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我国能源消费以煤炭为主,约占能源消费总量的60%[1]。研究表明,开采活动加快地下水流速,使水文地球化学作用发生改变,导致矿区地下水环境发生巨大变化[2-6]。Galhardi等[7]对巴西菲盖拉市煤矿研究发现,煤矿开采产生的酸性矿山废水影响该区地下和地表水环境。Raj等[8]对煤矿区土壤重金属污染的研究发现,煤炭开采及运输使周边土壤中Hg、Pb、Mn等重金属的浓度严重超标。随着我国能源基地建设的推进,大型煤矿开发建设数量与日俱增,对土壤及地下水环境造成了不同形式的生态影响与环境污染[9]。我国山西、内蒙等众多地区存在土壤与地下水的铁锰离子污染问题,矿区产生的一系列社会经济与生态环境问题亟待解决,矿区土壤与地下水的修复问题一直是环境工程领域的热点问题。
微生物对外界环境变化灵敏,监测微生物在采矿阶段的规律,对微生物修复技术应用于污染矿区环境治理有重要作用[10]。Ma等[11]通过测序技术分析煤矿废水处理厂的微生物群落特征,利用废水中的微生物群落进行废水的管理。Emenike等[12]发现利用微生物的自然修复能够有效降低重金属离子毒性,使破坏的矿区土地恢复利用。吴海维等[13]通过培养耐铅菌株,发现菌株对铅污染土壤的去除机理包括表面吸附和胞内累积或转化2种形式,对污染土壤中铅离子的去除效率极高。Evanise等[14]借助镉污水污泥中提取的微生物处理镉重金属污染土壤,通过将其固化来解决镉污染土壤修复问题。Cabral等[15]发现假单胞菌能够将甲基汞分解为毒性更低的Hg2+。Malik等[16]发现煤矿中分离出的微生物具有出色的煤炭增溶潜力,它们能够成功用于原位甲烷生产,以满足未来的能源需求。大多数学者致力于研究矿区复垦土壤的微生物特征变化和土壤微生物对矿区重金属浓度的响应,研究矿区开采状态下矿区微生物的空间分布特征和特征金属污染离子之间的关联性研究较少。
本文通过16S rDNA和PCR-DGGE分子生物学技术研究伊敏矿区煤层、地下水、土壤体系中微生物的垂向分布规律和伊敏矿区、生活区、核心采矿区域微生物的横向分布规律,全面探索煤矿开采对矿区土壤与地下水中的微生物尤其是对铁锰微生物的影响,进一步探究铁锰细菌与铁锰元素分布的空间响应关系;再通过为期两个月铁锰溶液对矿区土壤及煤层的淋溶实验,研究矿区中铁锰微生物的种属及其变化规律,以及优势菌株空间分异规律,为更好地发挥微生物在矿区铁锰污染环境中的净化作用,同时为矿区铁锰污染地下水、土壤修复技术和微生物原位修复技术大应用提供参考。
矿区土壤与地下水中铁锰菌群分布规律及成因解析
Distribution and genetic analysis of iron and manganese microbial community in soil and groundwater of mining area
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摘要: 针对大量煤矿区地下水环境和土壤中铁锰富集的现象,采用16S rDNA联合DGGE分子生物学方法测定某矿区煤层-潜层地下水-土壤层微生物的垂向分布规律和生活区-矿区边界-核心采矿区中微生物的横向分布规律,与铁锰离子浓度分布进行对比分析,研究铁锰细菌不同种属在空间上的变化规律,揭示区域地下水环境和土壤环境质量铁锰超标的水文地质原因与变化规律。最后通过两个月的铁锰溶液室内驯化实验,全面分析矿区铁锰细菌的生物多样性与种属分类特征。结果显示,矿区地下水中铁锰细菌有硫细菌(Chloroflexi bacterium)、芽孢杆菌(Psychrodurans)、假单胞菌(Pseudomonas),微生物优势条带检出结果表明煤层品位越高的区域铁锰细菌所占比例越高,同时说明铁锰微生物的分布与铁锰元素的分布正向相关。煤层中铁锰细菌优势明显,占到84%左右。矿区土壤中铁锰细菌亦为优势种群,但占比远低于煤层中。非煤区土壤中铁锰细菌则优势不明显,仅占土壤优势菌群的20%。且在核心采区煤层水中发现了兼性菌——绿弯菌门(Chloroflexus),说明长期疏干煤层地下水改变了煤层微生物种群,使得微生物的多样性发生变化。室内驯化实验,得到4种均为芽孢杆菌菌属(Bacillus)的铁锰优势菌,对铁锰离子浓度耐受性较强并且能够进行生物富集,具有成为土壤修复工程菌的潜质。本研究可为矿区土壤与地下水铁锰污染治理提供一定的理论指导。Abstract: Aiming at the phenomenon of iron and manganese enrichment in groundwater environment and soil in a large number of coal mining areas, the vertical distribution of microorganisms among coal-submersible Groundwater-soil Layer in a mining area and the horizontal distribution of microorganisms in a residential mining area boundary-core mining area was determined and analyzed with 16S rDNA combined DGGE molecular biology method. Iron and manganese ion concentration distribution were analyzed. Based on