复配菌群降解煤产甲烷的宏基因组与宏转录组分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0050

摘要/Abstract

摘要：

【目的】芳香化合物代谢是微生物降解煤产甲烷的限制因素。为了提高煤的生物甲烷产量，经过富集、驯化获得煤降解产甲烷菌群（RI）和菲降解功能菌群，并通过二者配伍获得复配菌群（CM）。【方法】采用宏基因组与宏转录组相结合方法分析CM与RI的菌群结构及代谢途径的异同。【结果】复配后菌群的甲烷产量明显提高，增产114.55%。复配显著提高了芳香化合物降解菌的占比，如Pseudomonas的占比高达63.49%；同时提高了优势菌的代谢活性以及芳香化合物代谢途径中各关键酶的合成和表达。CM中芳香族化合物降解途径的基因丰度是RI的1.65倍，基因表达丰度是RI的6.34倍（P<0.05）。其中，关键酶EC:1.13.11.2基因丰度和表达丰度分别是RI的2.24、62倍。这些酶表达丰度的增加促使更多的芳香族化合物代谢为丙酮酸。复配同时增强了丙酮酸代谢为乙酰辅酶A过程的基因表达，该代谢途径中关键酶EC:1.2.4.1的表达丰度在CM中可达到RI的14.70倍。CM中各产甲烷途径的基因表达丰度也高于RI，是RI的2.66-7.10倍。【结论】复配富集了芳香化合物降解菌，并显著提高了芳香化合物降解产甲烷整个代谢途径中基因丰度，尤其是基因表达丰度，从而提高甲烷产量。

关键词: 煤层气, 复配菌群, 芳香族化合物, 宏基因, 宏转录

Abstract:

【Objective】Aromatic compound metabolism is considered a limiting factor for the generation of biogenic coalbed methane. To increase biomethane production from coal, the compounded microflora（CM）was obtained by compounding raw inoculum（RI）with ability of producing methane by degrading coal and phenanthrene-degrading functional microflora enriched from produced water. 【Method】A combined metagenome and metatranscriptome approach was used to analyze the differences in the community structure and metabolic pathways of CM and RI. 【Result】The methane yield of CM increased significantly by 114.55%. Compounding significantly increased the proportion of aromatics-degrading bacteria, such as Pseudomonas, accounting for 63.49%, and also increased the metabolic activity of the dominant bacteria as well as the synthesis and expression of the key enzymes in the metabolic pathway of aromatic compounds. Gene abundance of the aromatics-degrading pathway in CM was 1.65 times higher than that of RI, and the gene expression abundance was 6.34 times higher than that of RI（P<0.05）. The gene abundance and expression abundance of EC:1.13.11.2 were 2.24 and 62 times higher than those of RI, respectively. The increased expression abundance of these enzymes prompted the transformation of aromatic compounds to pyruvate. Compounding also enhanced the gene expression during the process of pyruvate metabolism to acetyl-CoA. The expression abundance of EC:1.2.4.1 reached 14.70-fold of the RI in CM. The gene expression abundance of the respective methanogenic pathways in CM was 2.66 to 7.10-fold of the RI. 【Conclusion】The compounding enriched the aromatics-degrading bacteria and significantly increased gene abundance, especially gene expression abundance, in the whole metabolic pathway of degrading aromatics to methane generation, resulting in the increased methane production.

Key words: coal bed methane, compounded microflora, aromatic compounds, metagenomic, metatranscriptomic

刘丁瑞, 郭红光, 弓凯仪. 复配菌群降解煤产甲烷的宏基因组与宏转录组分析[J]. 生物技术通报, 2024, 40(9): 270-281.

LIU Ding-rui, GUO Hong-guang, GONG Kai-yi. Metagenomic and Metatranscriptomic Analysis of Methanogenesis from Coal Degradation by Compounded Microflora[J]. Biotechnology Bulletin, 2024, 40(9): 270-281.

