微生物学报  2021, Vol. 61 Issue (4): 946-968   DOI: 10.13343/j.cnki.wsxb.20200448.
http://dx.doi.org/10.13343/j.cnki.wsxb.20200448
中国科学院微生物研究所,中国微生物学会,中国菌物学会
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文章信息

承磊, 马诗淳, 巫可佳, 张辉, 邓宇. 2021
Lei Cheng, Shichun Ma, Kejia Wu, Hui Zhang, Yu Deng. 2021
厌氧微生物培养分离:过去、现在和未来
Cultivation and isolation of Anaerobes: past, present and future
微生物学报, 61(4): 946-968
Acta Microbiologica Sinica, 61(4): 946-968

文章历史

收稿日期:2020-07-12
修回日期:2020-09-22
网络出版日期:2020-09-24
厌氧微生物培养分离:过去、现在和未来
承磊1,2,3 , 马诗淳1,2,3 , 巫可佳1 , 张辉1 , 邓宇1,2,3     
1. 农业农村部农村可再生能源开发利用重点实验室, 农业农村部沼气科学研究所, 四川 成都 610041;
2. 中国厌氧微生物资源管理中心, 四川 成都 610041;
3. 国家农业微生物双流观测实验站, 四川 成都 610213
摘要:厌氧菌是地球上数量最多、物种最丰富的微生物,也是分类上报道最少的微生物。它们对氧气敏感、生长条件苛刻,不容易培养分离。本文简要总结了厌氧微生物的研究历史,分析了限制厌氧微生物培养分离的主要因素,讨论了厌氧微生物培养分离的策略和方法,回顾了国内外厌氧微生物的系统分类学现状,并展望了厌氧微生物培养分离的发展趋势。
关键词厌氧微生物    分离培养    高通量筛选    定向分离    
Cultivation and isolation of Anaerobes: past, present and future
Lei Cheng1,2,3 , Shichun Ma1,2,3 , Kejia Wu1 , Hui Zhang1 , Yu Deng1,2,3     
1. Key Laboratory of Development and Application of Rural Renewable Energy, Ministry of Agriculture and Rural Affairs, Biogas Institute of Ministry of Agriculture and Rural Affairs, Chengdu 610041, Sichuan Province, China;
2. China Collection of Anaerobic Microorganisms, Chengdu 610041, Sichuan Province, China;
3. National Agricultural Experimental Station for Microorganisms, Shuangliu, Chengdu 610213, Sichuan Province, China
Abstract: Anaerobes are the largest number of microorganisms on earth but rarely reported from the point view of taxonomy. Owing to oxygen-sensitive and slow-growth, cultivation and isolation of fastidious anaerobe faces a great challenge. In this review, we introduce the research history of anaerobes, and analyze the factors affecting the isolating efficiency of anaerobes. Furthermore, we discuss the current status of isolation and cultivation methods and strategies, and summarize the research progress on systematic taxonomy of anaerobes. In the end, we give an outlook for isolating and cultivating anaerobes.
Keywords: anaerobes    isolation and cultivation    high-throughput screening    targeted isolation    

根据微生物对氧气的利用和耐受程度,科学家曾经将它们分为好氧、微好氧、严格厌氧、兼性厌氧和耐氧微生物(表 1)。厌氧微生物无法在有氧条件下生长。兼性厌氧微生物在有无氧条件下都可以生长繁殖。耐氧微生物则能够在厌氧条件下生长,也能耐受一定浓度的氧气。过去认为氧气浓度达到巴斯德点(Pasteur point),即氧分压达到当前大气氧分压水平(PAL)的1% (大约相当于94 μmol/L),是好氧微生物和厌氧微生物生长的分界点[1-2]。但Baughn等发现厌氧菌Bacteroides fragilis利用nmol/L级的氧气生长[3]。这类厌氧菌被定义为“纳级厌氧菌(Nanaerobe)”[3]。Stolper等发现某些好氧微生物也可以在氧气浓度 < 3 nmol/L条件下生长[4]。严格厌氧微生物对氧气的适应和生长耐受机制还有待进一步研究。

表 1. 根据氧气利用特征区分的微生物类型[5] Table 1. Types of microorganism according to oxygen utilization properties[5]
Microorganism types Oxygen required for growth Oxygen-free for growth Notes
Aerobe +
Microaerobe + Grow with low concentration of O2
Strict anaerobe + O2 is toxic to strict anaerobe
Facultative anaerobe + + Grow both with O2 or no O2
Aerotolerant microorganism +