that, the comparison between the microorganisms` distribution rules and the iron-manganese concentration was studied. The results of the spatial variation of different species of ferromanganese(Fe-Mn) bacteria revealed the regional groundwater quality hydrogeological reasons for excess iron manganese and their changing rules. Finally, the biological diversity and species classification characteristics of Fe-Mn bacteria in the mining area were analyzed by indoor domestication experiments for two months. The results show that the iron and manganese bacteria in the groundwater of the mining area include sulfur bacteria, Bacillus, and Pseudomonas. The detection results of the dominant microbial bands show that the higher the coal seam grade is, the higher the proportion of iron and manganese bacteria. It also shows that the distribution of iron and manganese microorganisms is positively correlated with the distribution of iron and manganese. Iron and manganese bacteria have apparent advantages in coal seams, accounting for about 84%. The iron and manganese bacteria in the mining area soil are also the dominant species, but their proportion is much lower than that in the coal seam. The advantages of iron and manganese bacteria in non-coal soil are not obvious, accounting for only 20% of the dominant soil bacteria. Also, the facultative bacteria-Chloroflexus phylum was found in the coal seam water of the core mining area, indicating that the long-term drainage of coal seam groundwater has changed the coal seam microbial population, resulting in changes in microbial diversity. Indoor domestication experiments revealed four dominant iron and manganese bacteria of the genus Bacillus. They a have strong tolerance to iron and manganese ion concentrations and can be bio-enriched, and have the potential to become soil remediation engineering bacteria. This study can provide certain theoretical guidance for the treatment of iron and manganese pollution in soil and groundwater in mining areas.
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表 1 采样点详细信息
Table 1. Sample point details
矿区编号Mining area number 位置Location 备注Note 样品编号Sample number 水样Water sample 2# 11号超降井 煤层水 W1 6# 新打井 矿区边界水 W2 10# 五牧场 生活区浅水 W3 土样Soil sample 8、9、10# 翻斗连王文立家、加油站、五牧场 生活区土壤 S1 13# 新建排水井旁边 矿区边界土壤 S2 1、2、3、4# 三采区 开采区土壤 S3 煤样Coal sample 1、2# 三采区西南 煤层 C1 3、4# 三采区东北 煤层 C2 表 2 培养基主要配置试剂
Table 2. Main medium configuration reagents
