酸性矿山废水对成熟期水稻根区理化因子及固氮微生物的影响

doi:10.13560/j.cnki.biotech.bull.1985.2023-1146

摘要/Abstract

摘要：

【目的】 酸性矿山废水（acid mine drainage, AMD）是一类低pH、高硫酸盐浓度和重金属富集的废水。探究成熟期水稻根区土壤固氮菌群落的丰度和组成及其对AMD的响应，并阐明土壤固氮菌群落结构变化的主要驱动因素。【方法】 对取自安徽省铜陵市矿区受AMD污染和未受污染的稻田土壤进行水稻盆栽试验，设置3组不同处理（A：AMD浇溉污染土、B：清洁水浇灌污染土、CK：清洁水浇灌未污染土），采用nifH基因高通量测序技术分析不同处理下的成熟期水稻根区土壤固氮菌群落特征。【结果】 AMD污灌使得水稻根区土壤中SO₄^2-、NO₃^-、重金属的含量显著上升，土壤酸化且固氮菌群落的多样性下降。水稻根区土壤的优势固氮菌包括Anaeromyxobacter、Geobacter等，CK处理中富集的固氮菌群数量显著高于A、B处理，且B处理中主要富集疣微菌门，CK处理中主要富集变形菌门。pH和重金属Cu、Pb、Zn是驱动水稻根区土壤固氮菌群落结构的主要因素。具有硫还原功能的Desulfovibrio和Desulfurivibrio对氮的变化贡献明显。【结论】 AMD对水稻根区土壤化学性质和固氮菌产生显著影响，恢复清洁水灌溉可促进固氮菌的恢复。

关键词: AMD, 水稻根区土壤, 根区固氮微生物, 氮, 重金属

Abstract:

【Objective】 Acid mine drainage（AMD）is a kind of wastewater with low pH, high sulfate concentration and heavy metal enrichment. This study is aimed to investigate the abundance and composition of azotobacter community in the root zone of rice at mature stage and its response to AMD, and to elucidate the main driving factors of the change of soil azotobacter community structure. 【Method】 Rice pot experiment was carried out on paddy soil contaminated and uncontaminated by AMD from Tongling mining area of Anhui province, and three different treatments were set up（A: AMD irrigated contaminated soil； B: clean water irrigated contaminated soil; CK: clean water irrigated uncontaminated soil）, nifH gene high-throughput sequencing technology was used to analyze the characteristics of azotobacter community in rice root zone soil under different treatments. 【Result】 The contents of SO₄^2-, NO₃^- and heavy metals in rice root zone soil increased significantly, the soil acidified, and the diversity of azotobacter community decreased. The dominant nitrogen-fixing bacteria in rice root zone soil included Anaeromyxobacter, Geobacter, etc. The number of nitrogen-fixing bacteria enriched in CK treatment was significantly higher than that in A and B treatments, and Verrucomicrobia were mainly enriched in B treatment, while Proteobacteria were mainly enriched in CK treatment. pH、Cu、Pb and Zn were the main factors driving the community structure of azotobacter in rice root zone. Desulfovibrio and Desulfurivibrio with sulfur reduction function have significantly contributed to the change in nitrogen. 【Conclusion】 AMD had significant effects on soil chemistry and azotobacter in the root zone of rice, and restoring clean water irrigation promoted the recovery of azotobacter. The above findings provide new insights and ideas for biological remediation of AMD-contaminated soil.

Key words: AMD, rice root zone soil, root zone nitrogen-fixing microorganism, nitrogen, heavy metal

田胜尼, 张琴, 董玉飞, 丁洲, 叶爱华, 张明珠. 酸性矿山废水对成熟期水稻根区理化因子及固氮微生物的影响[J]. 生物技术通报, 2024, 40(6): 271-280.

TIAN Sheng-ni, ZHANG Qin, DONG Yu-fei, DING Zhou, YE Ai-hua, ZHANG Ming-zhu. Effects of Acid Mine Drainage on Physicochemical Factors and Nitrogen-fixing Microorganisms in the Root Zone of Mature Rice[J]. Biotechnology Bulletin, 2024, 40(6): 271-280.

