Poly-3-Hydroxybutyrate Metabolism in the Type II Methanotroph Methylocystis parvus OBBP

Abstract

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.Poly-3-hydroxybutyrate (PHB) is a biologically produced, biodegradable polyester with properties similar to those of polypropylene and mechanical properties that can be tailored for different applications by changing the copolymer content of the polymer (1, 5, 27). Many bacteria accumulate PHB as a carbon storage polymer under conditions of unbalanced growth (i.e., nutrient deficiency and/or carbon excess) (1, 27). Although considerable efforts are currently being devoted to commercializing microbial PHB production, commercialization thus far has been limited because of the relatively high production cost of PHB compared to that of traditional petrochemical-based plastics, such as polyethylene and polypropylene (28). A major fraction of the production cost (30%) (7) is due to feedstocks, which are typically sugars such as glucose and sucrose (27).Methane is both an inexpensive feedstock for PHB production and a potent greenhouse gas; its use as a substrate for PHB production is therefore an effective means of carbon sequestration and an attractive alternative to sugar-based feedstocks. Methanotrophs have been shown to produce PHB (2, 18, 37, 41-44), and Wendlandt et al. (42) have reported PHB levels of up to 30 g liter Ϫ1 in a culture dominated by Methylocystis sp. GB 25 and maintained under nonaseptic conditions. While PHB production is well characterized in a variety of bacteria, comparatively few studies have investigated why bacteria accumulate PHB or the process of PHB consumption (16,20,33,36,41). In nonmethanotrophs, PHB consumption is linked to both short-term replication and long-term survival under carbon starvation. Handrick et al. (16) concluded that Ralstonia eutropha replicat...

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

“…Performance across replicate plates was consistent, allowing meaningful interpretation of trends across nutrient gradients. Growth rates in microtiter plates were only slightly reduced from those observed in serum bottles or bioreactors (14), with growth observed up to a maximum density of 6.9 g/liter total biomass. Higher gas transfer rates may be achievable by either adjusting the gas concentrations, increasing the rate of agitation, or developing pressurized incubation chambers, potentially allowing further increases in total biomass.…”

“…Higher-throughput alternatives to the GC method include Raman spectroscopy (10), BODIPY (borondipyrromethene) fluorescence (11), and Nile red fluorescence (12,13). Nile red fluorescence is particularly adaptable to highthroughput analysis, as the stain fluoresces only in the lipid phase, eliminating the need for additional washing steps, and the fluorescence can be quantitatively linked to P3HB content in bacterial cultivations (14). With modifications to sample preparation, this method is readily compatible with high-throughput flow cytometry or fluorescent plate reader systems.…”

Genome Sequence of the Methanotrophic Poly-β-Hydroxybutyrate Producer Methylocystis parvus OBBP

“…Methylocystis parvus OBBP (NCIMB 11129) is an obligate methylotroph originally isolated as a methane-utilizing bacterium and is considered the type species of the genus Methylocystis (16). This strain has industrial interest because it is able to produce poly-␤-hydroxybutyrate (PHB) from methane (9).…”

Formate Oxidation-Driven Calcium Carbonate Precipitation by Methylocystis parvus OBBP

“…Methylocystis parvus OBBP, a type II MOB, has been previously investigated for biotechnological applications. M. parvus OBBP is known to synthesize poly-3-hydroxybutyrate (PHB), a biopolymer that is used as a raw material for bioplastics (16). The strain accumulates PHB intracellularly when it is provided with an excess of carbon source and in the absence of sufficient essential nutrients (e.g., nitrogen, phosphorus, etc.)…”

“…The strain accumulates PHB intracellularly when it is provided with an excess of carbon source and in the absence of sufficient essential nutrients (e.g., nitrogen, phosphorus, etc.) (16,17). For bioremediation purposes, the particulate methane monooxygenase enzyme expressed by Methylocystis spp.…”