Role of Direct Interspecies Electron Transfer in the Methane Production of the Peatland under Warming

doi:10.13560/j.cnki.biotech.bull.1985.2025-1382

Abstract

Abstract:

Objective To explore the role of direct interspecies electron transfer (DIET) mechanisms in methane production from the Zoige peatland on the Qinghai-Tibet Plateau under warming conditions, and to provide DIET-based methane mitigation strategies in peatlands under global warming. Method Soils growing three dominant plant species in the Zoige peatland on the Tibetan Plateau were sampled to construct methanogenic and electrochemical enrichments, which were amended with pectin or cellulose and incubated at 18 ℃. By adding biochar (as an electron transfer shuttle) and electrochemical experiments, the effects of DIET on methane production from plant organic complexes and its response to warming (28 ℃) were investigated. Result The addition of biochar into the soil enrichments elevated methane production from cellulose and pectin by 1.1 to 9.8-fold and increased maximum methanogenic rates by 1.4 to 12.3-fold, and the temperature sensitivity of methanogenesis was doubled following biochar addition. Chronoamperometry detected extracellular electrons generated by soil bacteria in the electrochemically enriched cultures, with a current density of 33.7 μA/cm², and current consumption coupled methanogenesis by methanogenic archaea (21.2 μA/cm²) in electrochemical enrichments. 16S rRNA gene diversity analysis revealed that biochar selectively enriched the electroactive bacterial family Geobacteraceae, along with methanogenic archaea communities including the families Methanosarcinaceae, Methanomethylophilaceae (electroactive uncharacterized), and the uncultured methanogen group Rice Cluster Ⅱ. Co-occurrence network analysis further indicated significantly positive correlations between Geobacteraceae or Cellulomonadaceae and Rice Cluster Ⅱ, suggesting that they implemented DIET-based conversion of the plant organic complexes to methane. Conclusion DIET plays a main role in methane production from plant organic complexes in Zoige peatland, particularly under the warming scenarios, thereby inhibiting interspecies electron transfer should be considered as one of the key strategies for methane mitigation in peatlands.

Key words: cold peatland, methane production, electroactive microorganisms, electron transfer, warming

WANG Xue-meng, DONG Xiu-zhu, XUE Kai, LI Ling-yan. Role of Direct Interspecies Electron Transfer in the Methane Production of the Peatland under Warming[J]. Biotechnology Bulletin, 2026, 42(2): 77-88.

Figures/Tables 10

Table 1 Geographic characteristics of the soils sampled from Zoige peatland of the Tibetan Plateau in this study

优势植物

Dominating plant

经纬度

Longitude and latitude

土壤温度

Soil temperature （℃）

静水深度

standing water depth （cm）

刚毛荸荠 E. valleculosa

33°55′12″ N， 102°49′12″ E

17.7

28.7

西藏嵩草 K. tibetica

32°20′37″ N， 102°26′36″ E

18.3

28.7

木里苔草 C. muliensis

32°33′6″ N， 102°21′15″ E

15.5

30.4

Table 2 Inorganic element-vitamin-trace element solution

无机元素储液 Inorganic element solution		维生素储液 Vitamin solution		微量元素储液 Trace element solution
试剂 Reagent	浓度 Concentration （g/L）	试剂 Reagent	浓度 Concentration （g/L）	试剂 Reagent	浓度 Concentration （g/L）
NH₄Cl	6	生物素	0.002	FeCl₂·4H₂O	2
NaCl	6	叶酸	0.002	ZnCl₂	0.05
CaCl₂·2H₂O	0.2	VB₆	0.01	MnCl₂·4H₂O	0.05
MgCl₂·6H₂O	2	VB₂	0.005	CuCl₂·2H₂O	0.03
		VB₁	0.005	（NH₄）₆Mo₇O₂₄·4H₂O	0.05
		VB₃	0.005	AlCl₃	0.05
		VB₁₂	0.005	CoCl₂·6H₂O	0.2
		泛酸	0.005	H₃BO₃饱和溶液	1 mL
		对氨基苯甲酸	0.005	浓HCl	1 mL

Fig. 1 Anaerobic methanogenesis enrichment and cultures of the soils from Zoige peatlandA total of 2.5 g soils sampled from E. valleculosa, K. tibetica, and C. muliensis were inoculated into 25 mL pre-reduced basic medium (-) and amended with pectin or cellulose plus biochar (0.1% and 1.0%, W/V). The soil enrichments were incubated anaerobically at 18 ℃ for 180 d, CH4 in the headspace was measured. Data presented in the figure was acquired from E. valleculosa soil. About 10% (V/V) of the biochar was transferred from the enrichments of each generation for subculture. The group without biochar amended (0.0%) was subcultured with 10% (V/V) of the enrichment. The 4th generation soil enrichments were incubated anaerobically at 18 ℃ and 28 ℃ respectively. Soil enrichments from each generation were photographed at the 180-day incubation

