微生物学报  2019, Vol. 59 Issue (6): 1127-1142   DOI: 10.13343/j.cnki.wsxb.20190121.
http://dx.doi.org/10.13343/j.cnki.wsxb.20190121
中国科学院微生物研究所,中国微生物学会,中国菌物学会
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文章信息

佘伟钰, 冯灿, 杨渐, 蒋宏忱. 2019
Weiyu She, Can Feng, Jian Yang, Hongchen Jiang. 2019
三峡库区水体中固氮微生物多样性及其影响因素
Nitrogen-fixing microbial diversity and its influencing environmental variables in waters Three Gorges Reservoir
微生物学报, 59(6): 1127-1142
Acta Microbiologica Sinica, 59(6): 1127-1142

文章历史

收稿日期:2019-03-21
修回日期:2019-04-13
网络出版日期:2019-05-06
三峡库区水体中固氮微生物多样性及其影响因素
佘伟钰 , 冯灿 , 杨渐 , 蒋宏忱     
中国地质大学(武汉)生物地质与环境地质国家重点实验室, 湖北 武汉 430074
摘要[目的] 研究分析不同时空条件下三峡库区水体固氮微生物多样性,并探讨其与地球化学参数的相关性。[方法] 采集三峡库区不同时间(三月份和六月份)和空间(干流与支流)的水体样品,对其进行地球化学参数分析,并通过构建克隆文库分析样品中固氮功能基因(nifH)的多样性进而探讨其与水体地化参数的相关关系。[结果] 统计分析显示三峡库区水体固氮微生物α-多样性和群落组成具有时空差异。支流水体样品的固氮微生物α-多样性高于干流水体样品;六月水体样品的固氮微生物α-多样性高于三月水体样品。三峡库区三月水体样品中的固氮微生物群落以Proteobacteria(50.3%)和Firmicutes(40.0%)为主;六月水体样品的固氮微生物群落以Proteobacteria(48.4%)、Firmicutes(25.4%)和Cyanobacteria(19.0%)为主。Mantel检验结果显示:固氮微生物群落结构的差异与温度、pH和DIC等地球化学参数具有显著(P < 0.05)相关性,其中温度和pH的相关性系数最大。[结论] 三峡库区固氮微生物的种群结构和多样性具有时空差异,影响三峡水库水体中固氮微生物群落结构与多样性的主要环境因素为温度和pH,同时浊度、DIC、氨氮也对库区水体固氮微生物群落结构和多样性有一定的影响。
关键词三峡水库    固氮微生物    nifH基因    环境因素    
Nitrogen-fixing microbial diversity and its influencing environmental variables in waters Three Gorges Reservoir
Weiyu She , Can Feng , Jian Yang , Hongchen Jiang     
State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Wuhan 430074, Hubei Province, China
Abstract: [Objective] To explore the diversity of nitrogen-fixing microbial community and its response to geochemical parameters in the water of the mainstream and branches in the Three Gorges Reservoir (TGR) [Methods] A total of 18 water samples were collected from the mainstream and branches of the TGR before and after drainage (in March and June). The physical and chemical parameters of the samples were measured, and nifH gene diversity was analyized by using clone library-based phylogenetic analysis. The geochemical parameters of water bodies were correlated with the diversity and community composition of nitrogen-fixing microbial. [Results] The geochemical parameters differed significantly before (in March) and after (in June) drainage. The nitrogen-fixing microbes were dominated by Proteobacteria (50.3%) and Firmicutes (40.0%) the TGR waters in March (before drainage), in contrast with the dominance of Proteobacteria (48.4%), Firmicutes (25.4%) and Cyanobacteria (19.0%) in June (after drainage). Significant temporal and spatial variations were observed among the nitrogen-fixing microbial community composition and diversity as indicated by statistical analysis (Cluster and CCA analyses). The diversity of nitrogen-fixing microbes in the TGR waters after drainage (in June) was higher than before. The diversity of nitrogen-fixing microbes was higher in waters from tributaries than that from the main stream. Mantel test showed that the composition of nitrogen-fixing microbes was significantly correlated with the geochemical parameters of water. Water temperature was the most important factor affecting the community and variety of nitrogen-fixing microbes in the TGR waters. [Conclusion] The nitrogen-fixing microbial community compositions in the TGR waters show significant temporal and spatial variations, which could be ascribed to the different geochemical parameters, especially water temperature.
Keywords: Three Gorges Reservoir    nitrogen-fixing microbes    nifH    Environmental variables    

