黄粉虫（Tenebrio molitor）肠道中聚乳酸塑料降解菌的筛选及其降解特性

doi:10.3969/j.issn.1004-1524.2022.06.18

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (6): 1277-1287.DOI: 10.3969/j.issn.1004-1524.2022.06.18

黄粉虫（Tenebrio molitor）肠道中聚乳酸塑料降解菌的筛选及其降解特性

冯娟¹(), 朱廷恒²^,^*(), 罗春萍¹, 杨佳玥¹, 祝思瑜¹, 李彤¹

1.台州科技职业学院农业与生物工程学院,浙江台州 318020
2.浙江工业大学生物工程学院,浙江杭州 310014

收稿日期:2021-12-07 出版日期:2022-06-25 发布日期:2022-06-30
通讯作者: 朱廷恒
作者简介:*朱廷恒,E-mail: thzhu@zjut.edu.cn
冯娟（1984—）,女,浙江台州人,硕士,讲师,研究方向为微生物与分子。E-mail: mayim2006@126.com
基金资助:
台州市工业类科技计划(2003gy02);浙江省自然科学基金(LY18C140005)

Isolation and identification of polylactic acid degrading microorganisms from mealworm（Tenebrio molitor）gut

FENG Juan¹(), ZHU Tingheng²^,^*(), LUO Chunping¹, YANG Jiayue¹, ZHU Siyu¹, LI Tong¹

1. College of Agriculture and Bioengineering, Taizhou Vocational College of Science and Technology, Taizhou 318020, Zhejiang, China
2. College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China

Received:2021-12-07 Online:2022-06-25 Published:2022-06-30
Contact: ZHU Tingheng

摘要/Abstract

摘要：

聚乳酸（polylactic acid,PLA）材料是一种环境友好型的可降解塑料,在堆肥或高温条件下可以快速生物降解,但在自然条件下降解缓慢,产生大量的微塑料。本研究拟从黄粉虫肠道中筛选具有降解聚乳酸能力的微生物,鉴定降解菌的种类及测定降解菌的降解特性。采用PLA粉末为唯一食物喂养黄粉虫60 d,将其肠道提取液接种在以PLA为唯一碳源的固体培养基上进行富集、筛选及纯化降解菌。结合菌种形态观察、扫描电镜分析的方法和ITS序列序列信息构建系统进化树确定降解菌的分类;将筛选菌接入添加不同营养条件下的PLA液体发酵培养基,测定其降解效能。结果筛选出11株具有降解PLA塑料潜力的菌株,其中一株鉴定为真菌毛栓孔菌（Trametes hirsuta）FJ001菌株,接种在含有1.0%葡萄糖的PLA薄膜无机盐液体培养基中连续培养30 d,降解率可达20.1%。研究表明,黄粉虫肠道存在对PLA塑料有一定降解性的真菌,为降解聚乳酸材料微生物资源的开发提供了理论依据。

关键词: 黄粉虫, 聚乳酸, 肠道微生物, 生物降解, 筛选

Abstract:

Polylactic acid (PLA) is biodegraded rapidly under composting or thermophilic temperature but slowly under natural conditions with substantial microplastics generated. In this study, microorganisms with the ability to degrade PLA were screened from the gut of mealworm, and the degrading bacteria were identified and the degradation characteristics were determined. Mealworms was fed with PLA powder as the only food for 60 days, and the gut extract of mealworms was inoculated on the solid medium with PLA as the sole carbon source to enrich, screen and isolate PLA-degrading microorganisms. Isolated strains were identified through morphological, physiological and biochemical properties as well as ITS gene sequences analysis, the phylogenic tree was constructed to identify the taxonomic status of the strains. The screened bacteria were added to the PLA liquid fermentation medium under different nutrient conditions to determine their degradation efficiency. The results showed that 11 strains with PLA degradation potential were screened from the mealworms gut. A PLA-degrading strain (FJ001) was initially identified as the fungus Trametes hirsuta. After inoculating FJ001 in PLA thin film inorganic salt liquid medium containing 1.0% glucose for 30 days, the PLA degradation rate could reach 20.1%. In conclusion, Trametes hirsuta FJ001 was isolated from mealworms with capable of degrading PLA plastics effectively. The results suggested that strains which can degrade PLA plastics could be isolated from mealworms gut. The result showed that there were fungi that can degrade PLA in the gut of mealworm, which provided a theoretical basis for the development of microbial resources for degrading polylactic acid materials.

Key words: yellow mealworm, polylactic acid, gut microbiota, biodegradation, isolation

中图分类号:

Q93-331

冯娟, 朱廷恒, 罗春萍, 杨佳玥, 祝思瑜, 李彤. 黄粉虫（Tenebrio molitor）肠道中聚乳酸塑料降解菌的筛选及其降解特性[J]. 浙江农业学报, 2022, 34(6): 1277-1287.

FENG Juan, ZHU Tingheng, LUO Chunping, YANG Jiayue, ZHU Siyu, LI Tong. Isolation and identification of polylactic acid degrading microorganisms from mealworm（Tenebrio molitor）gut[J]. Acta Agriculturae Zhejiangensis, 2022, 34(6): 1277-1287.