氧气在细胞内能以氧自由基(O2/ROS)的形式存在,如O2–·、H2O2·OH和1O2,微生物细胞除了产生抗坏血酸和谷胱氨肽等小分子的抗氧化剂外,也可以产生抗氧化酶来消除氧自由基的胁迫,如超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和抗氧化蛋白(PXs)[6]。McCord等认为严格厌氧微生物不含有超氧化物歧化酶(SOD)和过氧化氢酶活性(CAT),而耐氧微生物则能利用超氧化合物氧化酶(SOR)抵御一定浓度的氧胁迫[7]。但是S’lesak等分析了100种严格厌氧微生物,发现93%的菌种中至少含有一种抗氧化酶[6]。从能量代谢的角度,厌氧微生物无法利用氧气作为末端电子受体获取ATP,只能通过底物水平磷酸化,或者利用硫酸盐等外源电子受体进行厌氧呼吸获得能量[8]。好氧微生物通过呼吸链传递电子并获取ATP,其中氧化酶作为关键功能酶,一度也被认为是好氧微生物独有的特征酶。Baughn等发现厌氧菌利用细胞色素bd氧化酶(cytochrome bd oxidase)利用nmol/L级的氧气生长[3]。进一步研究发现很多严格厌氧菌如Archaeoglobus fulgidusDesulfovibrio gigasMethanosarcina barkeriMoorella thermoacetica都含有类似的细胞色素bd氧化酶编码基因,可能都具有类似的低氧代谢能力[3, 9-10]。因此,通过厌氧微生物基因组中是否含有抗氧化酶和氧化酶基因,也难以准确区分它们是否属于厌氧微生物[1]

1 厌氧微生物培养分离的研究历史

早在300多年前,Antoni van Leeuwenhoek在静置培养3周后的胡椒水中,观察到有活的微生物存在,这是最早关于厌氧微生物培养的报道[11]。19世纪60年代,法国科学家Louis Pasteur开启了厌氧微生物学研究,首次根据微生物是否可以在有氧条件下生长,将微生物划分为好氧和厌氧微生物[12]。此外,他和合作者分离获得了第一个致病性厌氧菌,后被命名为Clostridium septicum[12]。巴斯德提出的煮沸或抽真空法制备预还原厌氧培养基是经典的厌氧操作技术之一。

在Hungate厌氧操作技术发明以前,科学家通常通过生物法(如共培养好氧细菌的方式消耗培养基中的氧气)、物理法(如煮沸、抽真空、惰性气体吹扫)和化学法(如添加铁、硫化物和碱性焦棓酸)等制备预还原培养基[13]。在厌氧培养的时候,通常是采用深层琼脂/液体培养基进行梯度稀释培养,并结合液封(如石蜡、原油、绵羊油、凡士林),或者橡胶塞、密封罐等方式密封[13-14]。这些操作繁琐、难以真正隔绝氧气,而且在挑取深层单菌落的过程中也容易被污染。这导致厌氧微生物研究进展缓慢,只能分离一些产芽孢厌氧菌、兼性厌氧菌或耐氧菌,如Clostridium[15-16]

1947年,美国微生物学家Robert E. Hungate通过深层琼脂法分离厌氧纤维素降解菌的时候,意外发现管内壁上层的薄层固体培养基上会形成单菌落[17]。随后他提出了滚管法分离厌氧菌的操作技术[18],经过不断的完善[19],该方法已成为厌氧微生物分离培养的经典方法[20]。该技术核心有3点:利用完全密封的玻璃管培养厌氧微生物;利用高温铜柱去除惰性气体中的残余氧,在无氧惰性气体保护下制备预还原培养基、转移厌氧微生物;利用滚管法制备贴内壁的固体培养基薄层,生长的单菌落很容易被挑取转移[18, 20]。这个操作技术所需要的设备简单,并能提供严格厌氧环境。Hungate在厌氧微生物学领域的奠基性工作,使其成为厌氧微生物学研究的先驱[21-22]。同时期有科学家开发了封闭的充满无氧气体的手套箱,实验人员可以通过手套进行厌氧操作[23-24],这种方法虽然比Hungate厌氧操作技术简单,但是手套箱中的残氧含量还是高于厌氧管,早期的厌氧手套箱无法控制洁净度,容易造成交叉污染,也难以大面积推广使用。我们课题组与企业联合开发的无菌无氧控温手套箱,氧含量最低小于10 mg/L,内部操作空间可以达到百级洁净,温度可以控制在10–25 ℃,适宜开展大规模的无菌操作[25]

