Influence of the Endogenous Storage Lipid Poly-beta-Hydroxybutyrate on the Reducing Power Availability during Cometabolism of Trichloroethylene and Naphthalene by Resting Methanotrophic Mixed Cultures

The role of the storage lipid poly-beta-hydroxybutyrate (PHB) in trichloroethylene transformation by methanotrophic mixed cultures was investigated. Naphthalene oxidation rates were used to assay for soluble methane monooxygenase activity. The PHB content of methanotrophic cells grown in reactors varied diurnally as well as from day to day. A positive correlation between the amount of PHB in the cells and the naphthalene oxidation rate as well as between PHB and the trichloroethylene transformation rate and capacity was found. Addition of beta-hydroxybutyrate increased the naphthalene oxidation rates significantly. PHB content in cells could be manipulated by incubation at different methane-to-nitrogen ratios. A positive correlation between the naphthalene oxidation rate and the PHB content after these incubations could be seen. Both the PHB content and the naphthalene oxidation rates decreased with time in resting methanotrophic cells exposed to oxygen. However, this decrease in the naphthalene oxidation rate cannot be explained by the decrease in the PHB content alone. Probably a deactivation of the methane monooxygenase itself is also involved.     

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.     

Evaluation of Toxic Effects of Aeration and Trichloroethylene Oxidation on Methanotrophic Bacteria Grown with Different Nitrogen Sources

In this study we evaluated specific and nonspecific toxic effects of aeration and trichloroethylene (TCE) oxidation on methanotrophic bacteria grown with different nitrogen sources (nitrate, ammonia, and molecular nitrogen). The specific toxic effects, exerted directly on soluble methane monooxygenase (sMMO), were evaluated by comparing changes in methane uptake rates and naphthalene oxidation rates following aeration and/or TCE oxidation. Nonspecific toxic effects, defined as general cellular damage, were examined by using a combination of epifluorescent cellular stains to measure viable cell numbers based on respiratory activity and measuring formate oxidation activities following aeration and TCE transformation. Our results suggest that aeration damages predominantly sMMO rather than other general cellular components, whereas TCE oxidation exerts a broad range of toxic effects that damage both specific and nonspecific cellular functions. TCE oxidation caused sMMO-catalyzed activity and respiratory activity to decrease linearly with the amount of substrate degraded. Severe TCE oxidation toxicity resulted in total cessation of the methane, naphthalene, and formate oxidation activities and a 95% decrease in the respiratory activity of methanotrophs. The failure of cells to recover even after 7 days of incubation with methane suggests that cellular recovery following severe TCE product toxicity is not always possible. Our evidence suggests that generation of greater amounts of sMMO per cell due to nitrogen fixation may be responsible for enhanced TCE oxidation activities of nitrogen-fixing methanotrophs rather than enzymatic protection mechanisms associated with the nitrogenase enzymes.     

Optimization of trichloroethylene oxidation by methanotrophs and the use of a colorimetric assay to detect soluble methane monooxygenase activity

Methylosinus trichosporium OB3b biosynthesizes a broad specificity soluble methane monooxygenase that rapidly oxidizes trichloroethylene (TCE). The selective expression of the soluble methane monooxygenase was followed in vivo by a rapid colorimetric assay. Naphthalene was oxidized by purified soluble methane monooxygenase or by cells grown in copper-deficient media to a mixture of 1-naphthol and 2-naphthol. The naphthols were detected by reaction with tetrazotized o-dianisidine to form purple diazo dyes with large molar absorptivities. The rate of color formation with the rapid assay correlated with the velocity of TCE oxidation that was determined by gas chromatography. Both assays were used to optimize conditions for TCE oxidation by M. trichosporium OB3b and to test several methanotrophic bacteria for the ability to oxidize TCE and naphthalene.Abbreviations A₆₀₀ absorbance due to cell density measured at 600 nm - HPLC high pressure liquid chromatography - NADH reduced nicotinamide adenine dinucleotide - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - sMMO soluble methane monooxygenase - TCE trichloroethylene     

Characterization of a methane-utilizing bacterium from a bacterial consortium that rapidly degrades trichloroethylene and chloroform.

