【作者】 郑龙玉；

【导师】 喻子牛；

【作者基本信息】 华中农业大学 ， 微生物学， 2012， 博士

【摘要】 亮斑扁角水虻(Hermetta illucens L.,简称水虻)可以可持续地处理动植物有机废弃物。水虻幼虫能够在大幅减少畜禽粪便等废弃物累积量的同时,消除废弃物携带的多种病原菌类、除臭及抑制家蝇滋生。通过转化废弃物营养得到的水虻虫体可以作为畜禽、水产动物等的优质蛋白饲料来源。本研究较全面地调查了水虻种群携带的细菌群落结构及其多样性,卵携带的微生物在水虻聚集产卵行为中的作用及其对水虻生长发育的重要性,最后对水虻虫体油脂作为生物柴油生产的新型生物质原料进行了初步探索,主要结果如下：(1)亮斑扁角水虻各连续生命阶段的细菌种群多样性利用16SrDNA-454焦磷酸测序技术,调查了水虻各个连续生命阶段的细菌多样性：从水虻各生命阶段一共获得了4852个PCR产物,从第一代卵、幼虫、预蛹、蛹、成虫和次代卵样品获得的序列数目分别为37、517、1181、2106、883和128。在≥80%自引支持度(bootstrap support)下,分类到门、纲、目、科、属水平的序列百分比分别为99.65%、97.34%、85.84%、69.31%和31.55%。各生长阶段完成分类的(99.67%)综合细菌多样性跨越了6个细菌门。拟杆菌门和变形菌门占到了所鉴定类群的三分之二,是最具优势的菌门。在水虻卵阶段发现的细菌中有大约15%为第一代卵和次代卵所共有,暗示着世代垂直传递的可能性。(2)卵表细菌介导亮斑扁角水虻的聚集产卵行为通过产卵地点选择测试(OSP),在室内考察了各潜在因素在水虻产卵行为中的作用。结果表明,收卵基质对水虻具有长距离吸引作用,同种新生虫卵可以刺激怀卵雌性聚集产卵；而后通过对消毒虫卵进行OSP测试,发现卵表细菌是产卵刺激效果的主要来源。在对卵分离细菌进行OSP测试后,确定起关键作用的是一个细菌复合体(BSF-4),其中有4株菌,分别属于戈登氏菌属(Gordonia sp.)、纤维单胞菌属(Cellulomonas sp.)、微杆菌属(Microbacterium sp.)和微球菌属(Micrococcus sp.),它们对水虻产卵都具有吸引作用,戈登氏菌作用最显著,但BSF-4复合体作用效果强于任何单株细菌,4株菌可能具有协同作用。另外,部分水虻虫卵分离细菌和异种昆虫(自然界潜在竞争者)源细菌对水虻产卵行为表现出抑制排斥作用。(3)卵表微生物对亮斑扁角水虻生长发育特性的影响通过对虫卵进行表面消毒获得了无菌幼虫,然后测定了在喂食相同无菌人工饲料条件下无菌幼虫与正常幼虫生活史特性的差异,并通过向无菌幼虫回接虫卵洗液和卵表分离细菌混合液验证微生物对于水虻生长发育的重要性。结果显示,无菌幼虫预蛹率和存活到成虫阶段的比率均显著低于正常幼虫,仅有57.5%能够成功羽化；无菌幼虫发育出现延迟,发育时间延长了50%以上,需近30d才能预蛹,预蛹重量降低；无菌幼虫的预蛹需更长时间才能羽化,得到的成虫均重显著低于正常幼虫,成虫平均寿命只有9d左右,显著短于正常幼虫。在回接卵洗液和混合菌液后,无菌幼虫各项指标均向正常幼虫靠拢,部分得到了显著改善,证实卵表细菌对于水虻幼虫生长发育至关重要。(4)亮斑扁角水虻虫体脂肪成分作为新型生物能源材料综合地评测了利用水虻幼虫降解转化固态餐厨剩余物来再次生产生物柴油的潜力。结果显示,1000头水虻幼虫(8d)接种到1kg固态餐厨剩余物中,经过7d的生长消化,最后可以生产得到大约23.6g的水虻基生物柴油,使得利用餐厨剩余物生产生物柴油的产率提高了近一倍(回收油脂：2.7%：水虻油脂：2.4%)。此水虻基生物柴油的大多数性能参数均已达到了欧盟关于生物柴油的标准(EN14214),其中包括密度(860kg/m3)、粘度(4.9mm2/s)、闪点(128℃)、十六烷值(58)、酯含量(96.9%)等。更多还原

