土壤生物多样性中参与顽固性物质降解的真菌的分离与筛选
Summary
在这里，我们提出了一个筛选土壤生物多样性的方案，以寻找参与顽固性物质降解的真菌菌株。首先，分离能够在腐植酸或木质纤维素上生长的真菌菌株。然后在酶测定和碳氢化合物和塑料等污染物上测试它们的活性。
Abstract
环境污染是一个日益严重的问题，识别生物修复过程中涉及的真菌是一项基本任务。土壤拥有令人难以置信的微生物生命多样性，可以成为这些生物修复真菌的良好来源。这项工作旨在通过使用不同的筛选测试来寻找具有生物修复潜力的土壤真菌。补充顽固物质作为唯一碳源的矿物培养基作为生长试验。首先，将土壤稀释液镀在培养皿上，用腐植酸或木质纤维素修饰矿物培养基。分离生长的真菌菌落并在不同的基质上进行测试，例如碳氢化合物（凡士林和用过的机油）的复杂混合物和不同塑料聚合物（PET，PP，PS，PUR，PVC）的粉末。定性酶测试与生长测试相关，以研究酯酶，漆酶，过氧化物酶和蛋白酶的产生。这些酶参与顽固物质的主要降解过程，并且它们被检查的真菌菌株的组成性分泌可能被利用用于生物修复。分离和测试了100多种菌株，并发现了几种具有良好生物修复潜力的分离株。总之，所描述的筛选测试是一种从土壤中鉴定具有生物修复潜力的真菌菌株的简单且低成本的方法。此外，还可以根据要求，通过在最少的培养基中添加其他顽固物质来定制不同污染物的筛选测试。
Introduction
土壤是地球上生命的基本组成部分，也是许多生态系统的基础。土壤中的矿物质，有机物和微生物可以被认为是一个系统，它们之间发生了密切的联系和相互作用。这些化合物的相互作用对陆地过程，环境质量和生态系统健康具有重要影响1。土壤污染在世界范围内造成了严重的环境问题。不分青红皂白、长期、过量地施用农药、石油产品、塑料等有毒物质，对土壤生态产生严重影响，从而改变土壤微生物群。土壤中的微生物群落由不同生理状态的各种生物组成，其中大多数是细菌和真菌。土壤中的许多污染物具有中长期稳定性，它们的持久性可以导致适应性机制的发展，使微生物能够利用顽固物质作为养分2，3。因此，这些微生物可以考虑用于生物修复技术。
生物修复试图通过利用微生物及其酶将废物降解或转化为毒性较小或无毒的化合物来减轻污染的影响。各种古菌，细菌，藻类和真菌都具有这种生物修复能力4。由于其特殊的生物降解作用，真菌是特别有前途的生物修复生物。它们可以使用其菌丝网络攻击不同的基质，使它们能够比其他微生物更有效地穿透土壤基质。此外，它们可以到达难以接近的间隙，其中污染物难以去除5，并且它们还可以承受低湿度水平6。此外，真菌合成不同的非特异性酶盒，通常降解天然顽固物质，如纤维素，木质素和腐植酸。那些缺乏目标基质的污染物可能参与各种顽固性污染物的降解，例如碳氢化合物，塑料和杀虫剂7，8，9，10。因此，尽管许多真菌物种已被报告为生物修复剂，但人们越来越有兴趣探索尚未研究的物种，以选择抗性污染物质生物修复的候选物种。已知具有生物修复特性的物种属于子囊菌门11，12，13，担子菌14，15和粘菌科。例如，众所周知，青霉属和曲霉属参与脂肪族烃13、不同塑料聚合物16、17、18、重金属19和染料20的降解。同样，对担子菌真菌（如Phanerochaete chrysosporium和Trametes versicolor）进行的研究表明，它们参与氧化顽固性物质，如芳香烃13和塑料21。参与生物降解过程的真菌的另一个例子是合子菌根瘤菌属，Mucor spp.和Cunninghamella spp.22，23。特别是，Cunninghamella能够氧化芳香烃，被认为是研究各种异种生物13产物解毒的模式生物。
有几种真菌酶参与顽固性物质24，25的主要降解过程，如酯酶，漆酶，过氧化物酶和蛋白酶。漆酶是在细胞中产生并随后分泌的含铜氧化酶，允许氧化各种酚类和芳香族化合物。它们可以降解邻位和对二酚，含氨基的酚，木质素和含芳基的二胺26。过氧化物酶使用过氧化氢作为介质来降解木质素和其他芳香族化合物。有许多不同的过氧化物酶，但降解有毒物质的最大潜力的是木质素过氧化物酶和锰过氧化物酶27。
酯酶和蛋白酶属于细胞外或外生细胞酶组，它们在其起源细胞外起作用，但仍与它们结合。这些酶可以催化大的顽固分子水解成较小的分子。由于其基质特异性低，这些酶可以在各种污染物的生物修复中发挥关键作用，例如纺织染料，纸浆和造纸工业释放的污水和皮革鞣制，石油产品，塑料和农药28，29，30。
