碳源对产甲烷菌群调控及产甲烷能力的影响

doi:10.13560/j.cnki.biotech.bull.1985.2024-1180

摘要/Abstract

摘要：

目的产甲烷菌是厌氧消化产甲烷过程的关键功能菌群。为了获得高效的产甲烷菌群，探究不同碳源对菌群产甲烷潜力的调控作用，以及对产甲烷菌丰度和群落结构的影响。方法设计A组（CH₃COONa和CH₃OH）、B组（CH₃COONa）和C组（CH₃COONa和HCOONa）3组不同碳源培养基，分别以厨余垃圾厌氧消化液（FW）和污泥悬浮液（SS）为接种物富集产甲烷菌。采用气相色谱、实时荧光定量PCR、荧光显微观察和高通量扩增子测序技术，分析产甲烷菌群的性能及其多样性组成。结果同一接种物下，A、B、C三组碳源富集的菌群产甲烷能力呈递减趋势。其中，A组的1A-2在49 d内累积CH₄产量最高，单位体积（L）培养基中，每克可用碳元素（C）产生的CH₄达到35.9 mL。mcrA基因拷贝数和荧光显微观察表明，产甲烷菌的丰度总体呈现A组˃B组˃C组。A组碳源对甲基营养型产甲烷菌具有定向富集作用；B组碳源的添加使乙酸营养型Methanothrix的相对丰度显著高于其他两组；而C组碳源则显著增加了氢营养型产甲烷菌的相对丰度。结论碳源种类显著影响富集菌群中产甲烷菌和细菌的多样性、丰富度及群落结构。以CH₃COONa和CH₃OH（A组）为碳源富集的菌群中产甲烷菌的丰度最高，主要包括Methanothrix、Methanoculleus、Methanomethylovorans、Candidatus Methanoplasma和Methanosarcina，且产甲烷能力最强。A组碳源通过富集互营细菌，协调产甲烷菌的代谢途径分配，促进直接种间电子传递（DIET）过程，显著提升CH₄产量。

关键词: 厌氧培养, 产甲烷菌, 实时荧光定量PCR, 荧光观察, 扩增子测序

Abstract:

Objective Methanogens play a pivotal role in methanogenesis during anaerobic digestion. To obtain an efficient methanogenic microbial community, the regulatory effects of different carbon sources on the methane production potential, methanogen abundance, and microbial community structure were investigated. Method Three distinct carbon source culture media (Group A: CH₃COONa and CH₃OH;Group B: CH₃COONa; Group C: CH₃COONa and HCOONa) were designed to enrich methanogenic communities by inoculating with anaerobic digestion liquid from kitchen waste (FW) and activated sludge suspension (SS). The performance and diversity composition of methanogenic microbial community were analyzed using gas chromatography, real-time quantitative PCR (qPCR), ﬂuorescence microscopy, and amplicon sequencing. Result The microbial communities enriched by carbon sources A, B, and C demonstrated a descending order of methane yield when inoculated with the same inoculum. Group 1A-2 achieved the highest cumulative methane yield over 49 d, with a methane yield of 35.9 mL per gram of available carbon (C) in a unit volume (L) of culture medium. The copy number of mcrA gene and fluorescence microscopy observations indicated a successive decrease in methanogen abundance in communities enriched by carbon sources A, B, and C. Specifically, carbon source A enriched methylotrophic methanogens, while carbon source B significantly boosted the relative abundance of aceticlastic Methanothrix compared to the other groups. Carbon source C notably enhanced the relative abundance of hydrogenotrophic methanogens. Conclusion The type of carbon source significantly influence the diversity, richness, and community structure of methanogens and bacteria in the enriched microbial communities. The microbial communities enriched with CH₃COONa and CH₃OH (Group A) showed the highest methanogen abundance, mainly including Methanothrix, Methanoculleus, Methanomethylovorans, Candidatus Methanoplasmaand Methanosarcina, and demonstrated the highest methanogenic capacity. Carbon source A promotes methane (CH₄) production by enriching syntrophic bacteria, balancing the metabolic pathway distribution of methanogens, and facilitating direct interspecies electron transfer (DIET).

Key words: anaerobic culture, methanogens, real-time quantitative PCR, fluorescence observation, amplicon sequencing

安苗苗, 蔺祥淏, 梁瑞娜, 赵国柱. 碳源对产甲烷菌群调控及产甲烷能力的影响[J]. 生物技术通报, 2025, 41(6): 355-366.

AN Miao-miao, LIN Xiang-hao, LIANG Rui-na, ZHAO Guo-zhu. Effect of Carbon Source in Methanogenic Communities Regulation and Methane Production Capacity[J]. Biotechnology Bulletin, 2025, 41(6): 355-366.

图/表 10

表1 产甲烷菌群富集培养物编号

Table 1 Identification numbers of methanogenic microbial community enrichment cultures

富集代数 Enrichment algebra	接种FW（代号1） Inoculated FW （code 1）			接种SS（代号2） Inoculated SS （code 2）
富集代数 Enrichment algebra	碳源A Carbon source A	碳源B Carbon source B	碳源C Carbon source C	碳源A Carbon source A	碳源B Carbon source B	碳源C Carbon source C
第一代（代号-1） First generation （code-1）	1A-1	1B-1	1C-1	2A-1	2B-1	2C-1
第二代（代号-2） Second generation （code-2）	1A-2	1B-2	1C-2	2A-2	2B-2	2C-2
第三代（代号-3） Third generation （code-3）	1A-3	1B-3	1C-3	2A-3	2B-3	2C-3

图1 产甲烷菌群富集培养物FW：厨余垃圾厌氧消化液；SS：污泥悬浮液

Fig. 1 Enrichment cultures of methanogenic microbial communityFW: Anaerobic digestion liquid from kitchen waste. SS: Suspension from activated sludge

