Reconstruction of a Genome Scale Metabolic Model of the polyhydroxybutyrate producing methanotroph Methylocystis parvus OBBP.

BACKGROUND: Methylocystis parvus is a type II methanotroph characterized by its high specific methane degradation rate (compared to other methanotrophs of the same family) and its ability to accumulate up to 50% of its biomass in the form of poly-3-hydroxybutyrate (PHB) under nitrogen limiting conditions. This makes it a very promising cell factory. RESULTS: This article reports the first Genome Scale Metabolic Model of M. parvus OBBP. The model is compared to Genome Scale Metabolic Models of the closely related methanotrophs Methylocystis hirsuta and Methylocystis sp. SC2. Using the reconstructed model, it was possible to predict the biomass yield of M. parvus on methane. The prediction was consistent with the observed experimental yield, under the assumption of the so called "redox arm mechanism" for methane oxidation. The co-consumption of stored PHB and methane was also modeled, leading to accurate predictions of biomass yields and oxygen consumption rates and revealing an anaplerotic role of PHB degradation. Finally, the model revealed that anoxic PHB consumption has to be coupled to denitrification, as no fermentation of PHB is allowed by the reconstructed metabolic model. CONCLUSIONS: The "redox arm" mechanism appears to be a general characteristic of type II methanotrophs, versus type I methanotrophs that use the "direct coupling" mechanism. The co-consumption of stored PHB and methane was predicted to play an anaplerotic role replenishing the serine cycle with glyoxylate and the TCA cycle with succinyl-CoA, which allows the withdrawal of metabolic precursors for biosynthesis. The stored PHB can be also used as an energy source under anoxic conditions when coupled to denitrification.

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

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

Methanotrophs mediated biogas valorization: Sustainable route to polyhydroxybutyrate production.

This study explores the ability of methanotrophs to convert biogas into biopolymers, addressing H2S as a limitation in the utilization of biogas as a carbon source for bioconversion. Transcriptomic analysis was conducted to understand the growth and changes in the expression patterns of Type I and II methanotrophs under varying H2S concentrations. Results suggested that Type II methanotrophs can possess a native H2S utilization pathway. Both Type I and II methanotrophs were evaluated for their growth and polyhydroxybutyrate (PHB) production from biogas. Methylocystis sp. MJC1 and Methylocystis sp. OK1 exhibited a maximum biomass production of 4.0 and 4.5 gDCW/L, respectively, in fed-batch culture, aligning with the transcriptome data. Furthermore, Methylocystis sp. MJC1 produced 2.9 g PHB/L from biogas through gas fermentation. These findings underscore biogas-based biotechnology as an innovative solution for environmental and industrial challenges with further optimization and productivity enhancement research expected to broaden the potential in this field.

Biosynthesis of polyhydroxybutyrate from methane and carbon dioxide using type II methanotrophs.

Methane (CH4) and carbon dioxide (CO2) are the dominant greenhouse gases (GHGs) that are increasing at an alarming rate. Methanotrophs have emerged as potential CH4 and CO2 biorefineries. This study demonstrated the synchronous incorporation of CH4 and CO2 into polyhydroxybutyrate (PHB) for the first time using 13C-labeling experiments in methanotrophs. By supplying substantial amounts of CO2, PHB content was enhanced in all investigated type II methanotrophic strains by 140 %, 146 %, and 162 %. The highest content of PHB from CH4 and CO2 in flask-scale cultivation reached 38 % dry cell weight in Methylocystis sp. MJC1, in which carbon percentage in PHB from CO2 was 45 %. Flux balance analysis predicted the critical roles of crotonyl-CoA carboxylase/reductase and phosphoenolpyruvate carboxylase in CO2 recycling. This study provided proof of the conversion of GHGs into a valuable and practical product using methanotrophic bacteria, contributing to addressing GHG emissions.

Genome sequence of the methanotrophic poly-ß-hydroxybutyrate producer Methylocystis parvus OBBP.

Methylocystis parvus OBBP is an obligate methylotroph considered the type species of the genus Methylocystis. Two pmoCAB particulate methane monooxygenase operons and one additional singleton pmoC paralog were identified in the sequence. No evidence of genes encoding soluble methane monooxygenase was found. Comparison of M. parvus OBBP and Methylocystis sp. strain Rockwell (ATCC 49242) suggests that both species should be taxonomically classified in different genera.

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.

Potassium deficiency results in accumulation of ultra-high molecular weight poly-beta-hydroxybutyrate in a methane-utilizing mixed culture.

AIMS: To investigate the effect of various single nutrient deficiencies on poly-beta-hydroxybutyrate (PHB) biosynthesis in a methane-utilizing mixed culture (dominant species Methylocystis sp. GB 25 DSM 7674). METHODS AND RESULTS: Poly-beta-hydroxybutyrate accumulation experiments were performed in 7 and 70 l bioreactors and initiated by potassium, sulfur or iron deficiency. After 24 h the PHB content reached levels of 33.6%, 32.6% and 10.4% respectively. Interestingly a polymer with an ultra-high average-weight molecular weight (M(w)) of 3.1 MDa was accumulated under potassium-limited conditions. When sulfur and iron were lacking M(w) were lower by 20.6 and 41.6%. Potassium-deficiency experiments were furthermore characterized by a maximum specific PHB formation rate 0.08 g g(-1)residual biomass (R) h(-1) and a yield coefficient of 0.45 g PHB g(-1) CH(4). CONCLUSIONS: Biosynthesis of an ultra-high M(w) PHB in a methane-utilizing mixed culture can be induced by potassium deficiency. SIGNIFICANCE AND IMPACT OF THE STUDY: This study greatly extends the knowledge in the field of bacterial biopolymer formation with gaseous substrates. The special system used here combines the use of methane a low-cost substrate available from natural and renewable sources with the possibility of employing a mixed-culture in an open system for the synthesis of a high-value product.