已经发表了许多用于选择生物修复真菌菌株的筛选方法。例如，秸秆基琼脂培养基已被用于筛选多环芳烃（PAH）降解31中具有高潜力的白腐真菌;并将小块腐烂的木材放在麦芽提取物琼脂（MEA）上以分离腐烂木材的真菌32。然而，已经提出的大多数方法选择非常特定的真菌作为其感兴趣的活性。本研究提出了一种更广泛的方法，用于选择具有更广泛作用范围的土壤真菌。该方法依赖于将连续稀释的土壤样品初始镀层到用腐植酸或与抗生素混合的木质纤维素修饰的培养基上，以选择具有降解这些天然顽固物质的能力的真菌。事实上，腐植酸和木质纤维素是具有极强生物降解性的物质，因为它们具有非常复杂的分子结构，这使得它们成为被测真菌降解能力的优良指标33，34。随后，对在第一次测试中选择的真菌进行筛选，以识别那些有可能降解特定污染物的真菌，例如凡士林，用过的机油和塑料。最后，进行定性酶测试以检测能够产生参与顽固性物质生物降解过程的酶的真菌菌株。为此，进行蛋白酶和酯酶测试，同时使用没食子酸和愈创木酚作为丙酶和其他木质素分解酶产生的指标35，36。之所以使用这些底物，是因为已经发现真菌将其氧化成棕色形式的能力与拥有木质素溶解能力37，38，39之间存在很强的相关性。
通过这些方案，可以直接从土壤样品中分离出具有高降解潜力和广泛作用的真菌菌株。这些真菌菌株的分离可以帮助找到用于生物修复目的的新候选者。
Protocol
1. 选择能够从土壤中降解顽固物质的真菌菌株 抗生素溶液的制备。 将青霉素（50毫克/升）、链霉素（40毫克/升）、金霉素（40毫克/升）、新霉素（100毫克/升）和氯霉素（100毫克/升）放入250毫升去离子无菌水中。 在向抗生素溶液中加入氯霉素之前，将其溶解在3mL≥99%乙醇中。 将抗生素溶液置于磁力搅拌器（无热量）上，并在溶液内用磁力搅拌棒10分钟?…
Representative Results
选择性培养基方法（协议第1节）允许筛选土壤丰富的生物多样性，并选择具有高生物修复潜力的真菌。用腐植酸和木质纤维素培养基，分离出100多种真菌菌株。这些真菌产生的酶参与天然顽固材料的生物降解，其化学结构类似于许多污染物。然而，用选择性培养基分离的真菌菌株需要进一步筛选。具体而言，选择性测试分离了能够降解腐植酸和木质纤维素的菌株，生长测试筛选了那些能够使用特…
Discussion
土壤丰富的生物多样性是真菌的丰富来源，具有许多代谢能力，其中一些可能是生物修复的潜在候选者。选择性培养基测试（协议的第1部分）是分离能够在天然复合聚合物上生长的真菌作为其唯一碳源的易于执行和有效的方法。真菌可以产生细胞外、非特异性水解酶和氧化还原酶30 ，例如木质素分解酶laccases和过氧化物酶31。这些酶可以降解木质纤维素和腐植?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢帕维亚大学的Scuola di Alta Formazione Dottorale（SAFD）和Solveig Tosi教授为这项工作提供了机会。
Materials
| 96 microwell plate | Greiner bio-one | 650185 | |
| Agar | VWR | 84609.05 | |
| Bushnell-Haas Broth | Fluka | B5051 | |
| CaCl2 | Sigma-Aldrich | C5670 | |
| Chloroamphenicol | Eurobio | GABCRL006Z | |
| Chlortetracycline | Sigma-Aldrich | Y0001451 | |
| CoCl2·6H2O | Sigma-Aldrich | C8661 | |
| CuCl2·2H2O | Sigma-Aldrich | C3279 | |
| Ethanol | VWR Chemicals | 20821.296 | |
| FeCl3·6H2O | Sigma-Aldrich | 236489 | |
| Filter 0.2 µm | Whatman | 10462200 | |
| gallic acid | Sigma-Aldrich | G7384 | |
| Glass cover slips | Biosigma | VBS634 | |
| Glass vials 15 mL | SciLabware | P35467 | |
| guaiacol | Sigma-Aldrich | G5502 | |
| High-density polyethylene (HDPE) | Sigma-Aldrich | 434272 | |