图2 富集菌群的累积CH4产量mL/（g∙L-1 C）：单位体积（L）培养基中，每克可用碳元素（C）对应的CH4产量（mL）

Fig. 2 Cumulative methane yields by enriched methanogenic microbial communitymL/(g‍∙‍L-1 C): The methane yield (mL) per gram of available carbon (C) in a unit volume (mL) of culture medium

图3 富集菌群mcrA基因PCR扩增结果

Fig. 3 PCR amplification results of mcrA gene in methanogenic microbial community

图4 富集菌群的mcrA基因定量分析A：重组质粒的阳性克隆筛选；B：基于mcrA基因的荧光定量PCR标准曲线；C：第二代富集菌群mcrA基因拷贝数copies/（g∙L-1 C）；copies/（g∙L-1 C）：单位体积（L）培养基中，每克可用碳元素（C）对应的mcrA基因拷贝数（copies）

Fig. 4 Quantitative analysis of mcrA gene in enriched methanogenic microbial communityA: Positive clone screening of recombinant plasmid. B: Standard curve of fluorescence quantitative PCR based on mcrA gene. C: Copy number of mcrA gene in the second generation of methanogenic microbial community enrichment cultures, i.e., copies/(g∙L-1 C). copies/(g∙L-1 C): The copy number of mcrA gene per gram of available carbon (C) in a unit volume (L) of culture medium

表2 不同稀释倍数下标准质粒的实时荧光定量PCR检测精度

Table 2 Real-time PCR detection precision of standard plasmids at different dilution ratios

稀释倍数 Dilution ratio	拷贝数 Copy number	C_t值（mean ± SD） Cycle threshold	变异系数 Coefficient of variation （%）
1.00×10^-7	6.53×10³	31.70 ± 0.70	2.21
1.00×10^-6	6.53×10⁴	27.65 ± 0.29	1.05
1.00×10^-5	6.53×10⁵	23.24 ± 0.66	2.84
1.00×10^-4	6.53×10⁶	17.96 ± 0.25	1.40
1.00×10^-3	6.53×10⁷	13.92 ± 0.19	1.36
1.00×10^-2	6.53×10⁸	10.78 ± 0.26	2.39
1.00×10^-1	6.53×10⁹	6.00 ± 0.00	0.00

图5 富集菌群在420 nm波长下的荧光显微观察结果

Fig. 5 Fluorescence microscopy-observed results of enriched methanogenic microbial community at 420 nm

表3 不同碳源富集菌群中产甲烷菌和细菌的Alpha多样性指数

Table 3 Alpha diversity indexes of methanogenic archaea and bacteria in microbial community enriched by different carbon sources

Category	Sample	Observed_species	Shannon	Simpson	Pielou	Chao1	ACE	Goods_coverage
产甲烷菌 Methanogens	1A-2	219	4.303	0.911	0.553	291.163	315.584	0.999
	1B-2	204	3.631	0.844	0.483	231.841	254.647	0.999
	1C-2	235	3.709	0.794	0.461	290.724	304.491	0.999
细菌 Bacteria	1A-2	1 644	6.622	0.905	0.620	1 646.686	1 664.078	0.999
	1B-2	1 010	4.234	0.731	0.424	1 028.896	1 062.259	0.997
	1C-2	975	5.925	0.944	0.597	1 046.697	1 079.658	0.995

图6 不同碳源富集菌群中产甲烷菌和细菌的物种组成基于Bray-Curtis距离的产甲烷菌（A）和细菌（B）群落PCoA分析；C：产甲烷菌群落在属水平的组成；D：细菌群落在门水平的组成；E：细菌相对丰度前100属的物种组成和系统发育树分析

Fig. 6 Species composition of methanogenic archaea and bacteria in microbial community enriched by different carbon sourcesPrincipal coordinate analysis (PCoA) of the methanogenic archaeal community (A) and bacterial community (B) based on Bray-Curtis dissimilarity. C: Distribution of methanogenic archaeal communities at the genus level. D: Distribution of bacterial communities at the phylum level. E: Cladogram describing the taxonomic community composition and relative abundances of bacteria at the level of the top 100 genera

表4 不同碳源富集菌群中产甲烷菌的相对丰度和营养类型占比

Table 4 Relative abundances and trophic type proportions of methanogenic archaea in microbial community enriched by different carbon sources

产甲烷菌营养类型 Methanogens trophic types	产甲烷菌 Methanogens	相对丰度 Relative abundance
产甲烷菌营养类型 Methanogens trophic types	产甲烷菌 Methanogens	1A-2	1B-2	1C-2
乙酸营养型 Aceticlastic type	Methanothrix	0.358	0.448	0.141
占比 Proportion （%）		35.8	44.8	14.1
氢营养型 Hydrogenotrophic type	Methanoculleus	0.108	0.296	0.282
	Methanobacterium	0.006	0.081	0.223
	Methanobrevibacter	0	0	0.197
	Methanospirillum	0.033	0.063	0.041
	Candidatus Methanogranum	0.023	0	0
	Methanothermobacter	0	0	0.009
占比 Proportion （%）		17.0	44.0	75.1
甲基营养型 Methylotrophic type	Candidatus Methanoplasma	0.107	0.016	0.066
	Methanomethylovorans	0.171	0.010	0.003
	Methanomethylophilus	0.012	0.003	0.001
	Methanomassiliicoccus	0.001	0	0
占比 Proportion （%）		29.2	2.9	7.0
多营养型 Multitrophic types	Methanosarcina	0.082	0	0
占比 Proportion （%）		8.2	0	0

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