Functional Microorganisms in Tomato Stalks/Maize Straws Co-compost Unveiled by Integrated Meta-omics
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摘要:
目的 探究番茄秸秆与玉米秸秆共堆肥发酵过程中的关键微生物及其功能，以进一步加速其发酵进程。 方法 以番茄秸秆和玉米秸秆为原料，二者按质量比3 ∶ 1混合进行条垛式堆肥，每周采集发酵堆20～50 cm深度样品检测木质纤维素酶活力的变化，以酶活力最高时期的代表性样品为研究对象，对其进行高通量焦磷酸测序和Orbitrap宏蛋白质组学研究。 结果 多数真菌隶属于子囊菌门Ascomycota，其中嗜热丝孢菌Thermomyces的相对丰度最高，约占真菌全部序列的70.5%，该属真菌主要分泌内切-1,4-β-木聚糖酶，是主要的半纤维素降解真菌。主要细菌为放线菌门Actinobacteria、变形菌门Proteobacteria和厚壁菌门Firmicutes，三者共占细菌总序列数的87.0%。其中，嗜热裂孢菌属Thermobifida和糖单孢菌属Saccharomonospora是主要的放线菌属，其相对丰度分别占全部细菌序列的16.5%和1.36%。Thermobifida是唯一的纤维素降解菌，鉴定到4种内切纤维素酶组分和3种外切纤维素酶组分；同时产生一种果胶裂解酶和一种β-木聚糖酶。Saccharomonospora分别产生一种β-木聚糖酶、两种丝氨酸蛋白酶和两种胰蛋白酶，在半纤维素的降解和蛋白质降解方面发挥重要作用。Idiomarina是变形菌门下的主要细菌属，平均相对丰度15.6%，主要参与蛋白质的降解。而厚壁菌门下的清野氏菌属Planifilum虽基因丰度不高（仅为1.23%），但却在半纤维素的降解过程中发挥重要作用。 结论 将高通量焦磷酸测序和Orbitrap宏蛋白质组学结合的整合宏组学方法在探明复杂生境中关键微生物的群落结构及功能方面具有可行性。 Abstract:Objective The integrated meta-omics was applied to reveal the functional microorganisms in the tomato stalks/maize straws compost. Method Sampling from the pile of a compost of tomato stalks and maize straws in the ratio of 3:1 (m: m) at the height of 20–50 cm was carried out once every week for lignocellulosic enzyme assay. When a peak enzymatic activity appeared, specimens were collected and the integrated meta-omic analysis that combined high-throughput pyrosequencing and Orbitrap studies was conducted. Result The greatest population of fungi in the compost belonged to the phylum Ascomycota, in which, Thermomyces was the most abundant accounting for 70.5%. Thermomyces is known to secret endo-1,4-β-xylanase that degrades hemicellulose in biomass. Of the total bacteria population, Actinobacteria, Proteobacteria, and Firmicutes phyla made up 87.0%, and the two genera of Actinobacteria, Thermobifida and Saccharomonospora, accounted for 16.5% and 1.36%, respectively. Thermobifida is capable of degrading cellulose with its 4 endoglucanases and 3 cellobiohydrolases. It also secrets enzymes associated with hemicellulose and pectate degradation. Saccharomonospora produces a β-xylanase, 2 serine proteases and 2 trypsins involved in the decomposition of hemicelluloses and proteins. Idiomarina was the major genus of Proteobacteria found in the compost with a relative abundance of 15.6%. It involves in protein degradation. Although low in abundance (merely 1.23%), Planifilum of Firmicutes phylum plays an import role in degrading hemicelluloses. Conclusion The integrated meta-omics that combined pyrosequencing and Orbitrap analysis provided a new tool for studying the community structure and functions of the microorganisms in a complex habitat such as a compost of different bio-materials. With the information obtained, ways to accelerate the composting process could be explored. -
Key words:
- Integrated meta-omics /
- tomato stalk /
- functional microorganism /
- Thermomyces
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图 1 堆肥温度（a）和主要酶活力（b）的变化
Figure 1. Changes on temperature （a） and activities of primary enzymes （b） in compost
图 2 第三周堆肥样品中细菌门（a）、真菌门（b）及主要属（平均相对丰度＞1%，c）组成
Figure 2. Microbes by bacterial phylum (a), fungal phylum (b) and genera with average relative abundance greater than 1% (c) in 3-week compost sample
图 3 不同时期所取堆肥样品在细菌（a）和真菌（b）属层次上的组成
注：横坐标中的W1、W2，…，W5分别代表第1、2…5周的样品。仅列出了平均相对丰度＞1%的属。
Figure 3. Genera of bacteria （a） and fungi （b） in samples collected at different times
Note: W1-W5 on X-axis represent samples taken in 1st, 2nd, … and 5th week. Genera with average relative abundances ＞1% are listed.