三峡工程是我国重要的水利水电工程,其在防洪、发电和航运等方面具有巨大的经济效益,但大坝建成后对库区的生态和水文环境问题造成了深远影响[1-2]。不同于世界上其他水库遵循的夏(或雨季)涨冬(或旱季)落蓄水方式,三峡水库采取夏落冬涨的蓄水方式[3-4]。即在每年汛期(6–9月)时降低水位至145 m,大量泥沙随排水流走;汛期过后将水位升至175 m,在冬季时(12–3月)保持高水位,拦蓄清水。由于大坝拦截作用,三峡库区河流的水文条件发生了巨大变化。例如,库区蓄水造成河面变宽,水流变缓,水体扩散能力减弱,和库区回水顶托等[5]。这些水文条件变化导致许多库湾和支流水体污染加重,同时造成局部水域的水体富营养化[6]。前人研究表明三峡库区水体富营养化程度存在时间和空间差异:春夏季发生水体富营养化的程度远高于秋冬季节;支流的水体富营养化程度明显高于干流[7-8]。以上研究表明高浓度的氮、磷营养盐是造成三峡库区水体富营养化问题的主要原因;且这些营养盐主要来自外源输入,包括上游来水、农田径流和城镇生活污水等[9-10]。尽管外源营养盐输入对水体富营养化具有重要贡献,但内源生物作用产生的营养盐(如生物固氮产生的氨态氮)对水体富营养化的贡献也不可忽视[11-13]

生物固氮作用是氮循环中重要的生物地球化学过程之一。它指微生物在固氮酶作用下,将大气中的气态氮转化为生物可利用氨的过程[14]。因此,微生物固氮作用产生的氨可能是水体氮营养盐的另一个重要来源[15]。作为固氮作用的执行者,固氮微生物的群落多样性敏感地响应环境条件变化,并进而影响水体环境氮循环[7]。因此研究三峡库区水体固氮微生物的群落结构和多样性及其影响因素将有助于我们全面理解库区水体氮营养元素的来源。近年来,已有大量报道环境中固氮微生物多样性的文献,其研究手段主要采用功能基因分析方法。即通过分析固氮酶关键功能基因(nifH)研究固氮微生物多样性及其分布规律。该方法已被广泛应用于各类自然环境,包括湖泊[16]、土壤[17-20]、海洋[21-23]、深海热液[24]、陆地热泉[25-27]、河口[19, 28]和水库[29]等。

综上,本研究拟基于nifH功能基因分析,研究三峡库区不同时间(春季和夏季)和空间(干流与支流)水体样品固氮微生物多样性和种群构成,并结合三峡库区水体的地球化学参数,借助CCA、PCA等统计学手段来揭示固氮微生物的时空分布特征。该研究结果将有助于进一步加深认识三峡库区水体氮循环过程,并可为评价库区环境提供基础研究数据。

1 材料和方法 1.1 采样点分布

本研究在湖北省三峡库区自下游向上游共选取9个采样点,如图 1。包括4个支流点(百岁溪、九畹溪、沙镇溪、神农溪)、5个干流点(所选支流与干流的河口交汇区),采样点选取与我们以前的研究一致[30]。分别于2017年三月(春)和六月(夏)在三峡库区共采集两批水体样品,三月水体样品标记为M1_M、M2_M、M3_M、M4_M、M5_M、B1_M、B2_M、B3_M、B4_M,六月水体样品标记为M1_J、M2_J、M3_J、M4_J、M5_J、B1_J、B2_J、B3_J、B4_J,编号前面的“M”代表干流(mainstream),“B”代表支流(branch);编号下划线后面的“M”代表三月(March),“J”代表六月(June)。