1977年,Carl R. Woese提出“三域学说”,即根据核糖体基因序列相似度来指示物种亲缘关系的理论[26],完全改变了人们对微生物物种多样性的认知。在DNA测序技术的推动下,科学家推测全球微生物数量达到了1030的量级,其中超过70%分布在陆相和海相深部缺氧沉积物中[27-28],这表明地球上的微生物仍然以厌氧微生物为主,其物种多样性可能远远超过好氧微生物。这也表明厌氧微生物资源是尚未充分开发的一块处女地,值得微生物资源与分类学家去关注。

2 厌氧微生物培养分离新策略 2.1 限制厌氧微生物培养分离的因素

微生物分离的经典思路是“先富集后分离”,通过选择性培养基让特定功能的微生物繁殖变为优势菌,重复在固体或液体培养基上梯度稀释培养,以纯化获取单菌落。这种传统思路的局限性也非常明显:(1) 分离过程的盲目性:环境中存在大量未培养微生物,但是不知道哪些微生物会在培养过程被选择性富集。(2) 分离的随机性:不同种类的环境微生物浓度可能相差3–5个数量级[29-30]。即使假定所有微生物都可以培养生长,理论上也需要随机挑取数百万级别的单菌落,才有可能分离到所有的微生物。而传统Hungate滚管法的分离通量很难超过2–3个数量级,难以挑取到所有微生物。(3)菌株生长的不确定性:人工设计的培养基和培养条件无法满足所有原位微生物的生长。如磷酸盐缓冲液与琼脂高温湿热灭菌后,会抑制微生物生长[31]。有些微生物的菌体生长浓度只有105个/mL甚至更低,难以通过选择培养的方式进行富集[32]。(4)菌株生理特征的未知性:厌氧微生物不仅生长缓慢,对氧气敏感,而且存在更为复杂的种间互作关系,如互营微生物依赖于产甲烷古菌的互营代谢关系[33]。如果不清楚这些未培养微生物的生理特征,也难以分离培养它们。

2.2 提高厌氧微生物培养分离的策略

鉴于厌氧微生物的特殊生理特征,我们总结国内外分离厌氧微生物的研究策略,主要通过测序引导分离、开发新装置提高分离效率、改善培养条件、预测未培养微生物的潜在代谢功能等策略,来提高厌氧微生物的分离效率(表 2)。

表 2. 厌氧微生物分离的策略 Table 2. Isolation strategies of anaerobic microorganisms
Isolation strategies Isolation methods Explanation of isolation methods References
Development of real-time observation technology 16S rRNA and functional genes sequencing The growth statue of targeted microorganisms was observed by 16S rRNA and functional gene sequencing [36-37]
FISH Real-time monitoring the growth condition of target microorganisms through FISH [34-35]
Development of equipment for high-throughput cultivation and identification Microplate Lowering cultivation volume to μL-nL and improving plates number to hundreds and thousands [42, 44]
Microfluidics Lowering cultivation volume to μL-nL and improving plates number to ten thousand [37, 43]
Microencapsulation Gradient dilution microorganisms and mixing low concentration agar to micro gel capsule and cultured in lipid medium [45]
Single cell sorting Using flow cytometry or Raman spectra technique to selectively pick microorganisms [40-41]
MALDI-TOF Use of protein profiles obtained by Matrix Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS) to for bacteria identification [38]
Optimization of cultivation condition Syntrophic cultivation Co-cultured incubation with hydrogenotrophic methanogens or syntrophic bacteria to isolate syntrophic bacteria and methanogens respectively [36, 46-47]
Parasitic cultivation Providing amino acids and cofactors to target microorganism by other microorganism [48]
Agar substitution Using phytagel (Glen glue) to prepare solid medium [31, 49]
Antibiotics addition Selective inhibition of anaerobe by different antibiotics addition [51]
Pretreatment with ethanol Pretreatment with ethanol to inactivate non-spore microorganisms to isolate spore-producing anaerobes [30]
Pre-reduced medium preparation by addition of different reductant solutions Titanium citrate or sodium ascorbate was chosen as reductants [52-53]
Selective addition of electron acceptors Selective enrichment and cultivation of different types of anaerobes through adding different electron acceptors [29]
Cultivation under low nutrition concentration Reducing medium or substrate concentration by 10-100 times [54-55]
In-situ cultivation Simulating in-situ environment, substance exchange and spatial separation to improve cultivability of microorganisms [70]
Continuous flow cultivation Isolation and cultivation of high abundance uncultured microorganism through continuous dynamic cultivation [56-57]
Culturomics High throughput isolation using different medium and incubation conditions High throughput isolation using different medium and incubation conditions [39, 66]
“Cocktail method” Treatment with multiple isolation methods Enrichment of targeted anaerobes by continuous flow cultivation, and cocultured incubation under low H2 pressure and low nutrition conditions [67]
Directional isolation Reverse genome method Using labelled antibody according to the speculating epitope from the membrane protein gene of uncultured microorganisms to screen and cultivate specific binding microorganism [69]

