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珠江河口滩涂沉积物中可培养细菌多样性及微塑料降解功能菌资源的挖掘  PDF

  • 王莹 1
  • 何欢欢 1
  • 卢春艳 1
  • 郑卓桓 1
  • 王森 1
  • 黄易 1
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  • 李文均 1,2
1. 中山大学 生命科学学院,水产动物疫病防控与健康养殖全国重点实验室,广东 广州; 2. 中国科学院新疆生态与地理研究所,荒漠与绿洲生态国家重点实验室,新疆 乌鲁木齐

最近更新:2025-04-09

DOI: 10.13343/j.cnki.wsxb.20240774

CSTR: 32112.14.j.AMS.20240774

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目录contents

摘要

目的

探究广东珠江河口滩涂沉积物中的可培养细菌多样性,并挖掘具有微塑料降解功能的菌株资源。

方法

使用5种培养基对微生物进行分离与纯化,采用MEGA-X进行系统进化分析。利用聚对苯二甲酸乙二醇酯(polyethylene terephthalate, PET)培养基筛选具有PET微塑料降解功能的菌株,并对相关基因进行功能注释。

结果

共分离得到265株细菌,分布于4门32科71属。其中,假单胞菌门(Pseudomonadota) 168株,占比63.40%;放线菌门(Actinomycetota) 38株,占比14.34%;芽孢杆菌门(Bacillota) 31株,占比11.70%;拟杆菌门(Bacteroidota) 28株,占比10.56%。基于16S rRNA基因序列的同源性分析,推测其中59株菌株可能为潜在新物种。同时,筛选获得1株具有潜在PET降解功能的菌株。

结论

本研究成功获得广东珠海香洲区滩涂特有的微生物菌株资源,并筛选得到1株能以PET作为唯一碳源进行生长的细菌。

微塑料(microplastics, MPs)是指尺寸小于5 mm的塑料纤维、颗粒或薄膜,包括聚对苯二甲酸乙二醇酯(polyethylene terephthalate, PET)、聚乙烯(polyethylene, PE)和聚苯乙烯(polystyrene, PS)等,它们主要来自塑料包装、化妆品和衣物纤维[

1]。随着人类对塑料使用量的增加,塑料垃圾已成为全球环境污染的主要因素之[2]。在自然侵蚀、机械损伤、紫外线照射等长期作用下,塑料垃圾会逐渐破碎降解为微塑[3]。微塑料通过风力、水流等途径广泛传播,不仅对陆地和海洋生态系统造成严重危害,还极易被环境中的生物体吸收,进而对生物的生长和繁殖产生不利影[4-5]。此外,微塑料能通过食物链或呼吸道进入人体,并在人体内累积,可能诱发炎症反应、内分泌干扰和细胞损伤等疾[6-8]

微塑料具有较强的稳定性,在自然环境中降解速度缓[

9]。然而,仍有特定的微生物能够降解微塑[10]。微生物降解是一种环境友好且可持续的微塑料处理方[11]。近年来,微生物学研究者已鉴定出众多具有微塑料降解能力的细菌分离株,这些细菌已成为当前研究的热点。微塑料降解细菌易于在微塑料表面定殖,并分泌塑料降解酶来加速其降[12]。目前,科学家们已围绕垃圾填埋场土壤、污水、污泥和海水等微塑料富集环境,开展了大量关于微塑料降解细菌的研[13-15]。Cao[16]采用文献计量分析法,系统总结了2012-2022年间关于微塑料生物降解及其机理的相关研究,结果表明,已报道的微塑料降解细菌主要隶属于假单胞菌门(Pseudomonadota)、芽孢杆菌门(Bacillota)、放线菌门(Actinomycetota)和拟杆菌门(Bacteroidota)。Dong[17]通过研究中国澳门周边4个河流采样站和4个近海采样站的地表水细菌多样性和种类组成,发现MPs表面的塑料降解细菌比例远高于水体中的塑料降解细菌,河流内MPs表层细菌以假单胞菌门和放线菌门为主,而近岸MPs表层芽孢杆菌门占比较高。

滩涂是由潮汐或河流侵蚀产生的沉积物在潮间带区域形成的沿海湿[

18]。根据组成成分的不同,滩涂可以分为岩滩、沙滩、泥滩三类,其组成物质从黏土大小的颗粒到粗沙,以及碳酸盐颗粒和有机物不[19]。当人类活动产生的微塑料进入海洋后,在潮汐、波浪等外力的作用下,微塑料会在海滩、近海以及大洋中迁移,漂浮在海岸附近的塑料很可能被冲上岸,而作为水陆交界区域的滩涂容易成为微塑料的富集[20-21]。因此,选择滩涂开展微塑料降解细菌的相关研究具有一定的现实意义。