培养基名称Medium name 培养基主要试剂Main reagent of medium 牛肉膏蛋白胨液体培养基 牛肉膏3 g、蛋白胨10 g、NaCl 15 g、去离子水1000 mL、pH 7.4—7.6、琼脂20 g 铁细菌培养基 柠檬酸铁铵10 g、MgSO4·7H2O 0.5 g、K2HPO4 0.5 g、CaCl2 0.2 g、NaNO3 0.5 g、NH4NO3 0.5 g、去离子水1000 mL、琼脂15 g、pH 6.6—7.0 锰细菌培养基 蛋白胨2 g、酵母膏0.5 g、琼脂15 g、MnSO4·4H2O 0.2 g、FeSO4·7H2O 0.001 g、过滤陈海水1000 mL 表 3 水样微生物多样性分析及戴斯系数图谱相似性分析
Table 3. Analysis of microbial diversity of water samples and similarity analysis of Dyce coefficient map
多样性指数
Diversity index水样Samples W1 W2 W3 香农指数H 2.74 2.79 2.76 均匀度E 0.95 0.98 0.96 丰度S 17 18 18 相似度/% W1 100 W2 57 100 W3 40.3 64.7 100 表 4 水样中微生物序列对比结果
Table 4. Comparison of microbial sequences in water samples
条带编号
Band No.最相似菌株
Most similar strain相似度/%
Similarity所属菌门
Mycoplasma登录号
Accession No.Band1 Flavobacterium marinum黄杆菌属 99 Bacteroidetes拟杆菌门 NR_109520.1 Band2 Flavobacterium marinum黄杆菌属 99 Bacteroidetes拟杆菌门 NR_109520.1 Band3 Flavobacterium sp.黄杆菌属 100 Bacteroidetes拟杆菌门 HG_934362.1 Band4 Flavobacterium antarcticum黄杆菌属 100 Bacteroidetes拟杆菌门 NZ_ATTM01000012.1 Band5 Bacteroidetes bacterium拟杆菌属细菌 100 Bacteroidetes拟杆菌门 KR_089390.1 Band6 Flavobacterium hydatis水生黄杆菌 96 Bacteroidetes拟杆菌门 NZ_JPRM01000023.1 Band7 Psychrobacillus psychrodurans耐冷嗜冷芽孢杆菌 100 Proteobacteria变形菌门 KP_836260.1 Band8 Psychrobacter pulmonis嗜冷杆菌 99 Proteobacteria变形菌门 KT_767781.1 Band9 Pseudomonas putida恶臭假单胞菌 99 Proteobacteria变形菌门 NZ_AMZE01000125.1 Band10 Marine bacterium海洋细菌 100 Proteobacteria变形菌门 KF_816561.1 Band12 Firmicutes bacterium厚壁菌门细菌 100 Firmicutes厚壁菌门 KR_089401.1 Band14 Flavobacterium rakeshii黄杆菌属 96 Bacteroidetes拟杆菌门 NR_109425.1 Band15 Comamonas aquatica丛毛单胞细菌属 100 Proteobacteria变形菌门 JX_081585.1 Band17 Comamonadaceae bacterium丛毛单胞细菌属 99 Proteobacteria变形菌门 EU_642395.1 Band18 Polaromonas naphthalenivorans极性单胞菌属 99 Proteobacteria变形菌门 NC_008781.1 Band20 Novosphingobium nitrogenifigens新鞘氨醇杆菌属 100 Proteobacteria变形菌门 NZ_GL876934.1 Band23 Sulfur bacteria硫细菌 100 Chloroflexi绿弯菌门 JN_038252.1 Band24 Firmicutes bacterium厚壁菌属细菌 100 Firmicutes厚壁菌门 AB451854.1 表 5 水样超标数据分析表
Table 5. Data Analysis Table for Water Sample Exceeding Standard
样品编号
Sample number编号 水样W1 水样W2 水样W3 测值 标准指数
Standard Index测值 标准指数
Standard Index测值 标准指数