【Abstract】 Black soldier fly (BSF), Hermetia illucens (L.), has been considered as a sustainable method for reducing animal and plant wastes. Larvae reduce dry matter, bacteria, offensive odor and house fly populations. The prepupae can be self-harvested and used as feedstuff for livestock and poultry.While some bacteria species have been cultured and identified from BSF, a true appreciation of fly associated bacterial diversity is not known. On the other hand, the significance of egg-associated bacteria on BSF larval development and what roles they play in the oviposition aggregation behaviour of gravid females are seldom reported up to now. Such information will provide insight into BSF biology and is needed to improve the current waste management system. To address these issues, this study was conducted accordingly. The potential of BSF larval grease to be used as a novel biomass feedstock for biodiesel production was also evaluated in the last chapter.The main results are as follows:(1) Bacterial diversity from successive life stages of black soldier fly.Using16S rDNA454pyrosequencing, we examined bacterial diversity associated with successive life stages of the BSF reared on a Gainesville diet. We obtained4852PCR products from the different life stages of the BSF. The number of sequences obtained from the first generation egg, larva, prepupa, pupa, adult, and second generation egg stages were37,517,1181,2106,883, and128, respectively. The percent of sequences which classified at the phylum, class, order, family, and genus levels were99.65%,97.34%,85.84%,69.31%, and31.55%, respectively, with≥80%bootstrap support. The combined diversity of bacteria classified (99.67%) across all life stages spanned six bacterial phyla with≥80%bootstrap support. Bacteroidetes and Proteobacteria were the most dominant phyla associated with the BSF accounting for two-thirds of the fauna identified. Of the bacteria found associated with the egg stage of development,15%were shared by first and second generation samples suggesting the possibility of vertical transmission and retention of bacteria through successive developmental stages.(2) Egg-associated bacteria-mediated oviposition aggregation by black soldier flies.Oviposition sites preference (OSP) tests were conducted under laboratory conditions to evaluate the roles of different potential factors in the oviposition aggregation behaviour of gravid female BSF. It is indicated that ovipositional substrates acted as a long distance attractant during oviposition site detection and fresh conspecific eggs had an effect of stimulation on oviposition behaviour. Further OSP tests of sterile eggs showed that egg-associated bacteria were the major source of this aggregation stimulus. Interestingly, among the bacteria isolated from BSF eggs, dramatical oviposition stimulation was only detected from BSF-4which is not a sole stain but a bacteria assemblage. Four stains of bacteria were identified as Gordonia sp., Cellulomonas sp., Micrococcus sp., and Microbacterium sp., respectively, by pyrosequencing and16S rDNA sequencing individually. The assemblage showed much stronger stimulation effect than individual ones in comparison OSP tests, although these four bacteria all can enhance oviposition separately. This suggests that synergy may exist in the bacteria assemblage. In addition, some bacteria off the BSF eggs or isolated from competitor insect species exhibited a repellent effect on oviposition of gravid females.(3) Effects of egg associated bacteria on the development of black soldier fly.Growth study with sterile BSF larvae obtained by egg surface sterilization was conducted to determine the changes of their life history traits compared with normal larvae provided with identical sterile artificial diet. The significance of bacteria to the development of BSF was further confirmed by bacteria replenishment to sterile larvae. The results demonstrated that percentage survivorship to prepupal and adult stages of sterile larvae were significantly lower than that of normal larvae. Only57.5%of sterile larvae successfully reached adult stage. Developmental delay was detected with sterile larvae; they took50%longer to develop to prepupal stage (30d). Additionally, sterile larvae also took longer to reach adult stage. Adults resulting from sterile larvae (9d) lived2d less than those from normal larvae. Prepupa and adults resulting from sterile larvae weighed significantly lighter. After bacteria replenishment by adding egg wash or mixed egg-associated bacteria suspension, some of the life history traits of sterile larvae were significantly improved, drawing close to that of normal larvae. It is shown that egg-associated bacteria are very important to the development of BSF larvae.(4) Exploring the potential of black soldier fly larval grease as a novel biomass feedstock for biodiesel production.This study was conducted to evaluate the potential of a secondary biodiesel production from the solid residual fraction of restaurant waste after typical grease extraction (SRF). About23.6g larval grease-based biodiesel was produced from approximately1000larvae grown on1kg of SRF. The weight of SRF was reduced by about61.8%after being fed by the black soldier fly larvae for7d. The amount of biodiesel yield from restaurant waste was nearly doubled (original restaurant waste grease, 2.7%; larval grease,2.4%). Most of the properties of this biodiesel met the specifications of the standard EN14214, including density (860kg/m3), viscosity (4.9mm2/s), flash point (128℃), cetane number (58) and ester contents (96.9%).更多还原