位于广东珠江河口西岸的珠海市香洲区,地理位置优越,区域内包含多个岛屿,如担杆岛、外伶仃岛、野狸岛、桂山岛等,海岸线绵长,自然资源丰[

22]。香洲区岛屿众多,滩涂广阔,生物多样性丰富,这为潜在功能菌株的挖掘提供了宝贵的资源库。本研究选取珠海市香洲区的野狸岛、桂山岛和外伶仃岛3个岛屿的滩涂作为研究对象,在5种不同的培养基条件下对滩涂中的可培养细菌进行分离和纯化,旨在探讨香洲区滩涂可培养细菌的多样性,同时通过筛选微塑料降解细菌实验,考察香洲区滩涂细菌的微塑料降解能力及其潜在的开发价值,以期为未来开发和利用香洲区滩涂微生物资源,以及挖掘微塑料降解功能菌株资源奠定基础。

1 材料与方法

1.1 材料

1.1.1 样本采集

采样地点位于广东省珠海市香洲区的野狸岛滩涂、桂山岛滩涂和外伶仃岛滩涂区域(表1)。在每个采样点采用采样柱(直径为9.8 cm)采集3个平均分布的表层(5-15 cm)滩涂沉积物(去除表层腐殖质)样品。每个采样点样品分别由3个表层样芯放入无菌采样袋混合而成。样品置于冰袋上运输回实验室,实验前储存在4 °C冰箱内。

表1  珠海市香洲区滩涂区域采样点及信息
Table 1  Location of samples in tidal flats of Xiangzhou District, Zhuhai
SampleDateIsland nameSample typeGeographical coordinates
XZ1 2024-01-02 Yeli Island Clayey 22°17′4″N, 113°35′2″E
XZ2 2024-01-02 Guishan Island Clayey 22°8′13″N, 113°48′52″E
XZ3 2024-01-02 Wai-lingding Island Sandy 22°6′18″N, 114°1′36″E

1.1.2 培养基

采用5种不同的培养基对珠海市香洲区3个滩涂中的可培养细菌进行分离和纯化。2216E、R2A和TSA培养基是用于微生物分离的常规培养基。2216EM培养基是在2216E培养基的基础上添加制霉菌素;2216EMN培养基在2216E培养基的基础上同时添加制霉菌素和萘啶酮酸。此外,采用无机盐培养基和PET培养基筛选具有PET微塑料降解功能的菌株。上述培养基的详细配方见表2

表2  培养基配方
Table 2  Media formulation
MediaFormulation (g/L)

2216E medium

(Zobell marine agar 2216)

Pronase 5.0, yeast extract 1.0, citric acid ferric 0.1, NaCl 19.45, MgCl2 5.98, Na2SO4 3.24, CaCl2 1.8, KCl 0.55, Na2CO3 0.16, KBr 0.08, SrCl2 0.034, H3BO3 0.02, Na2SiO3 0.004, NaF 0.002 4, NH4NO3 0.001 6, K2HPO4 0.008, agar 18.0, pH 7.6±0.2

2216EM medium

(2216E with nystatin)

Pronase 5.0, yeast extract 1.0, citric acid ferric 0.1, NaCl 19.45, MgCl2 5.98, Na2SO4 3.24, CaCl2 1.8, KCl 0.55, Na2CO3 0.16, KBr 0.08, SrCl2 0.034, H3BO3 0.02, Na2SiO3 0.004, NaF 0.002 4, NH4NO3 0.001 6, K2HPO4 0.008, nystatin 0.05, agar 18.0, pH 7.6±0.2

2216EMN medium

(2216E with nystatin, nalidixic acid)

Pronase 5.0, yeast extract 1.0, citric acid ferric 0.1, NaCl 19.45, MgCl2 5.98, Na2SO4 3.24, CaCl2 1.8, KCl 0.55, Na2CO3 0.16, KBr 0.08, SrCl2 0.034, H3BO3 0.02, Na2SiO3 0.004, NaF 0.002 4, NH4NO3 0.001 6, K2HPO4 0.008, nystatin 0.05, nalidixic acid 0.05, agar 18.0, pH 7.6±0.2

R2A medium

(Reasoner’s 2A agar)

Yeast extract 0.5, peptone 0.5, casein 0.5, glucose 0.5, dextrose 0.5, K2HPO4 0.3, MgSO4⋅7H2O 0.05, pyruvic acid sodium salt 0.3, agar 18.0, pH 7.5±0.2

TSA medium

(tryptic soy agar)

Tryptone 17.0, soybean peptone 5.0, NaCl 5.0, glucose 2.5, K2HPO4 0.5, agar 18.0, pH 7.5±0.2
Inorganic salt medium (NH4)2SO4 1.0, KH2PO4 0.7, K2HPO4 0.7, NaCl 2.0, MgSO4·7H2O 0.7, ZnSO4·7H2O 0.002, FeSO4·7H2O 0.002, MnSO4 0.001, agar 18.0
PET medium PET 0.005, (NH4)2SO4 1.0, KH2PO4 0.7, K2HPO4 0.7, NaCl 2.0, MgSO4·7H2O 0.7, ZnSO4·7H2O 0.002, FeSO4·7H2O 0.002, MnSO4 0.001, agar 18.0