A mixed culture of bacteria grown in a bioreactor with methane as a carbon and energy source rapidly oxidized trichloroethylene and chloroform. The most abundant organism was a crescent-shaped bacterium that bound the fluorescent oligonucleotide signature probes that specifically hybridize to serine pathway methylotrophs. The 5S rRNA from this bacterium was found to be 93.5% homologous to the Methylosinus trichosporium OB3b 5S RNA sequence. A type II methanotrophic bacterium, isolated in pure culture from the bioreactor, synthesized soluble methane monooxygenase during growth in a copper-limited medium and was also capable of rapid trichloroethylene oxidation. The bacterium contained the gene that encodes the soluble methane monooxygenase B component on an AseI restriction fragment identical in size to a restriction fragment present in AseI digests of DNA from bacteria in the mixed culture. The sequence of the 16S rRNA from the pure culture was found to be 92 and 94% homologous to the 16S rRNAs of M. trichosporium OB3b and M. sporium, respectively. Both the pure and mixed cultures oxidized naphthalene to naphthol, indicating the presence of soluble methane monooxygenase. The mixed culture also synthesized soluble methane monooxygenase, as evidenced by the presence of proteins that cross-reacted with antibodies prepared against purified soluble methane monooxygenase components from M. trichosporium OB3b on Western blots (immunoblots). It was concluded that a type II methanotrophic bacterium phylogenetically related to Methylosinus species synthesizes soluble methane monooxygenase and is responsible for trichloroethylene oxidation in the bioreactor.     

Characterization of a methane-utilizing bacterium from a bacterial consortium that rapidly degrades trichloroethylene and chloroform.

A mixed culture of bacteria grown in a bioreactor with methane as a carbon and energy source rapidly oxidized trichloroethylene and chloroform. The most abundant organism was a crescent-shaped bacterium that bound the fluorescent oligonucleotide signature probes that specifically hybridize to serine pathway methylotrophs. The 5S rRNA from this bacterium was found to be 93.5% homologous to the Methylosinus trichosporium OB3b 5S RNA sequence. A type II methanotrophic bacterium, isolated in pure culture from the bioreactor, synthesized soluble methane monooxygenase during growth in a copper-limited medium and was also capable of rapid trichloroethylene oxidation. The bacterium contained the gene that encodes the soluble methane monooxygenase B component on an AseI restriction fragment identical in size to a restriction fragment present in AseI digests of DNA from bacteria in the mixed culture. The sequence of the 16S rRNA from the pure culture was found to be 92 and 94% homologous to the 16S rRNAs of M. trichosporium OB3b and M. sporium, respectively. Both the pure and mixed cultures oxidized naphthalene to naphthol, indicating the presence of soluble methane monooxygenase. The mixed culture also synthesized soluble methane monooxygenase, as evidenced by the presence of proteins that cross-reacted with antibodies prepared against purified soluble methane monooxygenase components from M. trichosporium OB3b on Western blots (immunoblots). It was concluded that a type II methanotrophic bacterium phylogenetically related to Methylosinus species synthesizes soluble methane monooxygenase and is responsible for trichloroethylene oxidation in the bioreactor.     

Biodegradation of trichloroethylene by Methylosinus trichosporium OB3b

The methanotroph Methylosinus trichosporium OB3b, a type II methanotroph, degraded trichloroethylene at rates exceeding 1.2 mmol/h per g (dry weight) following the appearance of soluble methane monooxygenase in continuous and batch cultures. Cells capable oxidizing trichloroethylene contained components of soluble methane monooxygenase as demonstrated by Western blot (immunoblot) analysis with antibodies prepared against the purified enzyme. Growth of cultures in a medium containing 0.25 microM or less copper sulfate caused derepression of the synthesis of soluble methane monooxygenase. In these cultures, the specific rates of methane and methanol oxidation did not change during growth, while trichloroethylene oxidation increased with the appearance of soluble methane monooxygenase. M. trichosporium OB3b cells that contained soluble methane monooxygenase also degraded vinyl chloride, 1,1-dichloroethylene, cis-1,2-dichloroethylene, and trans-1,2-dichloroethylene.     

Biodegradation of trichloroethylene by Methylosinus trichosporium OB3b.