图/表 8

表1 水稻种植前供试土壤理化性质

Table 1 Physical and chemical properties of soil before planting rice

供试土壤 Tested soil	TN/ （g·kg^-1）	NO₃^-/（mg·kg^-1）	NH₄⁺/ （mg·kg^-1）	OM/ （g·kg^-1）	TP/ （g·kg^-1）	pH	SO₄^2-/ （mg·kg^-1）	Cu/ （mg·kg^-1）	Cd/ （mg·kg^-1）	Pb/ （mg·kg^-1）	Zn/ （mg·kg^-1）
污染土AMD contaminated soil	0.97±0.10	6.42±0.60	7.52±0.90	13.01±1.72	0.72±0.07	4.68±0.05	282.41±6.29	151.82±5.94	2.49±0.11	159.02±5.19	210.30±5.39
未污染土Uncontaminated soil	0.98±0.90	3.89±0.51	8.05±0.64	8.29±0.44	0.67±0.01	6.18±0.07	132.87±4.79	20.85±0.57	1.99±0.13	28.30±0.94	90.96±2.26

图1 试验示意图

Fig. 1 Schematic diagram of the test

图2 不同处理下土壤理化因子特征 A：AMD灌溉污染土壤；B：清洁水灌溉污染土壤；CK：清洁水灌溉未污染土壤。不同小写字母表示不同处理间差异显著（P < 0.05）。下同

Fig. 2 Characteristics of soil physical and chemical factors under different treatments A: AMD irrigated contaminated soil; B: clean water irrigated contaminated soil; CK: clean water irrigated uncontaminated soil. Different lowercase letters indicate significant difference between different treatments (P < 0.05). The same below

图3 不同处理下水稻根区土壤中固氮微生物的多样性和群落结构的差异 a：不同处理下水稻根区土壤中固氮菌的Chao1和Shannon指数多样性的箱型图；b：水稻根区固氮微生物群落的PcoA分析

Fig. 3 Differences in diversity and community structure of nitrogen-fixing microorganisms in rice root zone soil under different treatments a: Box plots of Chao1 and Shannon index diversity of azotobacter in rice root zone soil samples under different treatments; b: PcoA analysis of nitrogen-fixing microbial communities in rice root zone

图4 不同处理下水稻根区土壤中固氮微生物的群落组成差异 a：不同处理下水稻根区土壤中固氮菌分布circos图；b：LEfSe基于A、B、CK不同处理组间群落差异分析，只有LDA值大于2的类群被清晰显示

Fig. 4 Differences in community composition of nitrogen-fixing microorganisms in rice root zone soil under different treatments a: Distribution of azotobacter in rice root zone soil samples under different treatments; b: LEfSe was based on community difference analysis among different treatment groups A, B and CK, and only groups with LDA values > 2 were clearly displayed

图5 环境因子对OTUs水平固氮微生物的影响

Fig. 5 Effects of environmental factors on nitrogen-fixing microorganisms at OTUs level

图6 固氮菌各属的相对丰度与环境因子间的相关性

Fig. 6 Correlation between relative abundances of azotobacter genera and environmental factors *: P< 0.05; **: P < 0.01

表2 水稻根区土壤中的养分因子与各固氮菌属的逐步回归分析

Table 1 Stepwise regression analysis of nutrient factors in rice root zone soil and each azotobacter genus

养分因子Nutrient factor	回归方程Regression equation	R²	P
TN	Y=1.778-4225.623×Sinorhizobium-47.255×Desulfovibrio+456.497×Azoarcus-97.577×Azospira+1.902×Geobacter	1	0.000***
NO₃^-	Y=8.099-1661.173×Desulfurivibrio	1	0.003***
OM	Y=14.024-2588.656×Rhodopseudomonas	0.557	0.013**
TP	Y=89.211-97715.976×Xanthobacter-25902.225×Pelomonas-693.042×Desulfovibrio+1097.825×Rubrivivax+13487.397×Desulfobulbus	0.994	0.000***
SO₄^2-	Y=-357.746+614469.605×Frankia+91743.681×Sideroxydans	0.903	0.000***

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