1.1.3 主要仪器

微量分光光度计,ThermoFisher Scientific公司;PCR仪、凝胶成像仪,Bio-Rad公司;台式高压灭菌锅,Hirayama公司;台式pH计,Mettler Toledo公司。

1.2 样品预处理

称取5 g滩涂沉积物样品,放入已灭菌的250 mL三角瓶中(装有45 mL无菌生理盐水和小玻璃珠),置于恒温摇床28 ℃、200 r/min培养30 min,取出后静置5 min,用于后续的稀释涂布。

1.3 可培养细菌分离、纯化与保藏

将预处理后的滩涂样品悬浮液使用无菌生理盐水分别稀释至10-2、10-3、10-4这3个不同的稀释度,从每个稀释度中分别吸取100 μL样品,使用无菌涂布棒将其均匀地涂布在2216E、2216EM、2216EMN、R2A、TSA这5种不同的分离培养基上。涂布完毕后,对每个平板做好标记,使用封口膜将平板密封,并小心地装入保鲜袋中,于28 ℃的恒温培养箱中倒置培养1-4周。在分离培养周期内,定期观察平板上的菌落生长情况,根据菌落的大小、颜色、干湿度等形态特征挑取具有代表性的菌株,转接至四分板中继续培养,待培养完成后使用无菌接种针将菌株以三区划线法在新的琼脂平板上进行培养纯化,这一过程需要重复进行,直到获得单一的菌落。纯化后的菌株加入体积分数为20%的甘油保藏于-80 ℃冰箱中。

1.4 PCR扩增与序列测序

在PCR管中加入100 μL质量浓度为100 g/L的Chelex-100溶液,使用接种针将板子上的单菌落挑取出来,接着将其溶于对应的PCR管中,充分混匀,于99 ℃加热30 min后,PCR管中的上清液即为16S rRNA基因扩增模板。采用通用引物27F (5′-AGAGTTTGATCCTGGCTCAG-3′)和1492-R (5′-GGTTACCTTGTTACGACTT-3′)对分离纯化菌株的16S rRNA进行PCR扩[

23]。PCR反应体系(25 μL):2×Taq PCR StarMix 12.5 μL,DNA模板1 μL,引物27F和1492R (10 μmol/L)各1 μL,ddH2O 9.5 μL。PCR反应条件:94 ℃预变性5 min;94 ℃变性30 s,56.5 ℃退火30 s,72 ℃延伸2 min,32个循环;72 ℃终延伸10 min。使用质量浓度为10 g/L的琼脂糖凝胶电泳检测PCR产物,在凝胶成像仪上观察扩增结果。将PCR扩增成功的产物发送至金唯智(广州)生物科技有限公司进行16S rRNA双向测序。在测序结果成功拼接后,将拼接序列上传至NCBI网站,利用其在线BLAST功能进行相似性比对。最后使用MEGA-X软件构建系统发育[24]

1.5 微塑料降解菌筛选与鉴定

将分离纯化后的菌株分别在无机盐培养基和PET培养基中进行划线培养,置于28 ℃培养箱孵育5 d后,观察平板中菌株的生长情况。通过比较2种培养基上的菌落生长情况,若观察到菌株仅在以PET为唯一碳源的PET培养基上生长,而在无机盐培养基中未见生长的现象,则表明该菌株能够利用PET进行生长代谢,具备降解PET微塑料的能力。将筛选出的微塑料降解细菌的菌体样品送至金唯智(广州)生物科技有限公司进行DNA提取和全基因组测序,以获取细菌的完整基因组信息。测序完成后,将获得的基因组序列上传至KEGG数据库(https://www.genome.jp/kegg/)进行基因功能预测和代谢途径分析,同时通过PlasticsDB数据库(https://plasticdb.org/)进行塑料降解基因簇的比对。

2 结果与分析

2.1 可培养细菌多样性统计

通过稀释涂布及平板划线培养,分别在5种不同的培养基中,从来自广东省珠海市香洲区的野狸岛、桂山岛和外伶仃岛3处滩涂区域采集的3份样品中共分离获得265株细菌。从形态上观察,培养基中的菌落主要为圆形,颜色主要有白色、黄色、红色等。265株细菌分属于4门7纲22目32科71属105个种。如图1所示,菌株主要分布在4个门,其中168株分属于假单胞菌门,占总数的63.40%;放线菌门38株,占总数的14.34%;芽孢杆菌门31株,占总数的11.70%;拟杆菌门28株,占总数的10.56% (图1A)。

fig

图1  不同培养基分离培养的滩涂细菌多样性

Figure 1  Diversity of bacteria from tidal flats cultured in different media. A: Community structure of isolated bacteria at phylum level; B: Venn diagram of differential genera; C: Upset plot of differential genera (The intersection column of four media was marked in grey color and the numbers represent the count of total genera or intersections of each component); D: Community structure of isolated bacteria in five different media at phylum level; E: Community structure of isolated bacteria in five different media at genus level.