2.2.1 通过高通量测序技术等解决厌氧微生物分离过程的盲目性:

通过微生物分子生态学技术,实时跟踪不同处理和培养条件下目标菌的生长状态,降低厌氧菌分离的盲目性。Sekiguchi等采用荧光原位杂交技术(FISH)监测厌氧丝菌UN1-1的分离,后被鉴定为新属Anaerolinea[34-35]。类似的,Sakai设计产甲烷古菌RC-I的探针,用于指导产甲烷古菌新目Methanocella的分离[36]。Ma等在16S rRNA基因序列的引导下,结合滑动微流控芯片技术分离获得Ruminococcaceae的一个新属[37]。利用MALDI-TOF技术分析菌株核糖体蛋白的差异特征[38-39],菌株鉴定通量可以达到几百个菌株/小时,并能鉴定到种或属水平,这样能降低后续50%–80%的工作量。Witkowska等通过表面增强拉曼光谱谱技术可以识别菌株水平的微生物[40]。Bellais等通过特异性多克隆抗体反应,通过流式细胞仪识别和分选出来肠道Faecalibacterium prausnitzii[41]

2.2.2 开发新装置和设备提高厌氧微生物分离的通量:

随着高通量培养装备和检测技术的发展,将单个或少数几个微生物细胞分散到单孔中,也可以进行“先分离后富集”。开发厌氧微生物高通量分离培养装置,降低“培养瓶”的容积,可以提高微生物的分离效率。有科学家利用96或384微孔板,将培养体积降低到100–200 μL,相应的,“培养瓶”的数量能提高到数千个量级[32, 42]。微流控技术的应用进一步将培养孔的体积降低到nL甚至pL尺度。Ma等设计的微流控滑动芯片有3200个微孔能用于微生物培养,每孔容积只有6 nL左右[37]。Villa等应用微流控液滴技术,在厌氧手套箱中分离肠道厌氧微生物[43]。Ingham等构建的半透性芯片,最高可以布置近百万个芯片微孔,一次性可以扫描20多万个单菌[44]。Zengler等利用低浓度琼脂包裹的凝胶微囊,可以在流动性培养液中培养单菌[45]

2.2.3 通过优化培养条件改善厌氧微生物的可培养性:

添加伴生菌可以促进厌氧微生物的生长。如互营微生物是一类独特厌氧微生物,需要伴生菌消耗氢气并维持低氢分压才能生长。Qiu等通过添加外源氢营养型产甲烷古菌,分离获得了互营苯酚降解菌新属Syntrophorhabdus[46]。同时,他们课题组反其道而行之,通过互营丙酸降解菌产生的低浓度氢气,来“钓取”水稻根际甲烷排放的重要未培养产甲烷古菌新目Methanocellales[36-47]。Huber等发现嗜热古菌新门Cadidaus Nanoarchaeota依赖于嗜热化能自养古菌Ignicoccus才能生长[48]。Carbonero等发现难以在普通琼脂培养上生长的产甲烷古菌Methanothrix (原属名Methanosaeta),可以在0.3%结冷胶培养基上形成菌落[49]。国外科学家发现磷酸盐缓冲液与琼脂分开灭菌后可以提高微生物的可培养性[31, 49],我们研究也发现磷酸盐缓冲液会增加乙酸代谢产甲烷的延滞期,降低古菌群落的多样性[50]。添加抗生素选择性抑制厌氧微生物[51],添加乙醇获得产芽孢的厌氧菌[30],添加硫酸盐、铁锰氧化物作为电子受体,或添加不同的还原剂,如柠檬酸钛和抗坏血酸钠,也可以选择性富集不同类型的厌氧还原菌[29, 52-53]。这些都可以提高厌氧微生物新物种的分离概率[52-53]。此外,通过稀释培养基、降低培养基成分的浓度[54-55],在连续流反应器中动态驯化培养[56-57],也分离获得了厌氧微生物新种属[57]

2.2.4 通过宏组学技术预测未培养微生物的代谢功能:

高通量测序和生物信息学技术的快速发展,可以跳过微生物培养环节,拼接获得未培养微生物的全基因组序列,根据基因组序列特征推测它们的遗传代谢潜力。如Evans等直接提取地下煤层水中的总DNA,通过二代测序技术获得了宏基因组序列,再根据GC含量、片段测序深度和四核苷酸多态性等序列特征,组装拼接出2个未培养古菌的基因组序列。进一步分析发现这2个基因组中都含有依赖H2的甲基裂解产甲烷途径的编码基因,它们属于Bathyarchaeota(深古菌门),而不是传统认为的Euryarchaeota[58]。类似的,在VerstraetearchaeotaNezhaarchaeotaKorarchaeotaThaumarchaeota等未培养新门中都发现了新型产甲烷古菌[59-62]。通过富集培养、宏转录组测序和中间代谢产物分析,能发现未培养微生物的代谢新功能,如完全硝化细菌、降解短链烷烃的硫酸盐还原细菌和古菌等[54, 63-65]。标记培养和显微成像技术,如CARD-FISH和NanoSIMS结合,可以在单细胞水平鉴别未培养古菌的碳代谢功能。Chen等发现古菌Ca. Argoarchaeum细胞内的硫含量显著高于其他硫酸盐还原菌,推测原位硫酸盐还原降解乙烷主要发生在古菌而不是细菌细胞中[65]

由于微生物物种的多样性和生理特征的异质性,难以通过单一的策略和方法分离所有的微生物。组合多种培养基和培养条件,进行高通量分离筛选的“培养组学”应运而生[39, 66]。如Lagier等尝试了212种培养条件,从12个粪便样品中分离挑取了90万个单菌落,结合MALDL-TOF和16S rRNA基因测序,共获得了1057种微生物(其中新种以上的分类单元近200个)[39]。另外,Imachi等通过连续流驯化、共培养降低氢分压、低浓度生长和特异性序列监测的“鸡尾酒”法(综合多种分离策略和方法),并努力和坚持数十年,分离出厌氧古菌新门Asgard的纯培养物[67]。该研究可能会改写生命起源与进化认知[68]。Cross等通过宏基因组或单细胞测序技术获得未培养微生物的基因组序列,并预测未培养微生物的膜蛋白基因预测抗原表位,通过制备特异性的标记抗体,来特异性结合并筛选目标细菌,从而进行后续培养[69]。如果这个技术具有普适性,那么将突破活体微生物细胞的定向预分离技术,为后续的分离和原位生理功能研究奠定基础。

3 已分离厌氧微生物的物种多样性

截止本文修订时,全球有效发表的原核微生物(具有正确的拉丁文名)只有17304个种,分别属于38门、73纲、213目、52科、3563属(截止2020-8-15,https://lpsn.dsmz.de/text/numbers),其中厌氧微生物只有2151个种,低于好氧微生物一个数量级。科学家推测原核微生物的种水平多样性可能高达106–1012[71-74]。因此,从全球尺度看,可培养微生物比例不超过1%,其中厌氧纯培养物的可培养度不及0.1%,绝大部分厌氧微生物处于未培养状态。

20世纪80年代,我国开始厌氧微生物的分离培养,特别是产甲烷古菌的分离培养研究[75-80]。直到2003年,我国科学家(第一作者或通讯作者单位为国内科研机构)才首次正式提出厌氧微生物新物种[81]。截止2020年8月,国内科学家共提出92个新种,分布于11门、18纲、29目、47科、71属(表 3)。其中,穆大帅等发表的新纲Tichowtungiia是目前我国科学家提出的厌氧微生物的最高分类单元[82]。另外,我国科学家还提出了2个新目(ThermosediminibacteralesMoorellales)、9个新科,除中山大学李文均课题组提出的新科Xylanivirgaceae[83],其他均由我们课题组提出[84-86]。在92个新种中,只有9个属于古菌域,其中7个属于广古菌门,主要由东秀珠研究员和我们课题组提出,东老师课题组发现乙酸营养型产甲烷古菌新种Methanothrix harundinacea[87],后被证实具有利用电子还原二氧化碳产甲烷的功能[88]。我们课题组发现的甲基营养型产甲烷古菌新科Methermicoccaceae[85],后被证实具有直接降解煤炭中氧甲基化合物产甲烷的功能[89]