The methanotroph Methylosinus trichosporium OB3b, a type II methanotroph, degraded trichloroethylene at rates exceeding 1.2 mmol/h per g (dry weight) following the appearance of soluble methane monooxygenase in continuous and batch cultures. Cells capable oxidizing trichloroethylene contained components of soluble methane monooxygenase as demonstrated by Western blot (immunoblot) analysis with antibodies prepared against the purified enzyme. Growth of cultures in a medium containing 0.25 microM or less copper sulfate caused derepression of the synthesis of soluble methane monooxygenase. In these cultures, the specific rates of methane and methanol oxidation did not change during growth, while trichloroethylene oxidation increased with the appearance of soluble methane monooxygenase. M. trichosporium OB3b cells that contained soluble methane monooxygenase also degraded vinyl chloride, 1,1-dichloroethylene, cis-1,2-dichloroethylene, and trans-1,2-dichloroethylene.     

Mechanistic Analysis of Ammonium Inhibition of Atmospheric Methane Consumption in Forest Soils

Methane consumption by forest soil was studied in situ and in vitro with respect to responses to nitrogen additions at atmospheric and elevated methane concentrations. Methane concentrations in intact soil decreased continuously from atmospheric levels at the surface to 0.5 ppm at a depth of 14 cm. The consumption rate of atmospheric methane in soils, however, was highest in the 4- to 8-cm depth interval (2.9 nmol per g of dry soil per day), with much lower activities below and above this zone. In contrast, extractable ammonium and nitrate concentrations were highest in the surface layer (0 to 2 cm; 22 and 1.6 μmol per g of dry soil, respectively), as was potential ammonium-oxidizing activity (19 nmol per g of dry soil per day). The difference in zonation between ammonium oxidation and methane consumption suggested that ammonia-oxidizing bacteria did not contribute significantly to atmospheric methane consumption. Exogenous ammonium inhibited methane consumption in situ and in vitro, but the pattern of inhibition did not conform to expectations based on simple competition between ammonia and methane for methane monooxygenase. The extent of ammonium inhibition increased with increasing methane concentration. Inhibition by a single ammonium addition remained constant over a period of 39 days. In addition, nitrite, the end product of methanotrophic ammonia oxidation, was a more effective inhibitor of methane consumption than ammonium. Factors that stimulated ammonium oxidation in soil, e.g., elevated methane concentrations and the availability of cosubstrates such as formate, methanol, or β-hydroxybutyrate, enhanced ammonium inhibition of methane oxidation, probably as a result of enhanced nitrite production.     

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     

Effects of toxicity, aeration, and reductant supply on trichloroethylene transformation by a mixed methanotrophic culture

The trichloroethylene (TCE) transformation rate and capacity of a mixed methanotrophic culture at room temperature were measured to determine the effects of time without methane (resting), use of an alternative energy source (formate), aeration, and toxicity of TCE and its transformation products. The initial specific TCE transformation rate of resting cells was 0.6 mg of TCE per mg of cells per day, and they had a finite TCE transformation capacity of 0.036 mg of TCE per mg of cells. Formate addition resulted in increased initial specific TCE transformation rates (2.1 mg/mg of cells per day) and elevated transformation capacity (0.073 mg of TCE per mg of cells). Significant declines in methane conversion rates following exposure to TCE were observed for both resting and formate-fed cells, suggesting toxic effects caused by TCE or its transformation products. TCE transformation and methane consumption rates of resting cells decreased with time much more rapidly when cells were shaken and aerated than when they remained dormant, suggesting that the transformation ability of methanotrophs is best preserved by storage under anoxic conditions.     

Effects of toxicity, aeration, and reductant supply on trichloroethylene transformation by a mixed methanotrophic culture.

The trichloroethylene (TCE) transformation rate and capacity of a mixed methanotrophic culture at room temperature were measured to determine the effects of time without methane (resting), use of an alternative energy source (formate), aeration, and toxicity of TCE and its transformation products. The initial specific TCE transformation rate of resting cells was 0.6 mg of TCE per mg of cells per day, and they had a finite TCE transformation capacity of 0.036 mg of TCE per mg of cells. Formate addition resulted in increased initial specific TCE transformation rates (2.1 mg/mg of cells per day) and elevated transformation capacity (0.073 mg of TCE per mg of cells). Significant declines in methane conversion rates following exposure to TCE were observed for both resting and formate-fed cells, suggesting toxic effects caused by TCE or its transformation products. TCE transformation and methane consumption rates of resting cells decreased with time much more rapidly when cells were shaken and aerated than when they remained dormant, suggesting that the transformation ability of methanotrophs is best preserved by storage under anoxic conditions.     