2.2 不同培养基可培养细菌多样性

采用5种培养基对3份滩涂样品中的细菌进行分离培养。其中,2216EM培养基中获得的菌株最多,为129株(55属),其次是2216E、R2A、2216EMN和TSA培养基,分别获得86株(31属)、27株(12属)、12株(8属)和11株(8属)。5种培养基分离出的细菌在属水平上无共有的属,2216EM和2216E培养基共有的可培养细菌有13个属,而其他培养基共有的可培养细菌数目则相对较少(图1B、1C)。在门水平上,5种培养基分离出的细菌中都含有放线菌门细菌,假单胞菌门细菌在2216E和2216EM培养基上占优势(图1D)。不同培养基的分离类群也有明显差异,2216EMN和R2A培养基均未分离到芽孢杆菌门类群,TSA培养基未分离到拟杆菌门类群。在属水平,5种培养基分离出的细菌分别属于71个属,污物单胞菌属(Defluviimonas)的菌株数目最多(17株,6.42%),是可培养细菌中丰度最高的属(图1E)。

2.3 可培养细菌多样性在不同采样点之间的比较

在3份滩涂样品中,样品XZ1分离得到的菌株数量最多,共计126株(44属),而样品XZ2和XZ3分别分离得到59株(21属)和80株(29属),如表3所示。在门水平上,除样品XZ2中未分离到放线菌门细菌外,其他每个样品中均能分离到的假单胞菌门、放线菌门、芽孢杆菌门以及拟杆菌门细菌,且假单胞菌门细菌的占比最高(图2A)。在属水平上,仅污物单胞菌属和莱茵海默氏菌属(Rheinheimera)细菌在所有样品中均被分离得到。此外,袁其朋菌属(Qipengyuania,4.91%)为样品XZ1的优势类群,假单胞菌门的尹正勋菌属(Yoonia,4.15%)为样品XZ2的优势类群,微杆菌属(Microbacterium,3.02%)和别样玫瑰变色菌属(Aliiroseovarius,3.02%)为样品XZ3的优势类群(图2B)。

表3  不同样品中分离细菌的数目及多样性情况
Table 3  The number and diversity of bacteria separated from different samples
SampleThe number of isolatesThe number of genera/speciesThe number of potential novel strains
XZ1 126 44/58 27
XZ2 59 21/28 10
XZ3 80 29/39 22
fig

图2  香洲区不同滩涂样品分离的细菌多样性

Figure 2  Bacterial diversity of different mudflat samples in Xiangzhou District. A: Community structure of isolated bacteria in three mudflat samples at phylum level; B: Community structure of isolated bacteria in three mudflat samples at genus level; C: Venn diagram of differential genera; D: Community heatmap of differential genera.

不同样品在属水平上的可培养细菌丰度与多样性存在明显差异(图2C、2D)。XZ1样品采集自野狸岛的沿海软质泥滩,采样地点气候温暖湿润,植被茂盛,吸引了许多鸟类前来栖息,土壤肥沃且含水量较高,因此在XZ1样品中分离得到的菌株数量最多,且多数为适应潮湿土壤环境的细菌,该样品中特有的优势属包括农霉菌属(Agromyces)、海草球菌属(Phycicoccus)、伪芽孢杆菌属(Fictibacillus)、萨克利夫氏菌属(Sutcliffiella)等;XZ2样品采集自桂山岛的一湾沙滩,桂山岛位于咸淡水交汇处,因此既能分离到适应海洋环境的细菌,如假暗棕色杆菌属(Pseudophaeobacter)、海洋硫化单胞菌属(Marinosulfonomonas)、居海杆菌属(Maribacter)等,也能分离到适应淡水环境的细菌,如科迪单胞菌属(Kordiimonas)、斯尼斯氏菌属(Sneathiella)等;XZ3样品采集自外伶仃岛的沙滩泳场附近,该地多为疏松砂土,透水性强,养分含量低,因此特有的属包括亮杆菌属(Leucobacter)、砂杆状菌属(Arenitalea)、沃雷氏菌属(Olleya)、砂土杆菌属(Arenibacillus)、食氢产水菌属(Hydrogenophaga)、副透明桑枣形菌属(Paraperlucidibaca)等。以往研究表明,砂杆状菌属、沃雷氏菌属、砂土杆菌属、副透明桑枣形菌属等细菌常在海洋环境中被发现,而亮杆菌属、食氢产水菌属则更多地在土壤环境中生[

25-26]