表 3. 国内报道的厌氧微生物新物种 Table 3. Novel taxa of anaerobic microorganisms proposed by Chinese scholars
Domain Phylum Class Order Family Genus Species References
Archaea Euryarchaeota Methanobacteria Methanobacteriales Methanobacteriaceae Methanobacterium Methanobacterium beijingense [90]
Methanothermoba- cter Methanothermobacter crinale [91]
Methanomicrobiales Methanomicrobiaceae Methanoculleus Methanoculleus receptaculi [92]
Methanoculleus hydrogenitrophicus [93]
Methanosarcinales Methanosaetaceae Methanosaeta Methanosaeta harundinacea [87]
Methermicoccaceae Methermicoccus Methermicoccus shengliensis [85]
Methanosarcinaceae Methanolobus Methanolobus psychrophilus [94]
Thermococci Thermococcales Thermococcaceae Palaeococcus Palaeococcus pacificus [95]
Thermococcus Thermococcus eurythermalis [96]
Bacteria Actinobacteria Actinobacteria_c Actinomycetales Actinomycetaceae Actinomyces Actinomyces ruminicola [97]
Micrococcales Micrococcaceae Glutamicibacter Glutamicibacter ardleyensis [98-99]
Microbacteriaceae Microbacterium Microbacterium nanhaiense [100]
Aquificae Aquificae_c Aquificales Desulfurobacteriaceae Desulfurobacterium Desulfurobacterium indicum [101]
Bacteroidetes Bacteroidia Bacteroidales Acetobacteroides Acetobacteroides Acetobacteroides hydrogenigenes [102]
Porphyromonadaceae Paludibacter Paludibacter jiangxiensis [103]
Parabacteroides Parabacteroides chartae [104]
Parabacteroides acidifaciens [105]
Proteiniphilum Proteiniphilum acetatigenes [106]
Marinilabiliales Marinifilaceae Ancylomarina Ancylomarina psychrotolerans [107]
Ancylomarina longa [108]
Marinilabiliaceae Mangroviflexus Mangroviflexus xiamenensis [109]
Thermophagus Thermophagus xiamenensis [110]
Firmicutes Clostridia Clostridiales Anaerovirgula Anaeromicrobium Anaeromicrobium sediminis [111]
Inediibacterium Anaerophilus nitritogenes [112]
Salimesophilobacter Salimesophilobacter vulgaris [113]
Wukongibacter Wukongibacter baidiensis [114]
Clostridiaceae Clostridium Clostridium algifaecis [115]
Clostridium amylolyticum [116]
Clostridium beihaiense [117]
Clostridium bovifaecis [118]
Clostridium fermenticellae [119]
Clostridium guangxiense [120]
Clostridium huakuii [121]
Clostridium liquoris [122]
Clostridium luticellarii [123]
Clostridium neuense [120]
Clostridium prolinivorans [124]
Clostridium swellfunianum [125]
Haloimpatiens Haloimpatiens lingqiaonensis [126]
Oceanirhabdus Oceanirhabdus sediminicola [127]
Proteiniclasticum Proteiniclasticum ruminis [128]
Defluviitaleaceae Defluviitalea Defluviitalea phaphyphila [129]
Defluviitalea Defluviitalea raffinosedens [130]
Lachnospiraceae Cellulosilyticum Cellulosilyticum ruminicola [131]
Natronincola Alkaliphilus Alkaliphilus crotonatoxidans [81]
Alkaliphilus halophilus [132]
Oscillospiraceae Acetanaerobacterium Acetanaerobacterium elongatum [133]
Ethanoligenens Ethanoligenens harbinense [134]
Hydrogenoanaerobacterium Hydrogenoanaerobacterium saccharovorans [135]
Petroclostridium Petroclostridium xylanilyticum [84]
Saccharofermentans Saccharofermentans acetigenes [136]
Peptococcaceae Desulfotomaculum Desulfotomaculum ferrireducens [137]
Peptostreptococcaceae Romboutsia Romboutsia sedimentorum [138]
Sporacetigenium Sporacetigenium mesophilum [139]
Terrisporobacter Terrisporobacter petrolearius [140]
Syntrophomonadaceae Syntrophomonas Syntrophomonas cellicola [141]
Syntrophomonas curvata [142]
Syntrophomonas erecta [143]
Thermosyntropha Thermosyntropha tengcongensis [144]
Thermohalobacter Brassicibacter Brassicibacter mesophilus [145]
Brassicibacter thermophilus [146]
Caloranaerobacter Caloranaerobacter ferrireducens [147]
Proteiniborus Proteiniborus ethanoligenes [148]
Vallitaleaceae Vallitalea Vallitalea okinawensis [149]
Xylanivirgaceae Xylanivirga Xylanivirga thermophila [83]
Eubacteriales Eubacteriaceae Peptacetobacter Peptacetobacter hominis [150]
Rhabdanaerobium Rhabdanaerobium thermarum [151]
Halanaerobiales Anoxybacter Anoxybacter Anoxybacter fermentans [152]
Moorellales Zhaonellaceae Zhaonella Zhaonella formicivorans [86]
Thermoanaerobact-erales Thermoanaerobacter-ium Thermoanaerobacterium Thermoanaerobacterium calidifontis [153]
Thermosediminiba-cterales Tepidanaerobactera-ceae Biomaibacter Biomaibacter acetigenes [154]
Negativicutes Hydrogenispora_o Hydrogenispora Hydrogenispora Hydrogenispora ethanolica [155]
Selenomonadales Selenomonadaceae Mitsuokella Selenomonas bovis [156]
Pectinatus Pectinatus brassicae [157]
Tissierellia Tissierellales Tissierellaceae Gudongella Gudongella oleilytica [158]
Keratinibaculum Keratinibaculum paraultunense [159]
Tepidimicrobium Tepidimicrobium xylanilyticum [160]
Sedimentibacter Sedimentibacter Sedimentibacter hongkongensis [161]
Kiritimatiellaeota Tichowtungiia Tichowtungiales Tichowtungiaceae Tichowtungia Tichowtungia aerotolerans [82]
Planctomycetes Planctomycetia Planctomycetales / Thermopirellula Thermopirellula anaerolimosa [162]
Proteobacteria Deltaproteobact- eria Desulfuromonadales Geobacteraceae Geobacter Geobacter anodireducens [163]
Geobacter soli [164]
Desulfovibrionales Desulfovibrionaceae Pseudodesulfovibrio Pseudodesulfovibrio indicus [165]
Syntrophobacterales Syntrophobacteraceae Syntrophobacter Syntrophobacter sulfatireducens [166]
Epsilonproteoba-cteria Campylobacterales Sulfurimonas Sulfurimonas Sulfurimonas hongkongensis [167]
Gammaproteoba-cteria Alteromonadales Shewanellaceae Shewanella Shewanella decolorationis [168]
Synergistetes Synergistia Synergistales Synergistaceae Lactivibrio Lactivibrio alcoholicus [169]
Thermodesulfo-bacteria Thermodesulfob-acteria_c Thermodesulfobacteriales Thermodesulfobacte-riaceae Thermodesulfatator Thermodesulfatator autotrophicus [170]
Thermotogae Thermotogae_c Kosmotogales Kosmotogaceae Kosmotoga Kosmotoga shengliensis [172-173]
Kosmotoga pacifica [173]
Thermotogales Fervidobacteriaceae Fervidobacterium Fervidobacterium changbaicum [174]
Verrucomicrobia Spartobacteria Chthoniobacterales Chthoniobacteraceae Terrimicrobium Terrimicrobium sacchariphilum [175]