图 1 采样点位置 Figure 1 Sampling locations

1.2 样品采集

图 1每个取样点用孔径为0.22 μm的微孔滤膜(Whatman,英国)过滤500 mL表层水(–0.5 m处),滤液用于水样阴阳离子浓度检测,滤膜干冰存放运往实验室,用于后续提取DNA分析。根据《水质采样技术规程》(SL 187-96),分别采集用于测试溶解性有机碳(DOC)和溶解性无机碳(DIC)、总氮(TN)、氨态氮(NH4+)和总磷(TP)的水样。

1.3 水体理化参数测试

参照我们以前的研究方法[30-32],在现场测试水体理化参数,包括水体pH和温度(上海三信仪表/SX711)、水体浊度(HANNA instruments/HI 98703)、水体DIC和DOC浓度(Analytic Jena/ multi N/C 2100)。返回实验室后,测试的水体理化参数包括:溶解无机碳浓度(双指示剂中和法测量)[33],总磷(磷钼蓝光度法)[34],总氮(碱性过硫酸钾消解紫外分光法),铵根浓度(纳氏试剂紫外分光法)[35]

1.4 DNA提取和PCR扩增

使用美国MP公司的Fast DNA Soil-Direct Kit试剂盒提取样品DNA。采用引物nifH3 (5′-ATRTT RTTNGCNGCRTA-3′)和nifH4 (5′-TTYTAYGGN AARGGNGG-3′)对样品总DNA进行第一轮扩增;用引物nifH1 (5′-TGYGAYCCNAARGCNGA-3′)和nifH2 (5′- ADNGCCATCATYTCNCC-3′)对上述扩增产物进行第二轮扩增,反应体系为2.5 μL 10×PCR缓冲液(Promega),2 μL dNTPs,17.2 μL无菌水,正反向引物各1 μL,0.3 μL rTaq DNA聚合酶以及1 μL DNA模板,共计25 μL。PCR扩增反应条件为:94 ℃ 5 min;94 ℃ 1 min,50 ℃ 1 min (引物nifH3/nifH4)或者55 ℃ 1 min (引物nifH1/nifH2),72 ℃ 1 min,反复进行30个循环,循环完成后再保持72 ℃ 10 min[32]。PCR产物采用大约1%琼脂糖凝胶电泳检测,凝胶内添加溴化乙锭(EB)用于染色,紫外灯照射下切取约为400 bp长度的目标条带,使用美国Axy gen公司的胶纯化试剂盒对PCR产物进行纯化回收。

1.5 克隆文库构建和系统发育分析

参照文献[30, 36]方法构建克隆文库和系统发育分析,使用Mothur v1.34.1软件(Furthest neighbor方法)[37],以98%的序列相似性对测回序列进行划分分类操作单元(即Operational taxonomic unit,简称为OTU)。将所选取的代表性OTU序列翻译成氨基酸序列,并与NCBI蛋白数据库(http://www.ncbi.nlm.nih.gov)中的同源氨基酸序列进行比对,并选取出最高相似性的参考序列,使用BioEdit v7.0.9.0软件[38]中Clustal W将代表氨基酸序列和参考序列修剪整齐后,导入MEGA v7软件中迭代运算1000次构建系统发育树。该研究所测定的克隆序列的GenBank登录号为MF464113–MF464184。

1.6 数据分析

参照我们之前的研究[36],使用公式C=1–n/NnifH基因克隆文库的覆盖度进行计算(其中C为覆盖度,N为所构建文库的克隆总数,n为该文库中只出现1次的克隆数量)。使用PAST软件(http://folk.uio.no/ohammer/past/)进行稀释曲线(rarefaction curve)分析、聚类分析、Mantel检验、T检验、Kruskal-Wallis检验、群落组成相似性分析(Analysis of similarities,ANOSIM)和克隆文库多样性指数分析。使用R语言Vegan包进行环境变量的主坐标分析(principal component analysis,PCA)和固氮微生物群落组成典型对应分析(canonical correspondence analysis,CCA);使用SPSS 22.0分析固氮微生物群落多样性指数与三峡库区环境变量的Pearson相关性。

