基于啮食泡沫塑料黄粉虫肠道菌群中聚苯乙烯生物降解的探究
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摘要
【背景】石油基塑料垃圾污染具有高度的化学惰性而不易分解,严重威胁着生态和海洋环境,筛选其高效降解菌并利用生物法治理塑料污染具有重要意义。【目的】聚苯乙烯是其中一种不能降解且用途广泛的塑料。近期有研究报道黄粉虫幼虫可啮食聚苯乙烯泡沫塑料。本文以啮食聚苯乙烯白色泡沫塑料的黄粉虫幼虫为研究材料,以期从肠道菌群中分离聚苯乙烯降解菌。【方法】分别以聚苯乙烯泡沫塑料与麦麸(对照组)为唯一食物来源饲喂黄粉虫幼虫,经过60 d的驯化,采用多种培养基对幼虫肠道提取液及虫体粪便排泄物进行分离、培养;通过形态学特征观察、扫描电镜与16S rRNA基因、ITS序列分析扩增、测定,根据序列信息构建系统进化树确定菌株的分类地位;将真菌菌种分别接种于加有PS膜片与颗粒的液体培养基中振荡培养,对膜片的力学性能变化进行测定,并对聚苯乙烯颗粒失重率进行计算。【结果】从幼虫粪便中分离的2株好氧菌分别被鉴定为Bacillus anthracis Ames PSI-1和Enterobacter cloacae PSI-2,从肠道中分离的真菌被鉴定为Aspergillus niger KHJ-1;电镜检测PS膜片表面出现大量孔洞,并且膜片疏水性减弱而亲水性增强,膜片的断裂伸长率与拉伸强度等力学性能也显著下降;真菌KHJ-1对PS颗粒的60 d降解测定中,发现PS颗粒由5 g原始重量减少了214.8 mg,失重率为4.29%。【结论】黄粉虫肠道中存在可以降解聚苯乙烯塑料的菌株,具有开发为新型降解聚苯乙烯高分子塑料的潜力。
[Background] Plastic are synthetic polymers derived from fossil oil and largely resistant to degradation. Plastic litter have adverse effects on the environment and marine biota. Therefore, new solutions for petroleum-based plastic waste degradation are urgently needed. [Objective] Polystyrene(PS) is notoriously persistent plastic that is not degradable at appreciable rates for decades. However, an environment was identified in which PS is susceptible to biodegradation: yellow mealworm, the larvae of Tenebrio molitor Linnaeus, can damage PS packing by chewing and eating styrofoam. So we try to isolate PS-degrading microorganisms from the mealworms' gut. [Methods] The larvae fed with styrofoam as the sole diet could live as well as those fed with bran diet over several months. Based on morphological, physiological and biochemical properties as well as 16S rRNA gene and ITS gene sequences analysis, the phylogenic tree was constructed to identify the taxonomic status of the strains. [Results] Two bacterial strains capable of degrading styrofoam were isolated from worm fecula egested from styrofoam-feeding larvae, named as Bacillus anthracisPSI-1 and Enterobacter hormaechei PSI-2. A PS-degrading fungal strain was isolated from the gut of mealworms and identified as Aspergillus niger KHJ-1. Over a 14-day incubation period of the fungal train KHJ-1 on PS films, a large number of cavities were obviously observed on PS films surfaces, and the changes were measured about the film of mechanical properties. The results showed that hydrophobicity of the film was decreased dramatically, the elongation at break and the tensile strength became smaller. A suspension culture of KHJ-1 strain was able to degrade 214.8 mg/5 g of PS solid particles, the weight loss ratio of PS was 4.29% over a 60-day incubation period. [Conclusion] The results demonstrated the presence of PS-degrading bacteria and fungus in the guts of mealworms could degrade PS directly and efficiently and provided promising evidence for the biodegradation of PS in the environment.
