(157d) Biological Conversion of Methane into Polyhydroxybutyrate (PHB)

Methane is the second most important greenhouse gas, presenting a global warming potential 28 times higher than CO₂. It implies that methane is much more effective at trapping heat in the atmosphere, causing harmful effects to several ecosystems. On the other side, petro derivative plastics also represent a major threat to several ecosystems, especially when considered its large worldwide production and persistence in the environment due to their non-biodegradability. An alternative to solve both problems could be producing biodegradable plastics using methane as feedstock. Methanotrophs are a group of bacteria present in the environment that are capable of methane usage as carbon source, which results in natural accumulation of polyhydroxybutyrate (PHB) inside their cells as energy reserve granules. PHB is a biodegradable polymer that still presents increased production price which inhibits its entrance into the market. The main goal of this work is to find methanotrophs in the environment and evaluate the feasibility of PHB from methane. Methanotrophic bacteria were obtained after enrichment of sediment samples from mangrove sites (-23.91744, -46.21605) in 25% methane atmosphere periodically renewed for 40 days. Experiments performed to evaluate PHB production were carried out in two stages; the first one aimed at maximum cellular growth and the second one aimed at stimulating PHB production by nitrogen depletion. Nutrient deficiency in the presence of abundant carbon source is required to natural accumulation of PHB within bacterial cells. Experiments were carried out in 250 mL erlenmeyer flasks containing 50 mL of Nitrate Mineral Salts medium and bacterial inoculum of O.D. 0.1 (600 nm). The flasks were closed with rubber stoppers and incubated at 28 ºC and 200 rpm for 12 days. Methane atmosphere in air at a proportion of 1:8 was used to ensure complete methane oxidation, which was replenished every 48 hours to avoid substrate limitation. At the seventh day of experiment, cells were harvested by centrifugation at 7830g for 10 min and transferred to nitrate free NMS to stimulate PHB production. Methane consumption was monitored by gas chromatography (GC). Lyophilized cells were subjected to propanolysis and subsequently analysed by GC for PHB quantification and characterization. Consortium MGISED was obtained from the methanotrophic enrichment. Metagenomic analysis elucidated that this was a Methylosinus trichosporium dominated consortium. M. trichosporium is a Type II methanotrophic bacterium capable of PHB accumulation. Experiments were carried out for 120 h under nitrogen depletion and the consortium achieved 20% of PHB accumulation in cell dry weight (CDW), corresponding to 0.2 g L^-1. Methane consumption rate throughout the experiment was 0.21 g L^-1, in average. This corresponds to 42% of the total carbon source available. Conversion factors of substrate in biomass and bioproduct were calculated as 0.96 g g^-1 and 0.3 g g^-1, respectively. These findings will help elucidate the feasibility of the process of PHB production from methane. The data acquired from PHB production experiments using a methanotrophic consortia dominated by M. trichosporium provided preliminary results to study the feasibility of the process to large-scale. PHB production by the isolated strain will also be studied to achieve more accurate and profound results on this subject, but this strain has shown itself as promising for large-scale PHB production.