2.4 分离菌株中的潜在新分类单元鉴定

对纯化成功的菌株进行16S rRNA基因的双向测序,并对获得的16S rRNA基因序列进行了比对分析。为了探究这些菌株间的进化关系,采用MEGA-X软件,运用邻接(neighbor-joining, NJ)法构建了系统发育树(图3)。根据测序菌株的16S rRNA基因在NCBI上的比对结果,结合其在系统发育树中的分类位置,共鉴定出59株潜在新种(相似度≤98.65%)。在这些潜在新种中,假单胞菌门菌株数量最多,共有34株(57.63%);其次为拟杆菌门菌株12株(20.34%);放线菌门菌株11株(18.64%);而芽孢杆菌门菌株数量最少,仅有2株(3.39%) (表4)。这一发现为深入研究这些细菌的分类学和系统发育关系提供了重要线索。

fig

图3  珠海市香洲区滩涂可培养细菌基于16S rRNA基因序列的系统发育树

Figure 3  Phylogenetic tree of culturable bacteria from mudflat in Xiangzhou District, Zhuhai City based on 16S rRNA gene sequence. The color ranges of the innermost circle indicates the classification on the phylum level; The color of the outnermost circle indicates the classification on the class level.

表4  分离菌株中筛选出的潜在新种
Table 4  The potential new species of isolated bacteria
StrainsThe most similar strains (type strain)GenBank accession numberSimilarity (%)Phylum
SYSU HH007 Demequina soli HI12-122T NR_179185.1 98.65 Actinomycetota
SYSU HH013 Altererythrobacter xiamenensis LY02T NR_133694.1 98.41 Pseudomonadota
SYSU HH015 Streptomyces somaliensis DSM 40738T NR_025292.1 98.46 Actinomycetota
SYSU HH019 Metabacillus schmidteae AS-110s11T OR825855.1 98.23 Bacillota
SYSU HH054 Rheinheimera riviphila KYPC3T NR_169409.1 89.67 Pseudomonadota
SYSU HH056 Pontixanthobacter luteolus SW-109T NR_043151.1 98.09 Pseudomonadota
SYSU HH066 Arenibacter palladensis LMG 21972T NR_042188.1 98.42 Bacteroidota
SYSU HH075 Ruegeria conchae TW15T NR_109062.1 98.65 Pseudomonadota
SYSU HH079 Pseudomonas xionganensis R-22-3 w-18T NR_180878.1 98.53 Pseudomonadota
SYSU HH081 Microbacterium lacus GP23T MW510008.1 98.64 Actinomycetota
SYSU HH084 Pseudoruegeria aestuarii 174T OR675238.1 97.56 Pseudomonadota
SYSU HH090 Ruegeria conchae TW15T NR_109062.1 98.52 Pseudomonadota
SYSU HH093 Robertkochia sediminum 1368T NR_181889.1 97.64 Bacteroidota
SYSU HH095 Agromyces tropicus CM9-9T NR_112811.1 98.44 Actinomycetota
SYSU HH097 Altererythrobacter epoxidivorans CGMCC 1.7731T CP012669.1 98.40 Pseudomonadota
SYSU HH098 Kangsaoukella pontilimi GH1-50T NR_179959.1 97.49 Pseudomonadota
SYSU HH106 Agromyces tropicus CM9-9T NR_112811.1 98.31 Actinomycetota
SYSU HH115 Leucobacter tardus K70/01T NR_042694.1 97.73 Actinomycetota
SYSU HH121 Amylibacter cionae H-12T NR_158032.1 95.46 Pseudomonadota
SYSU HH133 Erythrobacter mangrovi EB310T MT522623.1 98.37 Pseudomonadota
SYSU HH136 Altererythrobacter epoxidivorans JCS350T NR_043706.1 98.12 Pseudomonadota
SYSU HH141 Arenimonas donghaensis HO3-R19T NR_043790.1 97.92 Pseudomonadota
SYSU HH150 Aliiroseovarius sediminilitoris M-M10T NR_109620.1 95.00 Pseudomonadota
SYSU HH151 Photobacterium gaetbulicola Gung47T NR_117271.1 97.57 Pseudomonadota
SYSU HH153 Arenimonas donghaensis HO3-R19T NR_043790.1 97.85 Pseudomonadota
SYSU HH161 Agromyces tropicus CM9-9T NR_112811.1 98.44 Actinomycetota
SYSU HH176 Rheinheimera aquimaris SW-353T NR_044068.1 98.32 Pseudomonadota
SYSU HH178 Agromyces tropicus CM9-9T NR_112811.1 98.23 Actinomycetota
SYSU HH179 Defluviimonas aquaemixtae CDM-7T NR_134014.1 98.07 Pseudomonadota
SYSU HH181 Defluviimonas aquaemixtae CDM-7T NR_134014.1 97.99 Pseudomonadota
SYSU HH182 Tabrizicola aquatica RCRI19T NR_117979.1 97.95 Pseudomonadota
SYSU HH188 Pontimicrobium aquaticum CAUT MK051221.1 95.86 Bacteroidota
SYSU HH189 Muriicola soli MMS17-SY002T NR_179812.1 98.09 Bacteroidota
SYSU HH190 Arenibacter troitsensis M151T OR999768.1 98.30 Bacteroidota
SYSU HH191 Arenibacter troitsensis M151T OR999768.1 98.49 Bacteroidota
SYSU HH195 Arenibacter palladensis LMG 21972T NR_042188.1 98.58 Bacteroidota
SYSU HH199 Leucobacter luti RF6T NR_042425.1 97.27 Actinomycetota
SYSU HH201 Microbacterium saccharophilum K-1T NR_114342.1 98.31 Actinomycetota
SYSU HH205 Yoonia litorea DPG-5T NR_118329.1 98.37 Pseudomonadota
SYSU HH207 Amylibacter cionae H-12T NR_158032.1 95.58 Pseudomonadota
SYSU HH216 Kangsaoukella pontilimi GH1-50T NR_179959.1 97.63 Pseudomonadota
SYSU HH223 Rheinheimera aquimaris SW-353T NR_044068.1 97.90 Pseudomonadota
SYSU HH227 Roseovarius aestuarii SMK-122T NR_044424.1 98.52 Pseudomonadota
SYSU HH228 Sneathiella litorea DP05T MN381954.1 97.82 Pseudomonadota
SYSU HH239 Rheinheimera aquimaris SW-353T NR_044068.1 97.97 Pseudomonadota
SYSU HH243 Sneathiella litorea DP05T MN381954.1 97.96 Pseudomonadota
SYSU HH244 Altererythrobacter xiamenensis LY02T NR_133694.1 98.26 Pseudomonadota
SYSU HH250 Rheinheimera aquimaris SW-353T NR_044068.1 98.14 Pseudomonadota
SYSU HH259 Arenibacter troitsensis M151T OR999768.1 98.08 Bacteroidota
SYSU HH262 Pontimicrobium aquaticum CAUT MK051221.1 96.68 Bacteroidota
SYSU HH265 Arenibacter troitsensis NBRC 101532T NR_114004.1 98.37 Bacteroidota
SYSU HH266 Arenibacter troitsensis M151T OR999768.1 98.37 Bacteroidota
SYSU HH270 Maribacter cobaltidurans PR1T OQ352841.1 97.81 Bacteroidota
SYSU HH296 Ruegeria conchae TW15T NR_109062.1 98.31 Pseudomonadota
SYSU HH302 Altererythrobacter xiamenensis LY02T NR_133694.1 98.41 Pseudomonadota
SYSU HH304 Bacillus carboniphilus JCM9731T NR_024690 92.70 Bacillota
SYSU HH311 Agromyces tropicus CM9-9T NR_112811.1 98.44 Actinomycetota
SYSU HH320 Microbulbifer okinawensis ABABA23T NR_112917.1 94.27 Pseudomonadota
SYSU HH323 Defluviimonas aquaemixtae CDM-7T NR_134014.1 97.79 Pseudomonadota

