Identification of a Polyethylene-degrading Strain and Optimization of Its Fermentation Conditions

ZHAO Changle; LIN Xin; XU Yaobo; YAN Shuwei; ZHANG Tingting; LI Xinyi; WEI Jing

doi:10.13718/j.cnki.xdzk.2023.04.012

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Polyethylene (PE) products are the most widely used general plastics, and the resulting "white pollution" has become a worldwide problem. In this study, a PE-degrading bacterium was isolated by enriching and screening the inorganic salt medium with PE as the sole carbon source from the soil covered with plastic film for a long time and named as SW2. The colony was round, with neat edge, moist and smooth surface. The bacterium is Gram stain negative, spherical or rod-shapedv The 16S rRNA gene sequence analysis showed that it had 99% identity with Acinetobacter strain SY23, It was positive for contact enzymes, negative for oxidase and reducing nitrate. Based on the above characteristics, SW2 was preliminarily identified as Acinetobacter sp. In the inorganic salt medium containing 2% PE, SW2 had obvious growth at 24 h of culture, and reached to the peak growing at 72 h, but did not grow in the inorganic salt medium without PE. SW2 was inoculated to the inorganic salt medium containing PE products (agricultural plastic film and fresh-keeping film). On the 30th day of culture, obvious damage of agricultural plastic film and fresh-keeping film was observed under light microscope, while the surface of the control group was intact. The results show that SW2 can effectively utilize PE and degrade PE products. Under different temperature, pH and PE concentration conditions, the biological mass of SW2 was detected. The results showed that the optimal growth conditions of SW2 are 30 ℃, pH 7.0, and 20% PE. This research enriched the strain resources of PE-degrading bacteria and laid a foundation for the further study of biodegradable PE products.

Corresponding author: WEI Jing

Received Date: 18/07/2021

Available Online: 20/04/2023

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Abstract: Polyethylene (PE) products are the most widely used general plastics, and the resulting "white pollution" has become a worldwide problem. In this study, a PE-degrading bacterium was isolated by enriching and screening the inorganic salt medium with PE as the sole carbon source from the soil covered with plastic film for a long time and named as SW2. The colony was round, with neat edge, moist and smooth surface. The bacterium is Gram stain negative, spherical or rod-shapedv The 16S rRNA gene sequence analysis showed that it had 99% identity with Acinetobacter strain SY23, It was positive for contact enzymes, negative for oxidase and reducing nitrate. Based on the above characteristics, SW2 was preliminarily identified as Acinetobacter sp. In the inorganic salt medium containing 2% PE, SW2 had obvious growth at 24 h of culture, and reached to the peak growing at 72 h, but did not grow in the inorganic salt medium without PE. SW2 was inoculated to the inorganic salt medium containing PE products (agricultural plastic film and fresh-keeping film). On the 30th day of culture, obvious damage of agricultural plastic film and fresh-keeping film was observed under light microscope, while the surface of the control group was intact. The results show that SW2 can effectively utilize PE and degrade PE products. Under different temperature, pH and PE concentration conditions, the biological mass of SW2 was detected. The results showed that the optimal growth conditions of SW2 are 30 ℃, pH 7.0, and 20% PE. This research enriched the strain resources of PE-degrading bacteria and laid a foundation for the further study of biodegradable PE products.

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聚乙烯(polyethylene，PE)由乙烯单体经加聚反应而成，其制品包括塑料薄膜(如农用地膜、保鲜膜)、食品袋、垃圾袋、药品包装瓶、牛奶瓶、电线电缆绝缘材料等，是当前使用最为广泛的通用塑料^[1]. 中国是目前世界上最大的PE消费国之一，据不完全统计，2019年中国生产PE制品达到了19亿t^[2]. PE制品具有疏水性高、表面能低等特性，极难被降解，由此造成的“白色污染”成为了世界性难题^[1-3]. 传统处理方式如焚烧、填埋，可造成二次污染，并挤占生物利用空间，因此，生态友好的微生物降解策略备受关注.

目前，国内外学者已相继从农田、海洋、垃圾堆置点、昆虫肠道等分离并鉴定出了PE降解菌，这些研究为PE制品的生物降解研究奠定了基础^[4-11]. 本研究采用以PE为唯一碳源的无机盐培养基，成功从被农用地膜覆盖的公园土壤中筛选获得了1株PE降解菌，初步鉴定其为不动杆菌，并对其发酵条件进行了优化.

1. 材料和方法

1.1. 土壤样品、PE和培养基

土壤样品：重庆马鞍溪湿地公园长期覆盖有塑料垃圾的土壤(106.415E，29.826N). PE：平均分子质量为4 000的分析纯PE粉末，美国西格玛奥德里奇公司；农用地膜，淼森塑业有限公司；保鲜膜，四川鸿昌塑胶工业有限公司；无机盐培养基与LB培养基，配方见参考文献[4].

1.2. PE降解菌的富集与筛选

称取8 g土壤样本，与100 mL无菌水混匀，取200 μL土壤混合液分别接种到含0%，2% PE的100 mL无机盐培养基中，于30 ℃，150 r/min培养7 d后，取200 μL富集液于新鲜培养基中进行富集，重复4次以上，最后采用平板划线法进行纯化培养，保存菌种.