2 结果和分析 2.1 水体理化性质分析

三峡库区三月和六月采样点(样点位置见图 1)表层水体的地球化学数据如表 1所示。PCA分析所有三峡库区水体样品的地球化学数据(图 2)显示:三月(春)和六月(夏)的地球化学参数差异显著(Kruskal-Wallis检验:P < 0.05),按照采样时间样点分别聚集在虚线两侧。同时可见三月干流样点聚集在一起,支流点较为分散;而六月的干流、支流样点之间均较为分散,无明显聚集规律。通过T检验分析三月与六月的各项地球化学参数发现,水体温度、浊度、DIC和氨氮浓度这4个环境参数季节性变化显著(P < 0.001)(图 3)。另外,干流和支流水体环境地球化学参数(包括温度、pH等)同样存在显著差异(Kruskal-Wallis检验,P < 0.05)(图 4)。

图 2 三峡库区三月份与六月份采样点水体地球化学参数的主成分分析二维散点图 Figure 2 Principal component analysis (PCA) on the geochemical parameters of the sampled waters in the TGR in March and June

图 3 三峡库区三月和六月水体样品(包括干、支流所有样品)地球化学参数之间的差异 Figure 3 The variation of geochemical factors among the studied TGR water samples between March and June

图 4 三峡库区干流和支流水体样品(包括三、六月所有样品)地球化学参数之间的差异 Figure 4 The variation of geochemical factors among the studied TGR water samples collected from the main stream and its corresponding tributary sites

表 1. 采样点水体地球化学参数 Table 1. Locations and geochemical parameters of the sampled waters in the Three Gorges Reservoir (TGR)
Sample site (March/June) M1 M2 M3 M4 M5 B1 B2 B3 B4
T/℃ 14.3/24.4 14.4/23.5 14.4/23.5 15.1/23.1 13.8/22.7 16.6/23.6 15.7/27.0 16.6/27.7 16.0/24.8
Turbidity/(NTU) 1.50/13.1 1.35/14.2 2.75/17.9 1.51/34.5 1.72/34.5 0.99/5.27 1.36/6.49 2.37/6.98 4.35/8.73
pH 8.08/8.52 8.14/7.86 8.16/7.85 8.14/7.97 8.17/7.88 8.56/8.01 8.30/8.79 8.72/8.87 9.34/9.08
Intensity/klux 69.8/29.0 76.9/36.7 63.9/53.3 55.4/97.1 3.5/12.5 78.4/25.0 23.1/57.9 42.3/110.0 1.4/53.6
Water chemical parameters
TN*/(mg/L) 3.38/7.70 3.36/2.84 3.44/1.96 3.53/2.80 4.04/1.88 3.75/8.11 3.91/1.91 3.39/2.04 2.39/2.04
TP*/(mg/L) 0.21/0.27 0.13/0.16 0.18/0.18 0.16/0.15 0.11/0.36 0.14/0.23 0.18/0.14 0.13/0.11 0.07/0.21
NH4+/(mg/L) 1.08/0.53 1.33/0.00 1.64/0.28 1.130/0.005 1.56/0.00 1.49/0.76 1.50/0.15 1.16/0.26 1.62/0.26
DOC*/(mg/L) 6.31/62.60 6.09/27.30 6.04/20.90 30.40/46.20 4.85/28.60 3.57/8.62 5.51/6.91 6.65/6.54 25.4/9.15
DIC*/(mg/L) 96.0/60.0 125.0/60.0 125.0/55.2 122.0/60.0 128.0/55.0 112.0/50.4 122.0/55.2 118.0/50.4 96.0/52.8
*TN: total nitrogen; TP: total phosphorus; DOC: dissolved organic carbon; DIC: dissolved inorganic carbon.