Fig. 2 Effects of biochar on methane production from pectin or cellulose enrichments of peatland soilsAbout 10% (V/V) of the biochar was transferred into fresh medium from the pectin- or cellulose-amended microcosms for subculture. The group without biochar amendment (0.0%) was subcultured with10% (V/V) of the enrichment as the control group. The soil enrichments were incubated anaerobically at 18 ℃ and CH4 in headspace of the soil enrichments from under E. valleculosa (A, D), K. tibetica (B, E), or C. muliensis (C, F) was measured during incubation. The maximum methanogenic rates (Vmax, μmol/d) were calculated from Equation 1. Triplicate cultures were assayed, and the average and standard errors (n=3) are shown

Fig. 3 Electrochemical experiments from peatland soil enrichmentsElectrochemical soil cultures were constructed by inoculating 9 g soil of E. valleculosa into 90 mL pre-reduced basic medium amended with pectin inside H-cells. The electrochemical chambers without soil inoculation were included as an abiotic control, and anaerobically incubated at 18 ℃. A: The cathodic chambers were set at a potential of -0.4 V (vs. Ag/AgCl), 10 mmol/L BrES was added at day 15 of the incubation (black arrow indicated), and none amendment was included as a blank control. B: The anodic chambers were set at a potential of +0.4 V (vs. Ag/AgCl), 1 mg/mL of ampicillin and 2 mg/mL of kanamycin were amended every six days during incubation, and none amendment (-) was included as a blank control. The contents of CH4 and H2 in headspace of enrichments, and current were monitored during the incubation. Triplicate cultures were assayed, and the average and standard errors (n=3) are presentverifying the effects of DIET on methanogenesis

Fig. 4 Relative abundances of methanogenic archaea and bacteria in soil pectin- and cellulose-enrichments of peatland methanogenic archaeaTotal DNA was extracted from the biochar in pectin- and cellulose-amended enrichments with the highest methane production, which were from the 2nd and 3rd generations in Figure 2. 16S rRNA gene amplicons were sequenced, and the relative abundances of methanogens and bacteria were analyzed. The methanogens family (A, B) and top 15 of the most abundant bacterial family (C, D) from the enrichments of E. valleculosa, K. tibetica, or C. muliensis are shown. -B: without biochar amendment; BS: the supernatant of biochar plus pectin soil enrichments; BP: the particle of biochar plus pectin soil enrichments. Triplicate cultures were assayed, and the average (n=3) is shown

Fig. 5 Co-occurred network analysis of methanogens and bacteria in peatland soilsTotal DNA was extracted from the biochar of pectin or cellulose amended soil enrichments, respectively, and were used for 16S rRNA amplicon sequencing. Co-occurrence network was constructed incorporating the methanogens family and top 15 of the most abundant bacterial family (n=96). Pink lines indicate significant positive correlations, while green lines indicate significant negative correlations

Fig. 6 Effects of temperature increase on biochar-mediated methane production by soil microorganisms in peatlandAbout 10% (V/V) of the biochar extracted from the third-generation soil enrichments of E. valleculosa (A, D), K. tibetica (B, E), and C. muliensis (C, F) were inoculated into fresh methane-producing culture, which then were incubated at 18 ℃ and 28 ℃, respectively. CH4 yield in the headspace of the soil enrichments was measured at given time. The data are present as Mean ± SE (n=3)

Table 3 Effects of biochar on methanogenic rate from pectin amended soil enrichments (μmol/d)

土壤富集物 Soil enrichments	18 ℃ 产甲烷速率 Methanogenic rate at 18 ℃			28 ℃ 产甲烷速率 Methanogenic rate at 28 ℃
土壤富集物 Soil enrichments	-	0.1%	1.0%	-	0.1%	1.0%
刚毛荸荠 E. valleculosa	1.11	2.95	4.07	1.13	5.85	9.24
西藏嵩草 K. tibetica	0.47	0.74	1.09	0.54	1.39	2.43
木里苔草 C. muliensis	1.05	1.27	2.34	1.32	2.66	4.97

Table 4 Effects of biochar on methanogenic rate from cellulose amended soil enrichments (μmol/d)

土壤富集物 Soil enrichments	18 ℃产甲烷速率 Methanogenic rate at 18 ℃			28 ℃ 产甲烷速率 Methanogenic rate at 28 ℃
土壤富集物 Soil enrichments	-	0.1%	1.0%	-	0.1%	1.0%
刚毛荸荠 E. valleculosa	1.67	1.92	2.67	2.82	4.57	5.86
西藏嵩草 K. tibetica	0.97	1.47	4.33	0.97	4.34	10.95
木里苔草 C. muliensis	0.94	1.11	1.59	1.01	2.25	3.26

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