表 1 堆肥原料的理化性质
Table 1. Physiochemical properties of raw materials for composting
原料
Materials含水率
Moisture content/%pH 有机质
Organic matter/%有机碳
Organic carbon/%总氮
Total nitrogen/%C/N 番茄秸秆 Tomato stalk 78.3± 1.5 8.56± 0.21 96.7± 0.9 56.1± 0.5 2.83± 0.01 19.8± 0.2 玉米秸秆 Wheat straw 10.5± 0.6 7.20± 0.07 97.1± 1.1 56.3± 0.6 0.63± 0.02 89.3± 1.7 
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表 2 第3周堆肥样品中主要细菌属和真菌属分泌蛋白的功能鉴定
Table 2. Functions of enzymes secreted by dominant bacterial and fungal genera in samples taken in 3rd week
登录号
Accession number家族
Family描述
Description微生物
Microorganism光谱数
Spectrum count分子量
Molecular weight/kDa等电点
Isoelectric point纤维素酶 Cellulase Q08166 GH9 内切纤维素酶 Thermobifida fusca +++ 104.5 4.40 Q9KH72 GH6 纤维二糖水解酶 Thermobifida fusca +++ 48.6 4.72 Q47NH7 GH48 纤维二糖水解酶 Thermobifida fusca ++ 107.1 4.59 A0A147KMV8 GH9 内切纤维素酶 Thermobifida cellulosilytica +++ 102.4 4.32 A0A147KIK5 GH48 纤维二糖水解酶 Thermobifida cellulosilytica + 106.3 4.51 A0A068CD09 GH5 内切纤维素酶 Thermobifida fusca + 48.2 4.81 Q7X2N2 GH5 内切纤维素酶 Thermobifida fusca + 67.7 4.54 半纤维素酶 Hemicellulase A0A1I2PGX8 GH10 内切-1,4-β-木聚糖酶 Planifilum fulgidum + 52.8 5.58 I1CXX6 GH10 β-木聚糖酶 Saccharomonospora glauca + 49.3 5.14 P74912 GH10 β-木聚糖酶 Thermobifida alba + 52.4 5.66 O43097 GH11 内切-1,4-β-木聚糖酶 Thermomyces lanuginosus + 24.3 4.92 蛋白酶 Protease A0A094IT45 peptidase S1 类DegP周质丝氨酸内切蛋白酶 Idiomarina atlantica + 46.9 4.68 C7MXV7 peptidase S8 类枯草菌素丝氨酸蛋白酶 Saccharomonospora viridis ++ 54.1 4.55 C7MV18 Peptidase S1 胰酶 Saccharomonospora viridis + 39.4 4.96 C7MTG2 Peptidase S1 胰酶 Saccharomonospora viridis + 36.7 4.53 A0A1V9AC37 Peptidase S1 丝氨酸蛋白酶 Saccharomonospora sp. + 39.0 4.81 其他 Others Q47MW8 PL1 果胶裂解酶 Thermobifida fusca ++ 53.6 6.86 Q47PB9 AA10, CBM2 纤连蛋白 Thermobifida fusca + 46.8 4.65 Q47QG3 AA10 推测分泌的纤维素结合蛋白 Thermobifida fusca + 25.4 7.64 注：蛋白的相对丰度以平均“光谱数”来表征。符号+、++和+++分别代表平均光谱数＜4、[4,7]和＞7。
Note: Relative abundance of enzymes as evaluated by mean value of "spectrum count". Symbols of +, ++ and +++ indicate mean "spectrum count" ＜4, ranging 4 to 7, and ＞7, respectively.
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表 3 主要微生物属和酶活性的相关性
Table 3. Correlations between dominant genera and enzyme activities
项目 Items 内切纤维素酶 Endocellulase 木聚糖酶 Xylanase 蛋白酶 Protease 芽孢杆菌 Bacillus 0.949* 0.582 0.921* 嗜热真菌 Thermomyces 0.823 0.918* 0.507 海源菌属 Idiomarina 0.565 0.120 0.904* 注：表中仅列出了平均相对丰度＞1%，且与酶活性显著正相关的微生物属。*代表显著相关。
Note: Only genera with average relative abundance greater than 1% and significant correlation with enzymatic activities are listed. * indicates significant difference at P ＜0.05.
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