Biodegradation of High Molecular Weight Alkanes by the Microbiome of Galleria mellonella Larvae

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This proposal is designed to train at least 2 PhD, 3 MSc and 15 UG students in a multidisciplinary program. Alkanes longer than nonane are considered to be long chain alkanes. While microorganisms have been isolated that can degrade C20-C50 alkanes, the degradation rate is very low and the mechanism of degradation is unclear. This proposal focuses on the study of the degradation of C20 or longer linear and branched alkanes by greater waxmoth (Galleria mellonella) larvae and microorganisms from its digestive tract. The research is based on the hypothesis that polyethylene (PE)-biodegraders can also degrade long chain alkanes. PEs are high molecular weight alkanes of varying branching and crystallinity. Recently, it was demonstrated that larvae of G. mellonella and related organisms can degrade PE and PE-degrading bacteria have been isolated from their gut. G. mellonella larvae will be grown in the lab using PE and, for the first time, a variety of linear and branched alkanes both to study the range of alkanes that can be degraded by these larvae and to generate inocula (ground-up digestive tracts and fecula) for microbial enrichment cultures. Under anaerobic conditions there is preferential loss of high molecular weight n-alkanes in petroleum-contaminated sites, and there is preferential degradation of branched over normal alkanes under sulphate reducing conditions. Therefore, enrichment culture conditions employed will be aerobic and, for the first time using G. mellonella inocula, anaerobic conditions with a variety of terminal electron acceptors. A range of 13C alkane substrates will be synthesized and spiked into microcosms under aerobic, nitrate-, sulphate-reducing and methanotrophic conditions to identify alkane degraders. Total community DNA will be recovered and separated by density gradient centrifugation to recover the degrading population with 13C-enriched DNA. PCR amplification and purification, and Illumina MiSeq sequencing and data processing will be employed to identify the degraders. The abilities of isolates to degrade a range of alkanes of different molecular weights and branching will be examined in pure culture. Degradation products, DSC analysis of crystallinity changes and surface tension data of liquid media will provide insight into degradation mechanisms. The proposed program sets the framework for a series of investigations into biodegradation of plastics and petrochemicals based on the microbiome of G. mellonella and related organisms. This work will add significantly to our knowledge of the possibilities for biodegradation of recalcitrant petroleum fractions and plastics. Identification and isolation of degraders creates the opportunity to use these organisms in bioaugmentation, as well as informing engineers as to the appropriate physiological conditions (redox, electron acceptor) required for degradation of recalcitrant alkanes.