinus trichosporium OB3b at Low Methane Concentrations

Methane oxidation by pure cultures of the methanotrophs Methylobacter albus BG8 and Methylosinus trichosporium OB3b was inhibited by ammonium choride and sodium nitrite relative to that in cultures assayed in either nitrate-containing or nitrate-free medium. M. albus was generally more sensitive to ammonium and nitrite than M. trichosporium. Both species produced nitrite from ammonium; the concentrations of nitrite produced increased with increasing methane concentrations in the culture headspaces. Inhibition of methane oxidation by nitrite was inversely proportional to headspace methane concentrations, with only minimal effects observed at concentrations of>500 ppm in the presence of 250 μM nitrite. Inhibition increased with increasing ammonium at methane concentrations of 100 ppm. In the presence of 500 μM ammonium, inhibition increased initially with increasing methane concentrations from 1.7 to 100 ppm; the extent of inhibition decreased with methane concentrations of > 100 ppm. The results of this study provide new insights that explain some of the previously observed interactions among ammonium, nitrite, methane, and methane oxidation in soils and aquatic systems.     

Cerium Regulates Expression of Alternative Methanol Dehydrogenases in Methylosinus trichosporium OB3b

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.     

Soluble Methane Monooxygenase Gene Clusters from Trichloroethylene-Degrading Methylomonas sp. Strains and Detection of Methanotrophs during In Situ Bioremediation

The soluble MMO (sMMO) gene clusters from group I methanotrophs were characterized. An 8.1-kb KpnI fragment from Methylomonas sp. strain KSWIII and a 7.5-kb SalI fragment from Methylomonas sp. strain KSPIII which contained the sMMO gene clusters were cloned and sequenced. The sequences of these two fragments were almost identical. The sMMO gene clusters in the fragment consisted of six open reading frames which were 52 to 79% similar to the corresponding genes of previously described sMMO gene clusters of the group II and group X methanotrophs. The phylogenetic analysis of the predicted amino acid sequences of sMMO demonstrated that the sMMOs from these strains were closer to that from M. capsulatus Bath in the group X methanotrophs than to those from Methylosinus trichosporium OB3b and Methylocystis sp. strain M in the group II methanotrophs. Based on the sequence data of sMMO genes of our strains and other methanotrophs, we designed a new PCR primer to amplify sMMO gene fragments of all the known methanotrophs harboring the mmoX gene. The primer set was successfully used for detecting methanotrophs in the groundwater of trichloroethylene-contaminated sites during in situ-biostimulation treatments.     

Inhibition of nitrous oxide reduction in forest soil microcosms by different forms of methanobactin

Copper plays a critical role in controlling greenhouse gas emissions as it is a key component of the particulate methane monooxygenase and nitrous oxide reductase. Some methanotrophs excrete methanobactin (MB) that has an extremely high copper affinity. As a result, MB may limit the ability of other microbes to gather copper, thereby decreasing their activity as well as impacting microbial community composition. Here, we show using forest soil microcosms that multiple forms of MB; MB from Methylosinus trichosporium OB3b (MB-OB3b) and MB from Methylocystis sp. strain SB2 (MB-SB2) increased nitrous oxide (N₂O) production as well caused significant shifts in microbial community composition. Such effects, however, were mediated by the amount of copper in the soils, with low-copper soil microcosms showing the strongest response to MB. Furthermore, MB-SB2 had a stronger effect, likely due to its higher affinity for copper. The presence of either form of MB also inhibited nitrite reduction and generally increased the presence of genes encoding for the iron-containing nitrite reductase (nirS) over the copper-dependent nitrite reductase (nirK). These data indicate the methanotrophic-mediated production of MB can significantly impact multiple steps of denitrification, as well as have broad effects on microbial community composition of forest soils.     

Effect of Copper Speciation on Whole-Cell Soluble Methane Monooxygenase Activity in Methylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) activity in Methylosinus trichosporium OB3b was found to be more strongly affected as copper-to-biomass ratios changed in a newly developed medium, M2M, which uses pyrophosphate for metal chelation, than in nitrate mineral salts (NMS), which uses EDTA. When M2M medium was amended with EDTA, sMMO activity was similar to that in NMS medium, indicating that EDTA-bound copper had lower bioavailability than pyrophosphate-bound copper. EDTA did not limit the association of copper with the cells; rather, copper was sequestered in a form which did not affect sMMO activity.     

Surface layers of methanotrophic bacteria

Structural and functional characteristics of the regular glycoprotein layers in prokaryotes are analyzed with a special emphasis on aerobic methanotrophic bacteria. S-Layers are present at the surfaces of Methylococcus, Methylothermus, and Methylomicrobium cells. Different Methylomicrobium species either synthesize S-layers with planar (p2, p4) symmetry or form cup-shaped or conical structures with hexagonal (p6) symmetry. A unique, copper-binding polypeptide ‘CorA’/MopE (27/45 kDa), which is coexpressed with the diheme periplasmic cytochrome c peroxidase ‘CorB’/Mca (80 kDa) was found in Methylomicrobium album BG8, Methylomicrobium alcaliphilum 20Z, and Methylococcus capsulatus Bath. This tandem of the surface proteins is functionally analogous to a new siderophore: methanobactin. Importantly, no ‘CorA’/MopE homologue was found in methanotrophs not forming S-layers. The role of surface proteins in copper metabolism and initial methane oxidation is discussed.     