PAH Degradation and Bioaugmentation by a Marine Methanotrophic Enrichment

Methanotrophic bacteria were enriched from marine sediments and screened for their ability to biotransform polycyclic aromatic hydrocarbons (PAHs). Characterization of the methanotrophic enrichment showed that it was dominated by a Type I methanotroph, although significant amounts of 18:1 fatty acids were detected, suggesting the presence of Type II methanotrophs in marine systems. The methanotrophic enrichment degraded phenanthrene, anthracene, and fluorene to below detectable levels in 15 days. Partial transformation of fluoranthene occurred over 15 days, but pyrene was not transformed. Radiolabeled phenanthrene was oxidized to carbon dioxide with significant production of polar intermediates. The oxidation was inhibited by acetylene, an inhibitor of methane monooxygenase. The addition of the methanotrophic enrichment to a marine culture grown on PAHs as the sole carbon sources increased the transformation rate of phenanthrene, anthracene, and fluorene. The highest removal rates were obtained with a mixture containing 90% methanotroph enrichment and 10% PAH-degrading enrichment (by biomass). Fluoranthene and pyrene degradation rates by the PAH-degrading enrichment were not significantly increased by the addition of the methanotrophic enrichment. A possible mechanism for the increased transformation rate was the rapid oxidation of PAHs by methane monooxygenase, forming an intermediate that is more bioavailable for utilization by the PAH-degraders.     

Characterization of the methanotrophic bacterial community present in a trichloroethylene-contaminated subsurface groundwater site.

Groundwater, contaminated with trichloroethylene (TCE) and tetrachloroethylene (PCE), was collected from 13 monitoring wells at Area M on the U.S. Department of Energy Savannah River Site near Aiken, S.C. Filtered groundwater samples were enriched with methane, leading to the isolation of 25 methanotrophic isolates. The phospholipid fatty acid profiles of all the isolates were dominated by 18:1 omega 8c (60 to 80%), a signature lipid for group II methanotrophs. Subsequent phenotypic testing showed that most of the strains were members of the genus Methylosinus and one isolate was a member of the genus Methylocystis. Most of the methanotroph isolates exhibited soluble methane monooxygenase (sMMO) activity. This was presumptively indicated by the naphthalene oxidation assay and confirmed by hybridization with a gene probe encoding the mmoB gene and by cell extract assays. TCE was degraded at various rates by most of the sMMO-producing isolates, whereas PCE was not degraded. Savannah River Area M and other groundwaters, pristine and polluted, were found to support sMMO activity when supplemented with nutrients and then inoculated with Methylosinus trichosporium OB3b. The maximal sMMO-specific activity obtained in the various groundwaters ranged from 41 to 67% compared with maximal rates obtained in copper-free nitrate mineral salts media. This study partially supports the hypothesis that stimulation of indigenous methanotrophic communities can be efficacious for removal of chlorinated aliphatic hydrocarbons from subsurface sites and that the removal can be mediated by sMMO.     

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.     

Quantitative Determination of the Biodegradable Polymer Poly(β-hydroxybutyrate) in a Recombinant Escherichia coli Strain by Use of Mid-Infrared Spectroscopy and Multivariative Statistics

Fourier transform infrared (FTIR) spectroscopy in combination with the partial least squares (PLS) multivariative statistical technique was used for quantitative analysis of the poly(β-hydroxybutyrate) (PHB) contents of bacterial cells. A total of 237 replicate spectra from 34 samples were obtained together with gas chromatography-determined reference PHB contents. Using the PLS regression, we were able to relate the infrared spectra to the reference PHB contents, and the correlation coefficient between the measured and predicted values for the optimal model with a standard error of prediction of 1.49% PHB was 0.988. With this technique, there are no solvent requirements, sample preparation is minimal and simple, and analysis time is greatly reduced; our results demonstrate the potential of FTIR spectroscopy as an alternative to the conventional methods used for analysis of PHB in bacterial cells.     