(待续)

2.5 微塑料降解细菌的筛选及分析

将分离纯化后的菌株分别在无机盐培养基和PET培养基中划线培养5 d后,观察到

SYSU HH014菌株能在以PET为唯一碳源的PET培养基上生长,而在无机盐培养基中未见生长,而其余菌株在PET培养基和无机盐培养基中均不生长,表明SYSU HH014菌株能够利用PET进行生长代谢,具备降解PET微塑料的潜能。如图4A所示,从样品XZ1中成功筛选出能以PET为唯一碳源生长的菌株SYSU HH014。SYSU HH014菌株在无机盐培养基中无法生长,但在PET培养基中却能良好生长,这一结果表明该菌株能够利用PET进行生长代谢,具备降解PET微塑料的能力。通过基因组分析,确定SYSU HH014菌株属于芽孢杆菌属(Bacillus)的卡布氏芽孢杆菌(B. cabrialesii),并构建了该菌株的基因组系统发育树(图4B)。将SYSU HH014菌株的基因组在KEGG (图4C)和PlasticsDB (表5)数据库进行比对分析,发现该菌株含有多个与塑料降解相关的基因簇,如PET水解酶(PET depolymerase, PETase)[

27]、3-羟基戊酸脱氢酶(3-hydroxyvalerate dehydrogenase, 3HV dehydrogenase)[28]、聚乳酸解聚酶(polylactic acid depolymerase, PLA depolymerase)[29]基因簇等,这些基因簇在PET降解过程中可能起着关键作用,然而其具体功能和作用机制还需在后续研究中进一步探讨。

fig

图4  微塑料降解细菌的筛选与鉴定

Figure 4  Screening and identification of microplastic-degrading bacteria. A: Comparison of the growth of SYSU HH014 strain in inorganic salt medium (left) and PET medium (right); B: Phylogenetic analysis of SYSU HH014 strain based on genome; C: Annotation results of the SYSU HH014 strain genome on the KEGG.

