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

大蜡螟幼虫微生物组对高分子量烷烃的生物降解

• 批准号: RGPIN-2018-06166
• 负责人: Ramsay, Bruce
• 金额: $ 3.79万
• 依托单位: Queen's University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=747182
• 关键词: Biodegradation; Molecular; Weight; Alkanes; Microbiome

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.

这项提案旨在培养至少2名博士、3名硕士和15名UG学生参加多学科项目。长于正庚烷的烷烃被认为是长链烷烃。虽然已经分离出能降解C20-C50烷烃的微生物，但降解率很低，降解机理尚不清楚。这项建议侧重于研究大蜡蛾(Galleria Mellonella)幼虫和消化道微生物对C20或更长的直链和支链烷烃的降解。这项研究是基于这样一个假设，即聚乙烯(PE)生物降解剂也可以降解长链烷烃。PE是具有不同支化度和结晶度的高分子量烷烃。近年来，梅氏革兰氏菌及其相关生物的幼虫对PE具有降解作用，并从其肠道中分离到了PE降解菌。梅隆杆菌幼虫将在实验室中使用PE和第一次使用各种直链和支链烷烃进行培养，以研究这些幼虫可以降解的烷烃的范围，并产生用于微生物培养的接种物(地面上的消化道和粪便)。在厌氧条件下，石油污染区高分子量正构烷烃优先损失，硫酸盐还原条件下支链烷烃优先降解。因此，所采用的增菌培养条件将是好氧的，并且首次使用梅隆革兰氏菌接种，将是具有各种末端电子受体的厌氧条件。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型宇宙中，以识别烷烃降解物。通过密度梯度离心法回收和分离群落总DNA，恢复富含13C的退化种群。将采用聚合酶链式反应扩增和纯化，以及Illumina MiSeq测序和数据处理来鉴定降解物。分离株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测。降解产物、结晶度变化的DSC分析和液体介质的表面张力数据将有助于深入了解降解机理。拟议的计划为一系列基于梅隆革兰氏菌和相关生物的微生物群的塑料和石化产品的生物降解研究奠定了框架。这项工作将大大增加我们对顽固石油馏分和塑料生物降解可能性的了解。对降解物的鉴定和分离创造了将这些微生物用于生物强化的机会，并向工程师通报了降解顽固性烷烃所需的适当生理条件(氧化还原、电子受体)。