Purification and Characterization of the Soluble Methane Monooxygenase of the Type II Methanotrophic Bacterium Methylocystis sp. Strain WI 14

Methane monooxygenase (MMO) catalyzes the oxidation of methane to methanol as the first step of methane degradation. A soluble NAD(P)H-dependent methane monooxygenase (sMMO) from the type II methanotrophic bacterium WI 14 was purified to homogeneity. Sequencing of the 16S rDNA and comparison with that of other known methanotrophic bacteria confirmed that strain WI 14 is very close to the genus Methylocystis. The sMMO is expressed only during growth under copper limitation (<0.1 μM) and with ammonium or nitrate ions as the nitrogen source. The enzyme exhibits a low substrate specificity and is able to oxidize several alkanes and alkenes, cyclic hydrocarbons, aromatics, and halogenic aromatics. It has three components, hydroxylase, reductase and protein B, which is involved in enzyme regulation and increases sMMO activity about 10-fold. The relative molecular masses of the native components were estimated to be 229, 41, and 18 kDa, respectively. The hydroxylase contains three subunits with relative molecular masses of 57, 43, and 23 kDa, which are present in stoichiometric amounts, suggesting that the native protein has an α₂β₂γ₂ structure. We detected 3.6 mol of iron per mol of hydroxylase by atomic absorption spectrometry. sMMO is strongly inhibited by Hg²⁺ ions (with a total loss of enzyme activity at 0.01 mM Hg²⁺) and Cu²⁺, Zn²⁺, and Ni²⁺ ions (95, 80, and 40% loss of activity at 1 mM ions). The complete sMMO gene sequence has been determined. sMMO genes from strain WI 14 are clustered on the chromosome and show a high degree of homology (at both the nucleotide and amino acid levels) to the corresponding genes from Methylosinus trichosporium OB3b, Methylocystis sp. strain M, and Methylococcus capsulatus (Bath).     

Product toxicity and cometabolic competitive inhibition modeling of chloroform and trichloroethylene transformation by methanotrophic resting cells

The rate and capacity for chloroform (CF) and trichloroethylene (TCE) transformation by a mixed methanotrophic culture of resting cells (no exogenous energy source) and formate-fed cells were measured. As reported previously for TCE, formate addition resulted in an increased CF transformation rate (0.35 day-1 for resting cells and 1.5 day-1 for formate-fed cells) and transformation capacity (0.0065 mg of CF per mg of cells for resting cells and 0.015 mg of CF per mg of cells for formate-fed cells), suggesting that depletion of energy stores affects transformation behavior. The observed finite transformation capacity, even with an exogenous energy source, suggests that toxicity was also a factor. CF transformation capacity was significantly lower than that for TCE, suggesting a greater toxicity from CF transformation. The toxicity of CF, TCE, and their transformation products to whole cells was evaluated by comparing the formate oxidation activity of acetylene-treated cells to that of non-acetylene-treated cells with and without prior exposure to CF or TCE. Acetylene arrests the activity of methane monooxygenase in CF and TCE oxidation without halting cell activity toward formate. Significantly diminished formate oxidation by cells exposed to either CR or TCE without acetylene compared with that with acetylene suggests that the solvents themselves were not toxic under the experimental conditions but their transformation products were. The concurrent transformation of CF and TCE by resting cells was measured, and results were compared with predictions from a competitive-inhibition cometabolic transformation model. The reasonable fit between model predictions and experimental observations was supportive of model assumptions.     

Poly-3-hydroxybutyrate metabolism in the type II methanotroph Methylocystis parvus OBBP

Differences in carbon assimilation pathways and reducing power requirements among organisms are likely to affect the role of the storage polymer poly-3-hydroxybutyrate (PHB). Previous researchers have demonstrated that PHB functions as a sole growth substrate in aerobic cultures enriched on acetate during periods of carbon deficiency, but it is uncertain how C(1) metabolism affects the role of PHB. In the present study, the type II methanotroph Methylocystis parvus OBBP did not replicate using stored PHB in the absence of methane, even when all other nutrients were provided in excess. When PHB-rich cultures of M. parvus OBBP were deprived of carbon and nitrogen for 48 h, they did not utilize significant amounts of stored PHB, and neither cell concentrations nor concentrations of total suspended solids changed significantly. When methane and nitrogen both were present, PHB and methane were consumed simultaneously. Cells with PHB had significantly higher specific growth rates than cells lacking PHB. The addition of formate (a source of reducing power) to PHB-rich cells delayed PHB consumption, but the addition of glyoxylate (a source of C(2) units) did not. This and results from other researchers suggest that methanotrophic PHB metabolism is linked to the supply of reducing power as opposed to the supply of C(2) units for synthesis.