厌氧微生物生长条件苛刻,对保藏技术和设施要求较高。目前,厌氧微生物模式物种主要保藏在德国、美国和日本(表 4)。其中,德国DSMZ收集保藏的厌氧微生物模式物种达到了1896个,是国际上最大的厌氧微生物模式物种保藏机构。2003年在“国家科技基础条件平台建设项目”的资助下,我国厌氧微生物资源保藏工作开始起步。目前,国内已保藏厌氧微生物1663株,模式物种515个(表 5,不同保藏中心可能存在相同物种)。依托农业部沼气科学研究所的中国厌氧微生物资源管理中心(前身为农业部厌氧微生物重点开放实验室),是国际菌种联盟(WFCC)的会员单位,具备新物种的保藏资质,目前已保藏厌氧微生物模式菌株334种,产甲烷古菌45种,厌氧微生物模式物种保藏量国内领先,在国际上排名第四(表 45)。

表 4. 全球厌氧微生物模式物种收集保存现状 Table 4. Summary of Anaerobic type strains in major culture collecting centers
Affiliations Abbreviations Type strain (Species)
German Collection of Microorganisms and Cell Cultures DSMZ 1896
Japan Collection of Microorganisms JCM 866
American Type Culture Collection ATCC 844
China Collection of Anaerobic Microorganismsa CCAM 334
Collection de L'Institut Pasteur Of Institut Pasteur CIP 228
Belgian Co-ordinated Collections of Microorganisms LMG/BCCM 137
Korean Collection for Type Cultures KCTC 126
NITE Biological Resource Center NBRC 122
Bioresources Collection and Research Center BCRC 52
China General Microbiological Culture Collection Centerc CGMCC 51
Culture Collection University of Gothenburgb CCUG 43
a Data from Global Catalogue Microorganisms website: http://gcm.wfcc.info/cc/ccam. b Data from CCUG website: https://www.ccug.se/collections/search?t=ANAEROBIC&collection=typestrains. c 47 type strains were collected from https://lpsn.dsmz.de/, another 4 (Methanolobus vulcani, M. oregonensis, M. bombayensis and Methanobacterium flexile) were from http://www.cgmcc.net/directory/index.html. Other data from LPSN website: https://lpsn.dsmz.de/