Soluble Methane Monooxygenase Production and Trichloroethylene Degradation by a Type I Methanotroph, Methylomonas methanica 68-1

A methanotroph (strain 68-1), originally isolated from a trichloroethylene (TCE)-contaminated aquifer, was identified as the type I methanotroph Methylomonas methanica on the basis of intracytoplasmic membrane ultrastructure, phospholipid fatty acid profile, and 16S rRNA signature probe hybridization. Strain 68-1 was found to oxidize naphthalene and TCE via a soluble methane monooxygenase (sMMO) and thus becomes the first type I methanotroph known to be able to produce this enzyme. The specific whole-cell sMMO activity of 68-1, as measured by the naphthalene oxidation assay and by TCE biodegradation, was comparatively higher than sMMO activity levels in Methylosinus trichosporium OB3b grown in the same copper-free conditions. The maximal naphthalene oxidation rates of Methylomonas methanica 68-1 and Methylosinus trichosporium OB3b were 551 ± 27 and 321 ± 16 nmol h^-1 mg of protein ^-1, respectively. The maximal TCE degradation rates of Methylomonas methanica 68-1 and Methylosinus trichosporium OB3b were 2,325 ± 260 and 995 ± 160 nmol h^-1 mg of protein^-1, respectively. The substrate affinity of 68-1 sMMO to naphthalene (K_m, 70 ± 4 μM) and TCE (K_m, 225 ± 13 μM), however, was comparatively lower than that of the sMMO of OB3b, which had affinities of 40 ± 3 and 126 ± 8 μM, respectively. Genomic DNA slot and Southern blot analyses with an sMMO gene probe from Methylosinus trichosporium OB3b showed that the sMMO genes of 68-1 have little genetic homology to those of OB3b. This result may indicate the evolutionary diversification of the sMMOs.     

Application of a Tetrazolium Salt with a Water-Soluble Formazan as an Indicator of Viability in Respiring Bacteria

The tetrazolium salt sodium 3′-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis (4-methoxy-6-nitro)benzene-sulfonic acid hydrate (XTT) was examined for use as a colorimetric indicator of viability in respiring bacteria. XTT was reduced to an orange, water-soluble formazan product by Methylosinus trichosporium OB3b, Pseudomonas putida, Escherichia coli, and Bacillus subtilis. Formazan production was proportional to live cell biomass, and XTT was reduced by all cultures in the absence of added electron-coupling agents. XTT reduction by M. trichosporium OB3b was linear over several hours and was stimulated by the presence of an exogenous substrate (methanol). Addition of cyanide to cultures incubated under oxic conditions gave an initial 10-fold increase in XTT reduction. Viability of bacteria incubated in the absence of exogenous carbon substrates was measured as XTT reduction and compared with viability estimates from plate counts. Results obtained with the two methods were generally comparable, but the XTT assay was superior when cell recovery on plates was low. Incubation of E. coli for 7 days in the absence of exogenous carbon substrates decreased viability by 90%, whereas the corresponding decreases for cultures of M. trichosporium OB3b, P. putida, and B. subtilis were less than 40%.     

A comparison of methanobactins from Methylosinus trichosporium OB3b and Methylocystis strain Sb2 predicts methanobactins are synthesized from diverse peptide precursors modified to create a common core for binding and reducing copper ions

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.     

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. Received: 3 November 1998 / Accepted: 1 March 1999     

Effect of Nitrogen Source on Growth and Trichloroethylene Degradation by Methane-Oxidizing Bacteria

The effect of nitrogen source on methane-oxidizing bacteria with respect to cellular growth and trichloroethylene (TCE) degradation ability were examined. One mixed chemostat culture and two pure type II methane-oxidizing strains, Methylosinus trichosporium OB3b and strain CAC-2, which was isolated from the chemostat culture, were used in this study. All cultures were able to grow with each of three different nitrogen sources: ammonia, nitrate, and molecular nitrogen. Both M. trichosporium OB3b and strain CAC-2 showed slightly lower net cellular growth rates and cell yields but exhibited higher methane uptake rates, levels of poly-β-hydroxybutyrate (PHB) production, and naphthalene oxidation rates when grown under nitrogen-fixing conditions. The TCE-degrading ability of each culture was measured in terms of initial TCE oxidation rates and TCE transformation capacities (mass of TCE degraded/biomass inactivated), measured both with and without external energy sources. Higher initial TCE oxidation rates and TCE transformation capacities were observed in nitrogen-fixing mixed, M. trichosporium OB3b, and CAC-2 cultures than in nitrate- or ammonia-supplied cells. TCE transformation capacities were found to correlate with cellular PHB content in all three cultures. The results of this study suggest that the nitrogen-fixing capabilities of methane-oxidizing bacteria can be used to select for high-activity TCE degraders for the enhancement of bioremediation in fixed-nitrogen-limited environments.