Effect of Microbial Agent on Reconstruction of Functional Microbes in Compost
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摘要: 堆肥产品不仅可以作为有机肥或土壤改良剂，还可以在包气带土层防护地下水污染的过程中起到微生物载体的作用.在堆肥中接种菌剂能够加速堆肥进程，促进堆肥材料的腐熟，但是也有研究认为接种剂与堆肥土著微生物的竞争会导致菌剂无法发挥作用.为了阐明菌剂与土著微生物之间的相互作用，采用宏蛋白质组学方法，分析餐厨垃圾堆肥接菌组（木质纤维素混合菌剂）和对照组（未接菌）中功能微生物群落和碳水化合物代谢途径的变化.结果表明：接菌组中假单胞菌目（Pseudomonadales）和散囊菌纲（Eurotiomycetes）菌群的相对丰度比对照组分别提高了12.5%和22.0%，成为优势细菌和真菌，二者在碳水化合物代谢活性上也成为优势菌群.菌剂主要是由芽孢杆菌目（Bacillales）和散囊菌纲的曲霉（Aspergillus）组成，曲霉因具有堆肥系统所需的木质纤维素分解能力而成为优势真菌，而菌剂中的芽孢杆菌虽然数量较多，但是缺乏堆肥系统所需的功能而无法成为优势细菌.餐厨垃圾中易降解物质分解过程中产生的有机酸会导致酸性环境，对照组中能够适应酸性环境的芽孢杆菌目和酵母菌纲（Saccharomycetes）是优势群落，添加菌剂后，堆肥系统中土著的假单胞菌目和散囊菌纲具有较高的碳水化合物代谢活性和多种有机酸转化通路，因此在与菌剂中的芽孢杆菌目和土著的芽孢杆菌及酵母菌的竞争中成为优势菌群.研究显示，外源菌剂与土著微生物之间以及各土著微生物之间都会发生竞争作用，能否成为优势菌群取决于是否适应堆肥底物新陈代谢的变化，因此只要选择好菌剂的功能和接种时机，菌剂就能够发挥原有的作用.Abstract: Compost products could be used as organic fertilizer or soil conditioner, and could play an important role in the protection of groundwater as microbial carrier in the vadose zone. The present study is aimed at exploring the mechanism of microbial agents for improving the composting process. Metaproteomics was employed to investigate the influence of microbial agents on the structure of functional microbial communities and carbohydrate metabolism in food waste composting. The results showed that the relative abundance of order Pseudomonadales and class Eurotiomycetes increased by 12.5% and 22.0% respectively, and they replaced Bacilli and Saccharomycetes as dominant microbial communities with respect to total protein abundance and carbohydrate metabolic activity in the treatment group. The microbial agent was composed of Aspergillus and order Bacillales, and the Aspergillus was the main player in lignocellulose decomposition in food waste composting. This suggested that the Aspergillus became the dominant communities due to its roles in lignocellulose degradation, whereas the order Bacillales could not become the dominant bacteria because of competition interaction between microbial agent and indigenous microorganisms. The organic acids produced by decomposition of easily degradable organic matter in food waste could result in an acidic environment. In the control group, the Bacilli and Saccharomycetes were able to adapt to the acidic conditions and became the dominant communities. Whereas in the treatment group, the indigenous Pseudomonadales and Eurotiomycetes had high carbohydrate metabolic activity and various metabolic pathways of organic acids, so they replaced Bacilli and Saccharomycetes as dominant communities. The effectiveness of exogenous microbial agents and indigenous microorganisms depends on their adaptation to the metabolism in the composting process.
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Key words:
- compost /
- microbial agent /
- metaproteomics /
- carbohydrate metabolism /
- lignocellulose degradation
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图 1 试验反应器示意
注：1—气泵；2—气体流量计；3—反应堆；4—温差控制装置；5—恒温箱；6—气体测定仪；7—过滤装置.
Figure 1. Schematic diagram of experimental reactor
图 3 细菌和真菌群落组成和相对丰度
Figure 3. Bacterial and fungal community composition and relative abundance in the metaproteome
图 4 微生物群落的碳水化合物代谢路径图
注：仅在接菌组发现的酶用红色高亮数字序号表示，仅在对照组发现的酶用蓝色高亮数字序号表示，在接菌组和对照组都存在的酶用绿色高亮数字序号表示.