表 5. 我国厌氧微生物模式物种保藏现状 Table 5. Type species conservation of anaerobic microorganisms in mainland of China
Organization names Supporting institution Anaerobes Methanogens Main anaerobes resources Data sources
Strain number Type strain (species) Strain number Type strain (species)
China Collection of Anaerobic Microorganisms (CCAM) Biogas Institute of Ministry of Agriculture 630 334 73 45 Fermentative bacteria, Sulfate reducing bacteria, syntrophic bacteria and methanogens etc. http://gcm.wfcc.info/cc/ccam
China Center of Industrial Culture Collection (CICC) China National Research Institute of Food and Fermentation 309 19 0 0 Clostridia, Bifidobacterium http://sales.china-cicc.org/category.php?id=1&sh=go
Agricultural Culture Collection of China (ACCC)# China Institute of Agricultural Resources and Regional Planning 299 17 111 10 Methanogens and Anaerobic fermentation system and oil deposit Data from ACCC
China General Microbiological Culture Collection Center (CGMCC) Microbiology Research Institute of China Science Academy 143 51 12 11 Methanogens, fermentative bacteria http://www.nimr.org.cn/page/search/search1.jsphttps://lpsn.dsmz.de/
http://www.cgmcc.net/directory/mulu.php?number=&genus=&species=&yiming=%E7%94%B2%E7%83%B7&p=1
Guangdong Microbial Culture Collection Center (GDMCC) Guangdong Institute of Microbiology 168 48 0 0 Anaerobes form animal oral, gut or faeces Data from GDMCC
National Center For Medical Culture Collections (CMCC) National Institutes for Food and Drug Control 77 14 0 0 Clostridium tetani http://www.nimr.org.cn/page/search/search1.jsp
China Center for Type Culture Collection (CCTCC) Wuhan university 18 12 0 0 Bifidobacterium Saccharomyces http://www.nimr.org.cn/page/search/search1.jsp
Marine Culture Collection of China (MCCC) Third Institute of Oceanography, Ministry of Natural Resources 14 14 0 0 Marine anaerobes: hot aureus、oil spirochete etc. Data from MCCC
China Pharmaceutical Culture Collection (CPCC) Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College 5 5 0 0 Anaerobes from animal faeces Data from CPCC
The total only included anaerobic microorganisms, but not included aerotolerant and facultatively anaerobic microorganisms. #: Most of anaerobes come from CCAM.

4 展望

在上世纪60年代,知名微生物学家Roger Stanier悲观地认为“对细菌分类是无法实现的科学目标”[176]。但是Carl R. Woese根据核酸序列相似性提出的“三域学说”理论,近乎完美地解决了微生物的系统分类学问题[26]。基于这个思路揭示了地球上丰富的微生物多样性,发现了海量的“微生物暗物质”[28, 178]。这奠定了现代环境微生物学研究的基础,并开创了微生物分子生态学研究。当然,新认知也产生新的科学问题——微生物数量高达1030,物种数可能超过百万级的“微生物暗物质”是否可以被分离培养?笔者长期从事厌氧微生物资源与系统分类学研究,过去我们主要基于生理和形态学特征,并结合多种手段开展厌氧微生物的分离。例如,我们富集获得了65 ℃条件下降解甲醇产甲烷的富集培养物,但是没有如此高温甲基营养型产甲烷古菌的报道。因此,我们选择采用Hungate固体滚管技术,并降低到55 ℃培养1–2个月后挑取单菌落。从而顺利分离并鉴定到Methermicoccus shengliensis ZC-1[85]。后来采用先“分离”后“培养”的思路,先将菌液分分散到微孔中,再尝试添加不同类型的培养基进行富集培养,也先后获得了多个新属[86, 155]。但是,如果采用这些传统思路和方法,根据全球新物种的分离鉴定速度(102–3量级/年)来推测,那么似乎这又是“无法实现的科学目标”。

然而笔者相信,利用多学科交叉融合研发出的智能装备,开展厌氧微生物的全自动培养和分离将是未来的发展趋势;从群落、细胞、基因等多维度收集获得的大数据,用于训练开发人工智能,设计适用于未培养微生物的培养基和生长条件,动态监控并调整未培养微生物的生长状态,快速鉴定未培养微生物的功能。在微尺度、近原位和单细胞水平的微生物功能研究,将把传统的实验浓缩到一个芯片上完成。面对生长缓慢、对氧气敏感、营养条件苛刻的厌氧微生物的分离培养,耐心和坚持也是至关重要的。

致谢

感谢中国农业微生物菌种保藏管理中心的顾金刚和李世贵、中国海洋微生物菌种保藏管理中心的邵宗泽和李光玉、中国药用微生物菌种保藏管理中心刘红宇、广东省微生物菌种保藏中心邓名荣等老师提供厌氧微生物菌种保藏数据。感谢本课题组李江和杨敏所作的校对工作。

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