| Humic acids | Aldrich Chemistry | 53680 | |
| K2HPO4 | Sigma-Aldrich | P8281 | |
| KH2PO4 | Sigma-Aldrich | P5655 | |
| Lignocellulose | / | / | Sterilized bioethanol production waste |
| L-shaped cell spreader | Laboindustria S.p.a | 21133 | |
| magnetic stirrer | A.C.E.F | 8235 | |
| Malt Extract Broth | Sigma-Aldrich | 70146 | |
| MgSO4·7H2O | Sigma-Aldrich | M2643 | |
| Micropipette 1000 μL | Gilson | FA10006M | |
| Micropipette 200 μL | Gilson | FA10005M | |
| MnCl2·4H2O | Sigma-Aldrich | M5005 | |
| Na2MoO4·2H2O | Sigma-Aldrich | M1651 | |
| NaCl | Sigma-Aldrich | S5886 | |
| Neomycin | Sigma-Aldrich | N0401000 | |
| Penicillin | Sigma-Aldrich | 1504489 | |
| peptone | Sigma-Aldrich | 83059 | |
| Polyethylene terephthalate (PET) | Goodfellow | ES306031 | |
| Petri dishes | Laboindustria S.p.a | 21050 | |
| Petrolatum (Paraffin liquid) | A.C.E.F | 009661 | |
| Potato Dextrose Broth | Sigma-Aldrich | P6685 | |
| Polystyrene (PS) | Sigma-Aldrich | 331651 | |
| Polyurethane (PUR) | Sigma-Aldrich | GF20677923 | |
| Polyvinyl chloride (PVC) | Sigma-Aldrich | 81388 | |
| Sterile falcon tube | Greiner bio-one | 227 261 | |
| Sterile glass vials 20 mL | Sigma-Aldrich | SU860051 | |
| Sterile point 1000 μL | Gilson | F172511 | |
| Sterile point 200 μL | Gilson | F172311 | |
| Sterile polyethylene bags | WHIRL-PAK | B01018 | |
| sterile syringe | Rays | 5523CM25 | |
| Streptomycin | Sigma-Aldrich | S-6501 | |
| Tween 80 | Sigma-Aldrich | P1754 | |
| Used engine oil | / | / | complex mixture of hydrocarbons, engine additives, and metals, provided by an Italian private company |
| Vials 50 mL | Sigma-Aldrich | 33108-U | |
| ZnCl2 | Sigma-Aldrich | Z0152 |
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Cite This Article
Temporiti, M. E. E., Daccò, C., Nicola, L. Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials. J. Vis. Exp. (183), e63445, doi:10.3791/63445 (2022).