表5  SYSU HH014菌株基因组在PlasticsDB的比对结果
Table 5  The BLAST results of SYSU HH014 strain genome on the PlasticsDB
SequenceEnzymeStrainsPercent identityLength (bp)E-valueBitscore
00013 PU esterase Comamonas acidovorans 35.0 492 3.30E-57 208.8
00027 PLA depolymerase Paenibacillus amylolyticus 49.8 203 1.10E-52 182.2
00035 PEG aldehyde dehydrogenase Streptomyces sp. 41.7 468 1.50E-99 329.7
00040 Protease Parengyodontium album 39.6 321 2.60E-43 163.7
00071 3HV dehydrogenase Paracoccus denitrificans 42.7 255 1.30E-53 188.7
00079 Esterase Clostridium hathewayi 35.0 409 1.60E-54 196.8
00093 PVA dehydrogenase Stenotrophomonas rhizophila 41.7 120 1.40E-22 100.1
00152 Protease Lederbergia lenta 60.7 275 1.80E-99 321.2
00154 Protease (PLA) Bacillus licheniformis 65.1 381 1.30E-149 469.9
00157 Esterase (PLA; PBSA) Alcanivorax borkumensis 36.8 473 4.00E-72 251.1
00179 Hydrolase (PBAT; PBSA; PCL) Bacillus pumilus 75.1 209 3.00E-99 316.2
00185 PETase (PET) Uncultured bacterium 40.4 277 2.30E-52 186.8
00209 Oxidoreductase (PU; PBAT) Bacillus velezensis 66.0 47 2.30E-12 71.6

3 讨论

本研究对珠海市香洲区的野狸岛、桂山岛和外伶仃岛的滩涂区域样品进行了细菌的分离与培养。从3份滩涂样品中共分离获得265株细菌菌株,分别为168株(63.40%)假单胞菌门细菌、38株(14.34%)放线菌门细菌、31株(11.70%)芽孢杆菌门细菌和28株(10.56%)拟杆菌门细菌。这一结果与Couradeau[

30]对波多黎各沿海岩滩细菌类群的研究以及Lee[31]对济州岛沙滩细菌多样性的研究结果基本一致。

本研究中的优势属主要包括农霉菌属、芽孢杆菌属、污物单胞菌属、尹正勋菌属、袁其朋菌属、莱茵海默氏菌属和栖砂杆菌属(Arenibacter),这与李斌[

32]对我国沿海滩涂细菌资源多样性的研究结果相似。其中,污物单胞菌属细菌的相对丰度最高,且主要在XZ1、XZ2样品中分离得到(图2B)。在之前的研究中也发现污物单胞菌属细菌可在红树林滩涂、南海海滩等环境中分离获[33-34]。污物单胞菌属细菌具有多种代谢功能,能够将硫酸盐、硝酸盐和亚硝酸盐分别还原为硫化氢和氮气等物质,在废水处理中参与多种污染化合物如多环芳烃、氮污染物的降[35-36]。在分离出的11株栖砂杆菌属细菌中有9株疑似为新物种。栖砂杆菌属细菌在海洋生态系统中发挥着关键作用,具有积累碘的能[37],能够降解海洋中的有机物质,如淀粉,以及表面污染[38-40]。此外,它们还能直接或间接分解藻类细胞,促进生态系统的物质循[41]。后续工作将对栖砂杆菌属中的这9株疑似新种以及本次研究中成功分离的其余50株潜在新种进行详细的物种鉴定,以便进一步探索和解析它们的生物学功能和特性。

在获得的可培养细菌中,许多属具有应用潜能。例如,在样品XZ1中发现的罗伯特科赫菌属(Robertkochia)细菌能够分解木质纤维素,可用于制造生物燃料和化学制品,其产生的碱性磷酸酶可促进盐碱农田植物的生[

42-43]。在样品XZ1和XZ3中分离得到的袁其朋菌属细菌曾在多种滩涂环境中被分离得到,已有报道指出该属的一株菌Qipengyuania sp. 3-20A1M对于控制海洋生态系统中由鞭毛藻造成的水华污染具有一定潜在效[44-46]。在3份样品中都分离得到了莱茵海默氏菌属的细菌,该属的多个物种均具有抗菌活[47-48]。值得注意的是,近年来研究者们发现莱茵海默氏菌属的部分物种具有汞耐受性,可以有效地挥发和去除汞,为汞污染的生物处理提供了新的方[49-50]

在培养基方面,2216E培养基常用于海洋细菌的培[

51]。在本研究中,通过5种培养基对滩涂细菌进行分离培养,成功鉴定了4门71属的细菌。相比之下,2216E、2216EM、2216EMN这3种培养基表现出了优越的覆盖能力,共分离得到4个门66个属的细菌。这一结果表明,2216E系列培养基能够有效支持海洋滩涂微生物的分离与鉴定工作,为后续研究提供了有力的实验基础和技术手段。R2A培养基主要用于分离和培养在寡营养条件下生长的微生物,这类培养基更容易分离得到稀有和难培养的细菌类[52-53]。本研究使用R2A培养基虽然未发现更多新物种,但仅有该培养基分离到了食氢产水菌属、泰熙菌属(Taeseokella)的微生物。TSA培养基包含胰蛋白胨和大豆酪蛋白水解物,提供了维生素、矿物质等微量营养物质,常用于多种细菌的分离和培[54]。然而,在本研究中,TSA培养基的分离效果较差,仅分离出11株细菌。