1—内切葡聚糖酶；2—β-葡糖苷酶；3—漆酶；4—D-木糖还原酶；5—D-木酮糖激酶；6—1, 4-α-葡聚糖分支酶；7—葡萄糖-1-磷酸腺苷酰基转移酶；8—1, 3-β-葡聚糖合酶；9—葡萄糖-6-磷酸异构酶；10—6-磷酸果糖激酶；11—果糖二磷酸醛缩酶；12—磷酸丙糖异构酶；13—甘油醛-3-磷酸脱氢酶；14—磷酸甘油酸激酶；15—依赖2, 3-磷酸甘油酸的磷酸甘油酸变位酶；16—烯醇酶；17—磷酸烯醇丙酮酸羧激酶；18—磷酸烯醇丙酮酸羧化酶；19—果糖-1, 6-二磷酸酯酸；20—丙酮酸脱氢酶；21—丙酮酸脱氢酶；22—乙醛脱氢酶；23—磷酸乙酰转移酶；24—丙酮酸磷酸双激酶；25—丙酮酸羧化酶；26—柠檬酸合酶；27—乌头酸水合酶；28—异柠檬酸脱氢酶；29—2-酮戊二酸脱氢酶；30—琥珀酰辅酶A连接酶；31—琥珀酸脱氢酶；32—苹果酸脱氢酶；33—异柠檬酸裂合酶；34—苹果酸合酶；35—6-磷酸葡糖酸脱氢酶；36—转醛醇酶；37—磷酸戊糖变位酶；38—磷酸葡萄糖胺变位酶；39—UDP-葡萄糖-6-脱氢酶40—异丙醇脱氢酶；41—丙酮醛合酶；42—2-柠檬酸甲酯合成酶；43—乙酰乙酰辅酶A还原酶；44—肌糖-2-脱氢酶；45—磷脂酰肌醇-4-激酶.Figure 4. Depiction of the carbohydrate metabolic characteristics of microbial communities inferred from the metaproteome
图 5 菌剂群落与土著群落的相互作用
注：椭圆框表示优势菌群，虚线方框表示菌群功能.
Figure 5. The interaction of microbial communities in microbial agent and indigenous communities
表 1 蛋白质数据库搜索参数
Table 1. The search parameters of protein database
参数名称 参数设置 Enzyme(酶切信息) Trypsin Fixed modification (固定修饰) carbamidomethyl (C) Variable modification (可变修饰) Gln→Pyro-Glu (N-term Q)，Oxidation (M) Peptide mass tolerance (一级质谱精度) ±15 ppm Fragment mass tolerance (二级质谱精度) ±20 mmu Significance threshold (显著阈值) P < 0.05 
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表 2 堆肥样品中检测到的碳水化合物酶
Table 2. Number of carbohydrate metabolic enzymes which were detected in the metaproteome
微生物分类 碳水化合物酶数量/个 接菌组 对照组 Pseudomonadales 17 5 Enterobacteriales 12 5 other Gammaproteobacteria 10 8 Betaproteobacteria 3 3 Alphaproteobacteria 6 7 Bacilli 2 10 Clostridia 2 2 Actinobacteria 6 1 other Bacteria 5 5 Saccharomycetes 2 12 Schizosaccharomycetes 4 3 Eurotiomycetes 9 3 Sordariomycetes 1 2 Basidiomycota 1 0
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表 3 堆肥样品中检测到的木质纤维素酶
Table 3. Lignocellulolytic enzymes detected in the metaproteome
处理组 酶的名称 酶的种类 微生物名称 微生物分类(纲) 接菌组 木酮糖激酶 半纤维素酶 Aspergillus terreus Eurotiomycetes 内切葡聚糖酶 纤维素酶 Thermobifida fusca Actinobacteria β-葡糖苷酶 纤维素酶 Aspergillus clavatus Eurotiomycetes β-葡糖苷酶 纤维素酶 Aspergillus clavatus Eurotiomycetes α/β-葡糖苷酶 纤维素酶 Aspergillus fumigatus Eurotiomycetes β-葡糖苷酶 纤维素酶 Aspergillus nidulans Eurotiomycetes 漆酶 木质素酶 Melanocarpus albomyces Sordariomycetes 对照组 木糖还原酶 半纤维素酶 Aspergillus fumigatus Eurotiomycetes 内切葡聚糖酶 纤维素酶 Bacillus subtilis Bacilli
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