2.2 nifH基因克隆文库分析

三峡库区三月的9个nifH基因克隆文库共筛选出312个有效的克隆子,分属于61个OTU;统计分析克隆文库的覆盖度为82.6%–100% (表 2)。六月的9个克隆文库共筛选出279个有效克隆子,分属于74个OTU;覆盖度范围为70.8%–94.4% (表 2)。统计分析发现,六月水体样品的nifH基因OTU的平均丰度高于三月水体样品;支流水体样品的nifH基因OTU平均丰度高于干流水体样品(表 2)。相关性分析显示nifH基因文库多样性指数与环境变量没有显著相关性(数据未显示)。

表 2. 三峡库区nifH基因克隆文库的多样性指数 Table 2. Diversity indices of the nifH gene clone libraries retrieved from the studied TGR water samples
Libraries (March/June) Clones OTUs Coverage/% Simpson Shannon Chao-1
M1 35/38 7/12 94.3/70.8 0.76/0.88 1.58/2.27 7.3/19.0
M2 37/23 3/10 100.0/82.6 0.24/0.84 0.49/2.08 3.0/11.5
M3 37/41 7/17 86.5/80.9 0.59/0.85 1.17/2.39 17.0/26.0
M4 37/37 7/7 97.3/89.2 0.79/0.66 1.72/1.35 7.0/13.0
M5 36/38 6/11 88.9/78.9 0.60/0.79 1.12/1.82 12.0/39.0
B1 24/38 8/12 82.6/89.5 0.73/0.87 1.59/2.24 13.0/14.0
B2 48/18 17/6 83.3/94.4 0.89/0.78 2.47/1.65 22.6/6.0
B3 37/22 8/11 91.9/82.6 0.79/0.87 1.74/2.22 11.0/11.9
B4 22/38 7/15 86.4/86.8 0.70/0.90 1.53/2.50 8.5/16.7
“/” separated the data of March (left) and June (right).

系统发育分析结果显示,本研究三月、六月三峡库区取样点表层水体样品的固氮微生物主要分属于Proteobacteria、Firmicutes和Cyanobacteria门(图 5)。其中,研究样品中固氮微生物群落的优势OTU (在所有样品中的平均相对丰度大于5%)分别与α, β, γ-Proteobacteria、Synechococcale和Clostridia纲的参考菌株相近,其nifH基因的氨基酸相似性均高于95% (表 3)。另外,三月和六月的优势OTU具有显著差异:OTU1、OTU3、OTU5、OTU6、OTU8、OTU9和OTU10主要出现在三月样品;然而OTU2、OTU4和OTU7主要出现在六月样品。干流与支流样品的优势OTU同样存在差异:OTU1、OTU2、OTU3、OTU6、OTU7、OTU8和OTU9主要分布于干流样品;然而OTU4、OTU5和OTU10主要分布于支流样品(表 3)。

图 5 nifH基因蛋白系统发育进化树 Figure 5 Neighbor-joining tree result showing the phylogenetic relationships of the deduced nifH protein sequences translated from the nifH gene clone sequences obtained in this study. The nifH gene OTUs obtained from Cyanobacteria/Proteobacteria (A) and Firmicutes/Euryarchaeota (B) with their closely related sequences from the GenBank database. The scale bar represents the expected changes at each homologous position. Bootstrap values of > 50% are displayed

表 3. 三峡库区固氮微生物优势分类单元的分布差异(总平均相对丰度top10) Table 3. Different distribution of the dominant OTUs (relative abundance > 1%) of nitrogen-fixing microbes in the TGR
Units March/June Main stream/tributary Classification (Class) Similar strains Similarity/%
OTU 1 13.6%/0.6% 12.7%/0.0% α-Proteobacteria Bradyrhizobium japonicum 98
OTU 2 0.0%/12.3% 9.1%/2.0% γ-Proteobacteria Methylomonas sp. 99
OTU 3 9.6%/0.0% 8.6%/0.0% β-Proteobacteria Sideroxydans sp. 99
OTU 4 0.0%/7.7% 2.4%/4.6% Synechococcales Synechococcus sp. 98
OTU 5 6.1%/0.3% 0.3%/5.5% Clostridia Clostridium drakei 97
OTU 6 6.3%/0.0% 5.7%/0.0% Clostridia Clostridium sp. 97
OTU 7 0.0%/5.7% 5.1%/0.0% α-Proteobacteria Bradyrhizobium japonicum 99
OTU 8 5.4%/0.0% 4.9%/0.0% α-Proteobacteria Desulfobulbus elongatus 98
OTU 9 5.1%/0.0% 3.5%/1.1% Clostridia Clostridium sp. 97
OTU 10 4.8%/0.0% 0.0%/4.3% Clostridia Desulfosporosinus sp. 98