[Background] Plastic are synthetic polymers derived from fossil oil and largely resistant to degradation. Plastic litter have adverse effects on the environment and marine biota. Therefore, new solutions for petroleum-based plastic waste degradation are urgently needed. [Objective] Polystyrene(PS) is notoriously persistent plastic that is not degradable at appreciable rates for decades. However, an environment was identified in which PS is susceptible to biodegradation: yellow mealworm, the larvae of Tenebrio molitor Linnaeus, can damage PS packing by chewing and eating styrofoam. So we try to isolate PS-degrading microorganisms from the mealworms' gut. [Methods] The larvae fed with styrofoam as the sole diet could live as well as those fed with bran diet over several months. Based on morphological, physiological and biochemical properties as well as 16S rRNA gene and ITS gene sequences analysis, the phylogenic tree was constructed to identify the taxonomic status of the strains. [Results] Two bacterial strains capable of degrading styrofoam were isolated from worm fecula egested from styrofoam-feeding larvae, named as Bacillus anthracisPSI-1 and Enterobacter hormaechei PSI-2. A PS-degrading fungal strain was isolated from the gut of mealworms and identified as Aspergillus niger KHJ-1. Over a 14-day incubation period of the fungal train KHJ-1 on PS films, a large number of cavities were obviously observed on PS films surfaces, and the changes were measured about the film of mechanical properties. The results showed that hydrophobicity of the film was decreased dramatically, the elongation at break and the tensile strength became smaller. A suspension culture of KHJ-1 strain was able to degrade 214.8 mg/5 g of PS solid particles, the weight loss ratio of PS was 4.29% over a 60-day incubation period. [Conclusion] The results demonstrated the presence of PS-degrading bacteria and fungus in the guts of mealworms could degrade PS directly and efficiently and provided promising evidence for the biodegradation of PS in the environment.
引文
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[2]Yang J,Song YL,Qin XY.Biodegradation of polyethylene[J].Environmental Science,2007,28(5):1165-1168(in Chinese)杨军,宋怡玲,秦小燕.聚乙烯塑料的生物降解研究[J].环境科学,2007,28(5):1165-1168
[3]Ioakeimidis C,Fotopoulou KN,Karapanagioti HK,et al.The degradation potential of PET bottles in the marine environment:An ATR-FTIR based approach[J].Scientific Reports,2016,6:23501
[4]Lu CX,Liu L,Li JH,et al.Isolation and characterization of a microorganism degrading starch/polyethylene blends[J].Chinese Journal of Applied&Environmental Biology,2013,19(4):683-687(in Chinese)陆辰霞,刘龙,李江华,等.淀粉填充聚乙烯类塑料降解微生物的筛选和降解特性[J].应用与环境生物学报,2013,19(4):683-687
[5]Bai YY,Cheng JA.Nutritive value and rearing methods of Tenebrio molitor in China[J].Entomological Knowledge,2003,40(4):317-322(in Chinese)白耀宇,程家安.我国黄粉虫的营养价值和饲养方法[J].昆虫知识,2003,40(4):317-322
[6]Ye XQ,Liu DH,Hu C.Some factors’effects on the solubility of protein from yellow mealworm(Tenebrio molitor L)larvae[J].Journal of Zhejiang University(Science A),2001,2(4):436-438
[7]van Ooik T,Rantala MJ,Saloniemi I.Diet-mediated effects of heavy metal pollution on growth and immune response in the geometrid moth Epirrita autumnata[J].Environmental Pollution,2007,145(1):348-354