Standard Index温度/℃ 9.2 7.4 8.0 色度 6 0.4 5 0.33 5.5 0.37 浊度 2 0.67 2 0.67 2 0.67 pH 7.66 0.44 7.37 0.247 7.59 0.39 溶解氧 10.88 — 7.72 — 8.36 — ORP/mV -247.2 — -215.3 — -227.3 — 铁 0.677 2.26 1.167 3.89 ≤0.3 — 锰 0.849 8.49 0.335 3.35 ≤0.1 — 铜 <0.010 — <0.010 — <0.010 — 锌 <0.001 — <0.001 — <0.001 — 氨氮 <0.02 — <0.02 — <0.02 — 矿化度 354 — 407 — 399 — 总硬度 230 0.511 203 0.451 322 0.715 磷酸盐 0.068 — <0.02 — <0.02 — 亚硝酸盐氮 0.006 0.3 <0.001 — <0.001 — 重碳酸盐 226 — 379 — 562 — 溶解性总固体 355 0.355 456 0.456 728 0.728 化学需氧量(CODCr) <10 — <10 — <10 — 高锰酸盐指数 3.73 1.24 1.38 0.46 4.43 1.47 氟化物 0.51 0.51 0.65 0.65 0.5 0.5 氯化物 24.2 0.097 28.3 0.113 26.7 0.106 硝酸盐(以N计) 0.15 0.008 <0.05 — 16.5 0.825 硫酸盐 62.5 0.25 33.1 0.132 55 0.22 表 6 各水样地下水水质与微生物特征对比
Table 6. Comparison of groundwater quality and microbial characteristics of each water sample
水样
water sample水质特征
Water quality characteristics微生物特征
Microbial characteristicsW1 1.铁超标2.26倍;
2.锰超标8.49倍;
3.高锰酸盐指数超标1.24倍W1水样中出现了硫细菌(Chloroflexi bacterium)、芽孢杆菌(psychrodurans)、假单胞菌(Pseudomonas)等铁锰细菌,其中芽孢杆菌包括3个优势条带分别为Band7、8、10;假单胞菌包括两个优势条带Band9、18;加上硫细菌优势条带Band23,铁锰细菌优势条带在煤层水所检测出的优势条带中占比46.2%。 W2 1.铁超标3.89倍;
2.锰超标3.35倍W2水样中也出现了硫细菌(Sulfur bacteria)、芽孢杆菌(psychrodurans)、假单胞菌(Pseudomonas)等铁锰细菌,其中芽孢杆菌包括一个优势条带为Band10;假单胞菌包括一个优势条带Band18;加上硫细菌优势条带Band23,铁锰细菌优势条带在矿区边界水样中所检测出的优势条带中占比23.1%。 W3 高锰酸盐指数超标1.47倍 W3水样中只出现了假单胞菌(Pseudomonas)这一种铁锰细菌,优势条带为Band18,占生活区地下水中所检测出的优势条带的14.2%。 表 7 土样微生物多样性分析
Table 7. Analysis of microbial diversity of soil samples
土样Soil sample S1 S2 S3 香浓指数H 2.74 2.68 2.79 均匀度E 0.97 0.97 0.95 丰度S 17 16 19 表 8 戴斯系数图谱相似性分析
Table 8. Analysis of similarity analysis of Dyce coefficient map
相似度/% Similarity S1 S2 S3 S1 100 70.2 85.4 S2 70.2 100 63 S3 85.4 63 100 表 9 煤样微生物多样性分析及戴斯系数图谱相似性分析
Table 9. Analysis of microbial diversity of coal samples and similarity analysis of Dyce coefficient map
煤样 Coal sample C1 相似度/% Similarity C2 香浓指数H 2.57 87.8 2.57 均匀度E 0.95 0.95 丰度S 15 15 表 10 土样与煤样细菌鉴定序列比对分析结果
Table 10. Soil and coal sample bacterial identification sequence alignment analysis results
编号NO. 最相似菌株
Most similar strain登录号
Accession No.相似度/%
Similarity最相似类群
Most similar group土壤样品优势条带
Dominant bands of
soil samplesBand_3 Lactobacillus plantarum植物乳杆菌 NR_075041 100 Firmicutes厚壁菌门 Band_4 Lactobacillus plantarum植物乳杆菌 NR_042394 100 Firmicutes厚壁菌门 Band_5 Lactococcus lactis乳酸乳球菌 NR_040955 100 Firmicutes厚壁菌门 Band_6 Lactococcus lactis乳酸乳球菌 NR_040955 100 Firmicutes厚壁菌门 Band_10 Lactobacillus paracasei副干酪乳杆菌 NR_121787 100 Firmicutes厚壁菌门 Band_13 Pseudomonas monteilii蒙氏假单胞菌 NR_121767 98 Proteobacteria变形菌门 Band_15 Pseudomonas plecoglossicida变形假单胞菌 NR_024662 98 Proteobacteria变形菌门 Band_17 Lactobacillus paracasei副干酪乳杆菌 NR_121787 100 Firmicutes厚壁菌门 煤层样品优势条带