此外,三月与六月样品的固氮微生物群落组成具有差异。三月样品的主要固氮微生物群落为Proteobacteria (50.3%)和Firmicutes (47.1%),还有少量的Cyanobacteria (2.6%)。在Proteobacteria中,主要包括Alphaproteobacteria (17.0%)、Betaproteobacteria (17.6%)、Deltaproteobacteria (12.2%)、Epsilonproteobacteria (1.3%)和Gammaproteobacteria (2.2%) 5个纲;在Firmicutes中,仅有Clostridia1个纲;Cyanobacteria中仅有Nostocales 1个纲(图 6)。

图 6 三月和六月的三峡库区干流和支流水体固氮微生物群落构成结构图 Figure 6 Community composition of nitrogen-fixing microbes in the TGR waters in March and June

六月样品的主要固氮微生物群落为Proteobacteria (48.4%)、Firmicutes (28.3%)和Cyanobacteria (19.0%) 3个门。在Proteobacteria中,主要包括Alphaproteobacteria (11.8%)、Betaproteobacteria (5.0%)、Deltaproteobacteria (3.2%)、Epsilonproteobacteria (1.4%)和Gammaproteobacteria (26.9%) 5个纲;在Firmicutes中,以Clostridia (27.6%)为主和少量Negativicutes (0.7%);在Cyanobacteria中,有Synechococcales (8.6%)、Nostocales (6.5%)和Pleurocapsales (0.7%) 3个纲;此外,还有少量Euryarchaeota门的Methanobacteria (4.3%)(图 6)。

支流和干流水体样品的固氮微生物群落组成同样具有差异。在干流水体样品中,三月和六月均以Proteobacteria门固氮微生物种群为主;相较于三月干流样品,六月的干流样品中出现了属于Archaea和Cyanobacteria门的固氮微生物种群。在支流样品中,三月样品固氮微生物种群主要以Firmicutes为主,六月样品主要以Cyanobacteria为主(图 6)。

2.3 统计分析

CCA分析(图 7)显示:本研究样品的固氮微生物群落结构差异与温度、pH、浊度、铵根和溶解无机碳等地球化学参数具有显著(P < 0.05)相关性;另外,三月和六月样点在CCA图上完全分开。上述这一结果与群落相似性检验(ANOSIM)结果一致,即三月和六月样品的固氮微生物群落组成具有明显差异(R=0.455,P=0.001)。CCA分析还显示:三月样品干流水体样点均聚在一起,但是支流水体样点较为分散;然而六月干流和支流水体样点均较为分散,分布无明显规律(图 7)。群落相似性检验(ANOSIM)也进一步证实三月干流和支流水体样品中的固氮微生物群落存在显著差异(R=0.622,P=0.007),而六月干流和支流水体样品中的固氮微生物群落不存在显著差异(R=0.194,P=0.157)。

图 7 三峡库区所有水体样品nifH基因克隆文库与地球化学参数之间的CCA分析 Figure 7 CCA analysis showing correlation between the nifH gene clone libraries and geochemical parameters of the total TGR water samples. DIC: Dissolved inorganic carbon; Tur.: turbidity; Temp.: temperature; NH4: Ammonium

Mantel检验显示,温度对研究样品固氮微生物群落组成的影响最强(R=0.576,P=0.001)(表 4)。同时,pH、溶解无机碳和铵根浓度对研究样品固氮微生物群落分布也具有显著影响(P < 0.05) (表 4);另外,针对三月和六月水体样品单独进行Mantel检验发现,这两个季节水体样品的固氮微生物群落组成均主要受到温度和pH的显著影响(表 4)。