[8]Shen YH.Isolation of intestinal bacteria from T.molitor L and study on the phenomenon of plastic degradation[D].Shanghai:Master’s Thesis of East China Normal University,2011(in Chinese)沈叶红.黄粉虫肠道菌的分离和取食塑料现象的研究[D].上海:华东师范大学硕士学位论文,2011
[9]Yang Y,Yang J,Wu WM,et al.Biodegradation and mineralization of polystyrene by plastic-eating mealworms:Part1.Chemical and physical characterization and isotopic tests[J].Environmental Science&Technology,2015,49(20):12080-12086
[10]Yang Y,Yang J,Wu WM,et al.Biodegradation and mineralization of polystyrene by plastic-eating mealworms:Part2.Role of gut microorganisms[J].Environmental Science&Technology,2015,49(20):12087-12093
[11]Chen GZ,Zhang BL,Ji MM,et al.Gut microbiota of polystyrene-eating mealworms analyzed by high-throughput sequencing[J].Microbiology China,2017,44(9):2011-2018(in Chinese)陈冠舟,张白鹭,纪梦梦,等.高通量测序探究啮食聚苯乙烯泡沫塑料黄粉虫的肠道菌群结构[J].微生物学通报,2017,44(9):2011-2018
[12]Shen P,Chen XD.Microbiology Experiment[M].4th ed.Beijing:China Higher Education Press,2007:241(in Chinese)沈萍,陈向东.微生物学实验[M].4版.北京:高等教育出版社,2007:241
[13]Zhang Y,Shao MW,Zhang YL,et al.Inhibition of Amaranthus retroflexus by wasp gut fungal isolate Fusarium oxysporum MF06[J].Acta Microbiologica Sinica,2015,55(3):292-298(in Chinese)张蕴,邵明伟,张应烙,等.马蜂肠道菌抑制反枝苋的活性筛选及菌株MF06的活性代谢产物[J].微生物学报,2015,55(3):292-298
[14]Wang XP,Chen ZF,Ni HG,et al.Surface properties of Hydroxy-terminated polystyrenes[J].Chemical Journal of Chinese Universities,2005,26(9):1752-1756(in Chinese)王新平,陈志方,倪华钢,等.端羟基化聚苯乙烯的表面性质[J].高等学校化学学报,2005,26(9):1752-1756
[15]Buchanan RE,Gibbons NE.Bergey’s Mannual of Determinative Bacteriology[M].8th ed.Beijing:Science Press,1984:274-280(in Chinese)布坎南RE,吉本斯NE.伯杰氏细菌鉴定手册[M].8版.北京:科学出版社,1984:274-280
[16]Gerhardt PD,Lindgren DL.Penetration of packaging films:film materials used for food packaging tested for resistance to some common stored-product insects[J].California Agriculture,1954,8(6):3-4
[17]Riudavets J,Salas I,Pons MJ.Damage characteristics produced by insect pests in packaging film[J].Journal of Stored Products Research,2007,43(4):564-570
[18]Yun JH,Roh SW,Whon TW,et al.Insect gut bacterial diversity determined by environmental habitat,diet,developmental stage,and phylogeny of host[J].Applied and Environmental Microbiology,2014,80(17):5254-5264
[19]Atiq N,Ahmed S,Ali MI,et al.Isolation and identification of polystyrene biodegrading bacteria from soil[J].African Journal of Microbiology Research,2010,4(14):1537-1541
[20]Gilan I,Hadar Y,Sivan A.Colonization,biofilm formation and biodegradation of polyethylene by a strain of Rhodococcus ruber[J].Applied Microbiology and Biotechnology,2004,65(1):97-104
[21]Yang J,Yang Y,Wu WM,et al.Evidence of polyethylene biodegradation by bacterial strains from the guts of plastic-eating waxworms[J].Environmental Science&Technology,2014,48(23):13776-13784
[22]Bombelli P,Howe CJ,Bertocchini F.Polyethylene bio-degradation by caterpillars of the wax moth Galleria mellonella[J].Current Biology,2017,27(8):R292-R293
[23]Lu JJ.The analysis of Paenibacillus mucilaginosus KNP414stress resistance comparative genome and transcriptome under nitrogen starvation condition[D].Hangzhou:Master’s Thesis of Zhejiang Sci-Tech University,2014(in Chinese)卢景江.胶质类芽孢杆菌KNP414抗逆比较基因组及氮饥饿的转录组分析[D].杭州:浙江理工大学硕士学位论文,2014