Dominant seam
sample beltBand_5 Pseudomonas plecoglossicida变形假单胞菌 NR_024662 99 Proteobacteria变形菌门 Band_7 Pseudomonas monteilii蒙氏假单胞菌 NR_121767 98 Proteobacteria变形菌门 Band_8 Pseudomonas indoloxydans氧化吲哚假单胞菌 NR_115922 98 Proteobacteria变形菌门 Band_9 Lactococcus lactis乳酸乳球菌 NR_040955 100 Firmicutes厚壁菌门 Band_12 Pseudomonas entomophila虫媒假单胞菌 NR_102854 98 Proteobacteria变形菌门 Band_13 Pseudomonas monteilii蒙氏假单胞菌 NR_121767 98 Proteobacteria变形菌门 表 11 伊敏矿区重金属累积污染指数及综合污染指数
Table 11. The cumulative pollution index and comprehensive pollution index of heavy metals in Yimin mining area
样品名称
Sample name污染指数 Pollution index 汞Hg 镉Cd 铬Cr 砷As 铅Pb 铜Cu 铁Fe 锰Mn 重金属污染指数
Heavy metal pollution indexS1 0.04 0.14 0.21 0.21 0.04 0.09 0.32 0.24 0.118 W2 0.03 0.21 0.17 0.38 0.05 0.10 1.14 1.20 0.311 S3 0.15 0.22 0.20 0.25 0.05 0.07 0.78 0.64 0.229 表 12 矿区土壤微生物与内蒙古其它类型土壤对比表
Table 12. Comparison table between soil microorganisms in Yimin mining area and other types of soil in Inner Mongolia
土壤类型
Soil type多样性指数
Diversity index丰度
Abundance优势种群
Dominant population本次研究煤矿土壤
Yimin coal mine soil2.57—2.79 0.95—0.97 厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes),其中厚壁菌门占绝对优势[26]; 退化荒漠草原土壤
Degraded steppe soil2.68—2.84 0.95—0.97 优势菌群为拟杆菌门(Bacteroidetes),酸杆菌门(Acidobacteria),变型菌门(Proteobacteria)的γ、δ类群及厚壁菌门(Firmicutes),其中拟杆菌门占优势[33]; 林地
Woodland3.03—3.13 0.98—0.99 门水平优势细菌7个,分别为:变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)、酸杆菌门(Acidobacteria)、芽单胞菌门(Gemmatimonadetes)、硝化螺旋菌门(Nitrospira),其中变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)为多数样品的优势菌群[34-35]; 耕地
Arable land2.61—3.92 0.95—0.99 草地(非牧区)
Grassland (non-pastoral)2.39—4.08 0.96—0.98 草地(牧区)
Grassland (pastoral)3.19—3.57 0.98—0.99 煤矿排土场复垦土壤
Coal mine dumps reclaim soil2.50(复垦初期)—3.51(两年后) — 厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)、变形菌门(Proteobacteria)、酸杆菌门(Acidobacteria)、芽单胞菌门(Gemmatimonadetes),其中的放线菌门、拟杆菌门和变形菌门是最主要的细菌类群[36-37]。 表 13 驯化所得铁锰细菌序列比对结果
Table 13. Sequence alignment results of ferromanganese bacteria obtained from domestication
样品编号
Sample No.最相似菌株
Most similar strain相似度/%
Similarity登录号
Accession No.锰/土②Manganese/soil② Lysinibacillus xylanilyticus strain赖氨酸芽孢杆菌属 99 KP644237.1 锰/土④Manganese/soil④ Lysinibacillus macroides strain赖氨酸芽孢杆菌属 99 KR085803.1 铁/土①Iron/soil① Bacillus simplex strain芽孢杆菌属 99 GU201860.1 铁/土②Iron/soil② Brevibacillus agri strain短短芽孢杆菌属 99 NR_040983.1 -
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