图/表 10

图1 CM和RI的生物甲烷产量

Fig. 1 Biomethane yield in CM and RI

图2 RI和CM在域水平的分布图 A：RI和CM在宏基因域水平的分布图；B：RI和CM在宏转录域水平的分布图

Fig. 2 Distribution of RI and CM at domain level A: Distribution of RI and CM at the level of metagenomic domains; B: Distribution of RI and CM at the level of metatranscriptomic domains

图3 RI和CM三组平行样在门水平（A）、细菌属水平（B）和古菌属水平（C）的微生物组成图 MG：宏基因组；MT：宏转录组，下同

Fig. 3 Microbial composition maps of three parallel groups of RI and CM samples at phylum（A）, bacterial genus（B）, and archaeal genus（C） MG: Metagenomic; MT: metatranscriptomic,the same below

图4 生物反应器RI和CM中一级（A）和二级（B）代谢系统之间基因存在和表达绝对丰度 Y轴刻度代表每个类别的序列丰度（带注释的序列）的对数

Fig. 4 Absolute abundance of gene presence and expression between primary（A）and secondary（B）metabolic systems in bioreactor RI and CM The Y-axis scale refers to the logarithm of sequence abundance（sequences with annotations）for each category

图5 三级通路上RI和CM代谢通路差异性分析 A:三级通路中宏基因KEGG火山图；B:三级通路中宏转录KEGG火山图；C:煤降解产甲烷代谢途径宏基因丰度比较；D：煤降解产甲烷代谢途径宏转录表达丰度比较

Fig. 5 Differential analysis of RI and CM metabolic pathways on tertiary pathways A:Volcano plot of KEGG in metagenomic in tertiary pathways. B: Volcano plot of KEGG in metatranscriptomic in tertiary pathways. C: Comparison of abundance of coal-degrading methane-producing metabolic pathways in metagenomic. D: Comparison of abundance of coal-degrading methane-producing metabolic pathways in metatranscriptomic

图6 芳香族化合物代谢途径及基因丰度和基因表达丰度图柱状图代表每个基因丰度和表达丰度的对数，下同

Fig. 6 Aromatic compound metabolic pathways and plots of gene abundance and gene expression abundance The bar graphs refer to the logarithm of abundance and expression abundance for each gene, the same below

图7 丙酮酸代谢途径及基因丰度和基因表达丰度图

Fig. 7 Pyruvate metabolic pathways and plots of gene abundance and gene expression abundance

表1 乙酰辅酶A主要生成途径及途径基因丰度和基因的表达丰度

Table 1 Main production pathways of acetyl coenzyme A and abundance of genes and expression abundance of genes in the routes

模块Module	RI-MG	RI-MT	CM-MG	CM-MT	功能描述 Function
M00422	7578	1453.027	2252	6649.955	Acetyl-CoA pathway, CO₂ => acetyl-CoA
M00569	15455.33	114.273	34242	2434.298	Catechol meta-cleavage, catechol => acetyl-CoA / 4-methylcatechol => propanoyl-CoA
M00036	79514	704.217	98381.33	1709.537	Leucine degradation, leucine => acetoacetate + acetyl-CoA
M00013	10171.33	16.876	21789.33	957.577	Malonate semialdehyde pathway, propanoyl-CoA => acetyl-CoA
M00032	32807.33	343.8167	37454.67	2953.905	Lysine degradation, lysine => saccharopine => acetoacetyl-CoA
M00307	70726	1861.692	64192	3460.332	Pyruvate oxidation, pyruvate => acetyl-CoA
M00086	42996	780.0583	50578	1389.26	Beta-oxidation, acyl-CoA synthesis

图8 甲烷代谢途径及基因丰度和基因表达丰度图

Fig. 8 Methane metabolic pathways and plots of gene abundance and gene expression abundance

表2 产甲烷各个途径的基因含量及表达

Table 2 Gene content and expression of each pathway of methane production

样本名称Sample	M00357	M00356	M00567	M00563
RI-MG	72573	48854	50116	46484
RI-MT	7123	1821	2040	1703
CM-MG	63086	28250	30324	28090
CM-MT	18937	12936	11077	10457

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