Scoping acetogen diversity for biotechnological applications

Abstract

Acetogens are anaerobic microorganisms capable of utilising CO and/or H2 and CO2 as their sole source of carbon and energy. Ubiquitous throughout anaerobic environments, they are of particular interest in biotechnology as some species are capable of producing commercially valuable end-products including ethanol and butanol. The company LanzaTech has successfully developed the process of ethanol production from CO-rich gases. This project was a joint collaboration between The University of Auckland and LanzaTech. The aim was to develop methods for the exploration of acetogen diversity for further applications in biotechnology. A PCR method developed for the detection of acetogens in mixed samples was based on the CODH/ACS gene central to the Wood-Ljungdahl pathway of acetogenesis. This was used to screen environmental samples to identify sources of inoculants for enrichments. Termite hindgut and bovine rumen samples were identified as rich sources of acetogens. Anaerobic digested sludge samples did not amplify with CODH/ACS primers and were concluded to be a poor source of acetogenic bacteria. Bioreactor and serum bottle enrichment systems were established and operated under conditions that created high selective pressure for CO-utilising acetogens. The PCR method for CODH/ACS detection was used to screen enrichment samples for the presence of acetogens. Increases in microbial growth were observed in serum bottle enrichments, however DNA extractions for most serum bottle samples generated insufficient quantities of DNA for PCR analysis with CODH/ACS primers. Analysis of samples from bioreactor enrichments including OD, microscopy, PCR with CODH/ACS primers and HPLC showed increases in biomass and indicated the presence of actively growing microorganisms that could use CO as a growth substrate. Phylogenetic analysis of ACS amino acid sequences from environmental and experimental samples confirmed the detection of CODH/ACS and indicated a diversity of acetogens in each sample. Differences in acetogen communities between termite and rumen samples were observed. Many sequences from both environmental and experimental samples did not relate phylogenetically to any known isolates, suggesting the presence of novel acetogens. Further optimisation of enrichment parameters will be required to ensure ideal conditions for acetogen growth, however the established methodology provides a stable foundation for future work to isolate acetogens for biotechnology applications.