一株嗜热聚对苯二甲酸乙二醇酯降解菌的分离及其降解特性解析

Isolation and characterization of a thermophilic PET-degrading bacterium

摘要:

【目的】大量聚对苯二甲酸乙二醇酯(polyethylene terephthalate, PET)塑料作为废弃物被丢弃，严重危害生态健康。针对嗜热PET降解菌缺乏这一情况，本研究旨在获得能够降解PET的嗜热菌，并阐述其降解机制。【方法】采集云南腾冲热泉中的废弃PET瓶，分析其表面生物膜的微生物群落多样性，从中筛选能够以PET为营养源生长的嗜热菌，并基于16S rRNA基因序列加以鉴定；以菌株的定殖能力与生长曲线为指标，优选出降解能力较强的降解菌，并测定其最适pH、温度和NaCl浓度；降解能力较强的降解菌分别作用于PET及PET中间体双(羟乙基)对苯二甲酸酯[bis(hydroxyethyl) terephthalate, BHET]和对苯二甲酸单(2-羟乙基)酯[mono(2-hydroxyethyl) terephthalate, MHET)]，测定产物生成量与降解率；通过观察PET膜表面微观结构、活菌数、酯酶活性等探究降解菌与PET的互作过程。【结果】废弃PET瓶表面生物膜中的微生物群落多样性低；从生物膜中筛选出5株能够以PET为营养源生长的嗜热菌；其中，菌株JQ3以PET为唯一碳源生长最佳，作为降解能力较强的降解菌，被鉴定为嗜热淀粉芽孢杆菌(Bacillus thermoamylovorans)，其最适生长pH为7.0、最适生长温度为50 、最适生长NaCl浓度为0.5%；菌株JQ3以0.043 mg PET/d的速率降解PET，对苯二甲酸(terephthalic acid, TPA)产量在第7天达到峰值45.2 mmol/L；菌株JQ3对PET中间体降解效率显著，6 h可降解85.9%的BHET，60 h可降解50.1%的MHET。菌株JQ3能够定殖于PET表面并形成生物膜，侵蚀PET并造成开裂和剥落。【结论】B. thermoamylovorans JQ3作为一株嗜热PET降解菌，能够高温(60 降解PET及其中间体，为实现PET的有效降解提供了新策略。

关键词:塑料污染;聚对苯二甲酸乙二醇酯(PET)生物降解;嗜热淀粉芽孢杆菌;热稳定性;微生物筛选

Abstract:

[Objective] Polyethylene terephthalate (PET) plastics are usually discarded as waste, which seriously harms the ecological health. Considering the lack of thermophilic PET-degrading bacteria, this study aims to obtain a thermophilic PET-degrading bacterium and decipher its degradation mechanism. [Methods] We collected waste PET bottles from hot springs in Tengchong City, Yunnan Province and then extracted the biofilms from their surfaces to investigate the microbial diversity. We screened the thermophilic bacteria that could grow with PET as a nutrient source and identified them based on 16S rRNA gene sequences. According to the colonization ability and growth curve, we screened out a strain with strong degrading ability and determined the optimal pH, temperature, and NaCl concentration for its growth. Further, we investigated the degrading effects of the strain on PET and its intermediates, (bis(hydroxyethyl) terephthalate (BHET) and mono(2-hydroxyethyl) terephthalate (MHET), by measuring the product yield and degradation rate. In addition, we evaluated the interaction between the degrading bacterium and PET by observing morphology of the PET surface and determining the viable count and esterase activity. [Results] The microbial diversity was low in the biofilms of waste PET bottles. We isolated 5 thermophilic bacterial strains that could grow with PET as a nutrient source from the biofilms. Strain JQ3 using PET as the sole carbon source showed the best growth, which was identified as Bacillus thermoamylovorans. Its optimal growth conditions were pH 7.0, 50, and 0.5% NaCl. Strain JQ3 degraded PET at a rate of 0.043 mg PET/d, and the yield of terephthalic acid (TPA) peaked at 45.2 mmol/L on the 7th day. Moreover, strain JQ3 exhibited significant degradation effects on PET intermediates, degrading 85.9% of BHET within 6 h and 50.1% of MHET within 60 h. Strain JQ3 formed biofilm on the PET surface by colonization, which caused cracking and peeling of the PET surface. [Conclusion] The thermophilic PET-degrading strain B. thermoamylovorans JQ3 can degrade PET and its intermediates at high temperature (60), which provides a new strategy for PET degradation.

Key words:plastic pollution;polyethylene terephthalate (PET) biodegradation;Bacillus thermoamylovorans;thermostability;screening of microorganisms