图/表 7

图1 喂食PLA和喂食麸皮的黄粉虫粪便 A,麦麸喂养60 d的黄粉虫及其产生的粪便;B,350目PLA薄膜喂养60 d的黄粉虫及其产生粪便;C,黄粉虫啮食聚乳酸薄膜;D,PLA粉末喂食黄粉虫粪便放大160倍显微观察;E,未处理聚乳酸粉末放大160倍显微观察;F,麦麸喂养60 d的黄粉虫肠道提取物接种在PLA无机盐培养基微生物生长情况;G,PLA喂养60 d的黄粉虫肠道提取物接种在含有PLA无机盐培养基中生长情况。

Fig. 1 Fed on PLA or bran of mealworms A, Tenebrio molitor and its feces fed with wheat bran for 60 days; B, Tenebrio molitor fed with 350 mesh PLA film for 60 days and its feces; C, PLA film-feeding mealworm;D, Microscopic observation of feces of Tenebrio molitor fed with PLA powder at 160 times magnification;E, Microscopic observation of untreated polylactic acid powder at 160 times magnification;F, Microbial growth of Tenebrio molitor intestinal extract fed with wheat bran for 60 days inoculated in PLA inorganic salt medium; G, Growth of Tenebrio molitor intestinal extract inoculated in inorganic salt medium containing PLA for 60 days.

图2 十株菌株在PLA粉末固体培养基上培养情况

Fig. 2 Cutural characteristics of 10 strains on an inorganic salt medium with PLA as the only carbon source

表1 十一株筛选菌株在PLA粉末固体培养基上培养情况

Table 1 Cultural characteristics of the 11 strains on an inorganic salt medium with PLA as the only carbon source

菌株Strain	菌落形态Morphology		可溶性色素Soluble pigment	生长状况Growth status
LT-01	圆形/橘红Circle/Orange	无Spore-free	橘红Orange	稀少Sparse
LT-02	圆形/浓绿Circle/Dark green	浓绿孢子Dark green spore	无None	旺盛Vigorous
LT-03	不规则/橘红Orange/Wavy	无Spore-free	无None	一般Normal
LT-04	圆形/红色Circle/Pink	无Spore-free	红Red	旺盛Vigorous
YZL-01	圆形/灰Circle/Gray	灰色孢子Gray spore	无None	旺盛Vigorous
YZL-02	圆形/土黄Circle/Khaki	土黄孢子Khaki spore	无None	一般Normal
CMY-01	圆形/亮黄色Circle/Bright yellow	无Spore-free	亮黄色Bright yellow	一般Normal
CMY-02	不规则/黄Irregular/Chartreuse	黄绿孢子Chartreuse spore	黄绿Chartreuse	一般Normal
ZML-01	不规则/绿Irregular/Chartreuse	绿色孢子Green spore	绿Green	旺盛Vigorous
ZML-02	圆形/土黄Circle/Khaki	无Spore-free	无None	一般Normal
FJ001	圆形/白色Circle/White	无Spore-free	无None	旺盛Vigorous

图3 降解菌FJ001在培养基上的培养情况及菌丝观察 A,FJ001接种在PLA粉末固体培养基上培养10 d生长状态;B,FJ001接种在PLA薄膜液体培养基10 d摇瓶培养;C,FJ001接种在三乙酸甘油酯无机盐培养基生长状态;D、E,FJ001接种在PDA培养基培养10 d正反面;F,FJ001菌株在酪氨酸培养基形成的透明圈;G、H,电镜观察菌丝。A, FJ001 was inoculated on PLA inorganic salt medium for 10 days; B, Strain FJ001 in PLA film content shake for 10 days; C, FJ001 was inoculated on triacetyl glycerin medium for 10 days; D, E, FJ001 was inoculated on PDA medium for 10 days;F, FJ001 was inoculated on tyrosine medium for 4 days;G, H, Electron microscope observation.

Fig. 3 Developing conditions FJ001 strain in medium

图4 降解菌FJ001菌株系统发育树

Fig. 4 Phylogenetic tree of strain FJ001

图5 降解菌FJ001在不同营养条件下的降解性能

Fig.5 Degradation performance of strain FJ001 under different nutrient conditions

图6 PLA膜表面变化的扫描电镜照片 A,对照组PLA 膜（第10天）;B,对照组 PLA 膜（第30天）;C,FJ001作用下的 PLA 膜（第10天）;D,FJ001作用下的 PLA 膜（第30天）。

Fig. 6 Scanning electron microscope images of surface change of PLA film A, PLA film control on the 10th day; B, PLA film control on the 30th day; C, PLA film degraded by FJ001 on the 10th day; D, PLA film degraded by FJ001 on the 30th day.

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黄粉虫（Tenebrio molitor）肠道中聚乳酸塑料降解菌的筛选及其降解特性

Isolation and identification of polylactic acid degrading microorganisms from mealworm（Tenebrio molitor）gut

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