Rapid Enrichment of Cupriavidus necator in Mixed Microbial Cultures Using Autotrophic Growth: Mixed Microbial Cultures for Biodegradable Polymer Production Using CO2 and Organic Wastes

Abstract

Polyhydroxyalkanoate (PHA) is a biodegradable polymer that can replace petroleum-based plastics. Cupriavidus necator has been extensively utilized in industrial PHA production because the bacterium can grow and accumulate high amounts of PHA in their cells under heterotrophic and/or autotrophic growth conditions. Microbial PHA production using mixed cultures reduces the high PHA production costs and allows for the utilization of waste organics as carbon sources. However, C. necator cultivation in mixed microbial cultures has not been investigated before. In this work, we developed innovative methods for rapid and selective enrichment of C. necator in mixed cultures, utilizing the unique metabolic characteristics of C. necator (i.e., the ability to grow heterotrophically and/or autotrophically). Three enrichment methods were proposed: autotrophic-only, alternating autotrophic–heterotrophic, and heterotrophic-only cycles. These methods were examined with two wastewater sources as initial mixed mixtures. In the autotrophic-only method, the fraction of C. necator exceeded 92% of the total microbial population in only 60 h, whereas the alternating autotrophic–heterotrophic cycles showed >81.3% selection of C. necator. However, the heterotrophic-only method resulted in practically no enrichment of C. necator after five repeated feast–famine cycles. When tested for PHA accumulation, enriched cultures using the autotrophic and alternating cycles accumulated significant amounts of PHA, confirming the selective and rapid enrichment of C. necator over other microorganisms. Based on the experimental results, the autotrophic-based enrichment is a potential approach for the cost-effective cultivation of C. necator, which can reduce PHA production costs.