Optimizing the Methanotrophic Production of Polyhydroxyalkanoates Using Mixed Microbial Bacteria Cultures

The use of petroleum-based plastics has caused serious economic and environmental problems. It is imperative to develop sustainable ways to produce valuable and environmentally-friendly materials. Methanotrophic bacteria are known for assimilating methane while generating polyhydroxybutyrate (PHB) biopolymers, which are potential substitutes for conventional plastics. Additionally, biologically generated methane and volatile fatty acids (VFAs) are abundant and readily accessible through anaerobic digestion of organic wastes. Thus, optimizing the methanotrophic production will bring significant economic and environmental benefits. In section one, a feast-famine based cultivation system was adopted to produce PHAs using Hyphomicrobium, and Methylocysits dominated bacterial culture. The mixed bacterial culture was fed with acetic acid along with methane was subsequently evaluated for the enhancement of PHB production. Throughout the 27 cycles of cultivations, the mixed bacterial culture fed with methane alone produced the PHB in an average of 26.4 ± 3.5 wt%. By comparison, the PHB production was increased up to 34.8 ± 1.8 wt% when acetic acid was added at the concentration of 300 mg/L under pH-controlled conditions. Additionally, the characterization of PHB polymers (e.g., chemical composition and molecular weight) illustrated that the presence of acetic acid at optimal pH conditions facilitated the elongation of the polymer chain with higher molecular weight. In section two, the feasibility and suitability for scaling up PHAs production by using inocula from wastewater treatment plants (WWTPs) were explored. Methanotrophic enrichment cultures were cultivated by using various inocula from activated sludge and anaerobic digestion processes. Methanotrophs and PHA-accumulating bacteria were characterized by polymerase chain reaction (PCR) products of the pmoA and phaC functional gene. After 5 and 15 cycles of enrichment with methane as the sole carbon source, all cultures presented positive results of the pmoA and phaC gene, which indicate the accumulation of methanotrophs and PHA-accumulating bacteria. The metagenomic sequencing of the 16S rRNA gene from original sludges and enrichment cultures showed that the microbial community of enrichment narrowed down the operational taxonomic unit (OTU) for approximately 96.4%. After the enrichment processes, taxonomy diversity of culture with sludge inoculum from Village Creek Reclamation Facility displayed higher bacterial diversity compared with the cultures from Southside WWTP