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

该计划旨在培养至少2名博士，3名硕士和15名本科学生的多学科项目。比壬烷长的烷烃被认为是长链烷烃。虽然已经分离出能够降解C20-C50烷烃的微生物，但降解率很低，降解机制尚不清楚。本课题主要研究大蜡蛾（Galleria mellonella）幼虫及其消化道微生物对C20或更长的线性烷烃和支链烷烃的降解。这项研究是基于聚乙烯（PE）生物降解剂也能降解长链烷烃的假设。pe是具有不同分支和结晶度的高分子量烷烃。近年来，研究表明，mellonella及其相关生物的幼虫可以降解PE，并从其肠道中分离出PE降解菌。mellonella幼虫将首次在实验室中使用聚乙烯和各种线性和支链烷烃进行培养，以研究这些幼虫可以降解的烷烃范围，并产生用于微生物富集培养的接种物（磨碎的消化道和粪）。在厌氧条件下，高分子量正烷烃在石油污染部位的优先损失，在硫酸盐还原条件下，支链烷烃比正链烷烃更优先降解。因此，采用的富集培养条件将是好氧的，并且首次使用接种的葡萄球菌，厌氧条件具有多种终端电子受体。将合成一系列13C烷烃底物，并在好氧、硝酸盐、硫酸盐还原和甲烷营养条件下添加到微环境中，以鉴定烷烃降解剂。利用密度梯度离心法回收群落总DNA，恢复具有13c富集DNA的降解群体。采用PCR扩增纯化，Illumina MiSeq测序和数据处理鉴定降解物。***分离物降解一系列不同分子量和分支的烷烃的能力将在纯培养中进行检验。降解产物、DSC分析结晶度变化和液体介质表面张力数据将为深入了解降解机理提供依据。拟议的计划为一系列基于G. mellonella和相关生物体微生物组的塑料和石化产品生物降解研究设定了框架。这项工作将大大增加我们对难降解石油馏分和塑料生物降解可能性的认识。降解物的鉴定和分离创造了在生物增强中使用这些生物的机会，并告知工程师降解难降解烷烃所需的适当生理条件（氧化还原，电子受体）

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.**