1.3. PE降解菌的鉴定

按常规方法对分离菌进行菌落观察、革兰氏染色观察、扫描电镜(EVO LS10，Zeiss)观察，同时进行生理生化特性检测和16S rRNA基因系统发育分析，具体参见文献[4]. 16S rRNA基因通用引物序列为27 F 5′-AGAGTTTGATCCTGGCTCAG-3′和1 492 R 5′-TACGGCTACCTTGTTACGACTT-3′. PCR反应条件为95 ℃ 5 min；95 ℃ 30 s，55 ℃ 30 s，72 ℃ 100 s，35个循环；72 ℃ 10 min. PCR产物经纯化、回收、亚克隆、测序，样品序列在NCBI(https://www.ncbi.nlm.nih.gov/Blast.cgi)中进行比对，并用MEGA7.0软件进行系统进化分析.

1.4. 生长特性

将200 μL分离菌菌悬液分别接种到含0%，2% PE的100 mL无机盐培养基中，30 ℃，150 r/min培养，在第0，24，48，72，96，120 h分别取样1 mL，定性滤纸过滤去除PE颗粒，滤液测定吸光度OD₆₀₀，以检测分离菌在不同时间点的生长情况.

1.5. 降解特性

在无机盐培养基中加入不同PE制品(农用地膜、PE保鲜膜)，以作为培养基中的唯一碳源，将分离菌菌悬液接种到培养基中，30 ℃，150 r/min培养，于不同时间点显微观察PE制品的降解情况，同时以不接种菌的PE制品为对照，具体方法参照文献[4].

1.6. 发酵条件优化

1.6.1. 温度

将200 μL SW2菌液接种到含2% PE的100 mL无机盐培养基中(pH值为6.0)，分别在25，30，35，40 ℃的条件下150 r/min培养，于0，72，96 h测定培养液OD₆₀₀，每组重复3次.

1.6.2. pH值

将200 μL SW2菌液接种到不同pH值(4.0，5.0，6.0，7.0，8.0)的含2% PE的100 mL无机盐培养基中，30 ℃，150 r/min培养，在0，72，96 h时测定培养液OD₆₀₀，每组重复3次.

1.6.3. PE浓度

将200 μL SW2菌液接种到含不同浓度PE(0%，8%，12%，16%，20%，30%，40%)的100 mL无机盐培养基中，30 ℃，150 r/min培养，在0，72，96 h时测定培养液OD₆₀₀，每组重复3次.

1.7. 数据统计分析

采用SPSS 22.0对实验数据进行单因素方差分析，p＜0.05表示差异有统计学意义.

3. 讨论

本研究采用以PE为唯一碳源的无机盐培养基，从长期覆盖塑料薄膜的土壤中分离获得了1株PE降解菌，通过菌落形态观察、革兰氏染色观察、16S rRNA基因序列的系统分析、生理生化特性检测，初步鉴定分离菌株SW2为不动杆菌. 进一步研究表明，SW2可有效降解PE制品，并且对其最适发酵条件进行了优化. 不动杆菌属分布广泛，已有研究表明，该属菌株能够降解多种环境污染物，如石油烃、酚酸类化合物、氯氰菊酯等^[14-16]. 本研究我们成功分离获得了1株降解PE的不动杆菌属菌株SW2，不仅丰富了PE降解菌的菌种资源，而且为进一步研究其PE降解奠定了重要基础.

本课题组前期还从覆盖塑料薄膜的土壤中成功分离获得1株降解PE的诺卡氏菌(Nocardia sp.)SW1^[4]. 该菌对PE具有明显的浓度依赖，并且在PE浓度为2%时生长最快^[4]. 本研究中，SW2生长同样有PE浓度依赖，但其最适PE达到了20%(图 4c)，同时菌液OD₆₀₀值明显高于SW1，提示SW2可更有效地利用PE. 在对PE制品降解效能研究中，SW1在培养的第60 d光镜观察可见膜明显破损，出现空洞^[4]；钟越等^[5]报道从土壤中分离获得1株降解PE的放线菌属菌株，该菌在培养的第60 d，扫描电镜观察可发现膜片表面出现孔洞；在本研究中，SW2在第30 d光镜下即可观察到农用地膜、保鲜膜出现明显破损(图 3)，提示SW2可能具有更高效的PE制品降解效能.

在无机盐培养基中，PE颗粒悬于培养基的表面，一定程度影响了培养液OD₆₀₀的测定，因此我们采用了直接定性滤纸过滤法，以滤除培养液中的PE颗粒，然后测定滤液的OD值. 为了验证该方法的可靠性，我们通过提取已知浓度倍比稀释的培养液菌株基因组，测定吸光值OD₂₆₀以检测其核酸量. 结果表明，一方面，核酸量与菌液浓度具有很好的线性关系，其相关系数R值达到了0.99；另一方面，直接滤纸过滤法与菌液浓度相关系数R值略低于核酸量测定法，但其与菌液浓度同样具有良好线性关系，其R值达到了0.90. 因此，直接滤纸过滤法可用于PE降解菌在不同发酵条件下生物生长量的快速检测. 此外，SW2降解PE的关键酶如何? 影响因素有哪些? 其降解机制如何? 这些均有待进一步研究.

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Identification of a Polyethylene-degrading Strain and Optimization of Its Fermentation Conditions

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Identification of a Polyethylene-degrading Strain and Optimization of Its Fermentation Conditions

Corresponding author: WEI Jing