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

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

Methanotrophic production of polyhydroxybutyrate-co-hydroxyvalerate with high hydroxyvalerate content.

Type II methanotrophic bacteria are a promising production platform for PHA biopolymers. These bacteria are known to produce pure poly-3-hydroxybutyrate homopolymer (PHB). We isolated a strain, Methylocystis sp. WRRC1, that was capable of producing a wide range of polyhydroxybutyrate-co-hydroxyvalerate copolymers (PHB-co-HV) when co-fed methane and valerate or n-pentanol. The ratio of HB to HV monomer was directly related to the concentration of valeric acid in the PHA accumulation media. We observed increased incorporation of HV and total polymer under copper-free growth conditions. The PHB-co-HV copolymers produced had decreased melting temperatures and crystallinity compared with methanotroph-produced PHB.

Possibilities for controlling a PHB accumulation process using various analytical methods.

Poly-beta-hydroxybutyrate (PHB) and other polyesters can be produced by various species of bacteria. Of the possible carbon sources, methane could prove to be one of the most suitable substrates for the manufacture of PHB. The methanotrophic strain Methylocystis sp. GB 25 DSM 7674 was applied in order to accumulate PHB in a rapid, non-sterile process. Cultivation was performed in two stages: a continuous growth phase (dilution rate 0.17 h(-1)) and a PHB accumulation phase under deficiency conditions of an essential nutrient (e.g. phosphorus) in batch culture. The PHB content of the biomass was as high as 51%; efficiency was the highest during the first 5 h of the product formation process. The PHB produced is of very high quality, having a high molecular mass of up to 2.5 x 10(6) Da. In order to monitor and control the process, a rapid analysis method based upon turbidimetry in the visible range (438 nm) was applied. Moreover, the PHB content of the biomass was determined using an FT-IR-spectroscopic method with ATR sampling and multivariate calibration. We achieved a value of 1.4% as the best standard error of cross validation. The nitrogen content of the PHB final product (a product quality parameter) was estimated by spectroscopic method in the visible range.

Long-term cultivation of a stable Methylocystis-dominated methanotrophic enrichment enabling tailored production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

Methane (CH4) is a readily available feedstock for production of polyhydroxyalkanoates (PHAs). The structure and PHA production capacity of a Methylocystis-dominated methanotrophic enrichment was stable in long-term operation (>175 days) when grown exponentially under non-aseptic conditions in fill-and-draw batch cultures with ammonium as nitrogen source. Cells harvested in the draw step were incubated in the absence of nitrogen with various combinations of CH4 and valerate to assess capacity for synthesis of poly(3-hydroxybutyrate) (P3HB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). When fed CH4 alone, only P3HB was produced. When fed CH4 plus valerate, PHBV was synthesized. The mol% of 3-hydroxyvalerate (3HV) increased with added valerate. Oxidation of CH4 was required for valerate assimilation, and the fraction of CH4 oxidized increased with increased mol% 3 HV. By separating PHA accumulation from cell replication, tailored PHA-rich biomass can be generated by addition of co-substrate, while retaining a large inoculum for the next cycle of cell division.

[Features of solid materials' colonization by pure and mixed cultures of methanotrophs]. / Osobennosti kolonizatsii tverdykh materialov chistymi i smeshannymi kul'turami metanotrofov.

The process of colonization of hydrophilic (glass) and hydrophobic (polysterene) carriers by pure cultures of methanotrophs Methylocystis parvus UCM B-3490T, Methylococcus capsulatus UCM B-3030, as well as by their cultures mixed with Bacillus megaterium UCM B 5723T and Pseudomonas putida VKPM B-4188 under the conditions efficient for methanotrophic bacteria. M. parvus demonstrated the highest intensity of this process on the above carriers owing to high hydrophobic cell surface. Both methanotrophs colonized the glass surface more quickly with formation of microcolonies on carriers after 6 days of incubation in pure and mixed cultures with B. megaterium. The number of bacilli on these carriers quickly decreased. In the mixed cultures with P. putida the glass and polysterene colonization intensity decreased, while the amount of pseudomonas on carriers increased.

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

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

Characterizing a stable methane-utilizing mixed culture used in the synthesis of a high-quality biopolymer in an open system.

AIMS: To characterize a methane-utilizing poly-beta-hydroxybutyrate (PHB)-producing microbial community. METHODS AND RESULTS: Three different approaches based on microbiology, analytical chemistry and molecular biology were used to determine the composition of the mixed culture. The dominant species, Methylocystis sp. GB25, represents more than 86% of the total biomass. Seven accompanying bacterial species are present in the mixed culture of which two are methylotrophic bacteria and five are utilizers of complex carbon sources. Both these groups were found to be present at the same ratio with respect to each other. Results of fatty acid analysis and PCR-DGGE fingerprints reflect the stability of the mixed-culture composition in the open system during multiple continuous growth and polymer formation processes throughout a period of 29 months. The consistently high quality of the accumulated polymer further corroborates this finding. CONCLUSION: The methane-utilizing mixed culture has the potential of self-regulation resulting in a stable composition even under non-aseptic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Avoiding the necessity of sterile conditions, as demonstrated in this paper, is an important step towards the development of a viable large-scale process for the production of PHB using cheap substrates like methane from natural or renewable sources. This is the first report characterizing a bacterial mixed culture being used for the biotechnological production of a high-value product in an open system.