筛选出的PET降解细菌SYSU HH014呈现为圆形菌落,颜色为浅橘色。SYSU HH014菌株被鉴定为卡布氏芽孢杆菌,隶属于芽孢杆菌属。近年来,多个芽孢杆菌菌株被证实具备塑料降解活性。例如,周剑桥[

55]已证明嗜热淀粉芽孢杆菌(B. thermoamylovorans)能够降解PET及其中间体;Auta[56]发现蜡样芽胞杆菌(B. cereus)能够降解PE、PS以及PET等多种塑料;Nowak[57]首次证实解淀粉芽孢杆菌(B. amyloliquefaciens)具有降解土壤中低密度聚乙烯(low-density polyethylene, LDPE)的能力。这些实验结果说明芽孢杆菌属在微塑料降解研究中具有潜在价值。通过对SYSU HH014进行全基因组测序,获取了其基因组信息,并借助KEGG数据库通路进行了通路分析以及功能基因注释。KEGG通路分析显示,SYSU HH014菌株的大多数注释序列与代谢途径有关,包括氨基酸代谢、聚糖生物合成和代谢、碳水化合物代谢、脂质代谢、辅因子和维生素代谢、外源性生物降解和代谢、萜类化合物和聚酮类化合物的代谢等(图4C),其中,外源性生物降解和代谢通路包括将药物、色素、塑料前体等外源物质的降解过[58]。同时,该菌株在PlasticsDB中也比对到了许多已知的塑料降解基因,包括PETase、3HV dehydrogenase、PLA depolymerase等(表7)。这些结果表明,SYSU HH014菌株拥有多个关键的塑料降解酶基因簇,能够在其生长代谢过程中通过这些酶的相互作用来分解塑料中的聚合物链。后续实验将开展对SYSU HH014菌株的降解能力进行进一步的分析与研究,以期为开发新的微塑料降解技术提供可能性。

4 结论

本研究采用5种培养基对珠海市香洲区的野狸岛滩涂、桂山岛滩涂和外伶仃岛滩涂的细菌多样性进行了初步分析。从3份滩涂样品中共获得265株细菌,其中假单胞菌门的细菌数量最多。在所有使用的培养基中,2216EM培养基分离获得的菌株数目最多。在获得的可培养细菌中发现59株潜在新种,并成功筛选出1株具有微塑料降解能力的细菌。然而,在对微塑料降解细菌的筛选与分析过程中,本研究仍存在一定局限性。本研究仅基于PET微塑料进行了初步筛选,对于筛选出的微塑料降解细菌的降解特性尚需进一步深入和全面的探讨。从分离出的可培养菌株中发现共有73株属于红细菌目(Rhodobacterales)的细菌。目前,多个红细菌被证实具有塑料降解活性,如海岸沉积物别样玫瑰变色菌(A. sediminilitoris)[

59]、红球菌属细菌Rhodococcus sp. 36[60]等。除此之外本研究还筛选到了微杆菌属、假单胞菌属(Pseudomonas)、斯塔普氏菌属(Stappia)、食烷菌属(Alcanivorax)等属的细菌,这些属中的一些细菌能够降解其他类型的塑料,如微杆菌属的Microbacterium sp. 4-7细菌能降解PE;铜绿假单胞菌(P. aeruginosa)细菌可通过产生特定酶来分解聚氨酯塑料;泊库岛食烷菌(A. borkumensis)细菌能够在LDPE上形成厚生物膜并对其进行降解[40,61-63]。因此,在后续的实验中可以采用多类型微塑料无机盐培养基进行细菌筛选,以便于识别和分离出针对不同类型微塑料具有高效降解能力的特异性菌株。同时,应对筛选出的微塑料降解细菌进行详细的降解特性分析,并从基因组层面进行深入研究,揭示其在降解微塑料过程中所涉及的关键基因和代谢途径,以深入地理解微塑料降解细菌的降解机制,为开发新型生物降解技术、减少微塑料污染提供科学依据和技术支持,进而促进生态环境的可持续发展。

作者贡献声明

王莹:实验操作、数据分析和论文撰写;何欢欢:实验设计、实验操作、数据收集;卢春艳:实验设计、实验操作;郑卓桓:样本采集、菌株分离;王森:微塑料降解细菌筛选与鉴定;黄易:菌株分离;李冰雨:菌株纯化与保藏;董雷:实验设计、数据分析、论文审阅和修改;李文均:实验设计、数据分析、论文审阅和修改。

利益冲突

作者声明不存在任何可能会影响本文所报告工作的已知经济利益或个人关系。

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