表 4. Mantel检验本研究水体样品固氮微生物群落组成与环境参数相关性 Table 4. Spearman correlations between the nitrogen-fixing microbial community compositions in the studied samples and environmental variables as determined by Mantel tests
Environment factors All samples Samples in March Samples in June
R P R P R P
T/℃ 0.576 0.001 0.575 0.007 0.762 0.002
pH 0.329 0.001 0.587 0.002 0.763 0.001
NH4+ 0.394 0.001 0.145 0.265 0.050 0.313
DIC 0.461 0.001 0.177 0.231 0.127 0.182
DOC –0.024 0.595 –0.063 0.640 –0.130 0.791
Turbitidy 0.027 0.409 0.02 0.423 –0.128 0.78
TN 0.081 0.226 0.031 0.309 0.021 0.388
TP –0.031 0.624 –0.104 0.670 –0.166 0.827

3 讨论

本文研究结果显示三月(春)和六月(夏)三峡库区支流和干流水体中的固氮微生物群落组成存在明显差异(图 56)。该结果意味着三峡库区水体固氮微生物群落分布具有时空差异性。这些固氮微生物群落分布的时空差异主要由三峡库区水体环境条件在时间和空间上变化导致(图 34)。本文样品分别来自蓄水期高水位的三月份与排水期低水位的六月份,这两个季度的温度、光照和水动力条件具有明显差异:夏季的温度和光照强度高于春季;同时,三峡库区六月份处于排水期,由于雨季到来,水流冲刷岸边土壤带走土壤颗粒造成库区水体浊度增加,进而土壤中大量营养物质(溶解性有机碳和磷元素等)输入到库区水体,并提高水体营养条件[39-42]。这种高温度和营养的环境条件会极大促使微生物(包括固氮微生物)的大量繁殖,故而会导致六月样品的固氮微生物种α-多样性高于三月样品,同时六月样品的固氮微生物群落组成也与三月份样品具有显著差异(图 7)。另外,三峡库区干流与支流的水体理化性质也有明显差异。这主要是由于受大坝的拦截,干流水体理化性质和水动力条件直接受到人为因素(如航运排污和水位调控)的影响,然而支流水体理化性质主要受周围植被类型、外源输入和土壤条件影响[43]。因此,上述空间环境因素的差异会导致了三峡库区支流和干流水体中的固氮微生物群落结构和α-多样性差异。此外,三月份是三峡库区的蓄水期,库区干流水动力学条件更类似湖泊,水体理化参数相对均一,因而三月干流固氮微生物群落会比较相似(图 7);六月份由于三峡库区处于排水期,此时干流水体运移速度要高于支流,支流更加容易形成营养物质的富集,也更利于藻类的繁盛[44]。因此,在三峡库区六月份支流样品中存有大量Cyanobacteria门固氮微生物种群(图 6)。

Mantel检验结果显示,温度的时空差异是影响三峡水库水体中固氮微生物群落结构最为重要的环境因素(表 4)。温度是限制微生物生长和功能代谢的重要环境因素之一;不同微生物种群具有不同的生长和代谢温度范围。因此,不同温度条件下会有不同的固氮微生物群落结构和多样性[21, 45-46]。前人在黄石公园热泉和南海北部沉积物的固氮微生物研究中发现,温度升高会促使藻类(如Cyanobacteria)繁盛[45-46]。三峡库区三月份与六月份的平均气温相差~10 ℃。同时,干流水体温度比支流水体温度平均低2 ℃左右。这主要是由于三峡库区干流流域面积较广,江上风速较大,使得表层与底层水体相互交换以致温度偏低[47]。这种三峡库区三月和六月、支流与干流的水体温度差异可能是影响三峡库区固氮微生物群落结构的空间差异的重要原因。

除温度外,pH、铵根和溶解无机碳等水体指标也影响着三峡库区水体固氮微生物群落组成和分布。三峡库区水体三月份(春)的pH和DIC含量均高于六月份(夏)。pH和DIC浓度均可以影响和调节水体酸碱度,并进而影响微生物胞外酶的分泌和活性。故pH和DIC会显著影响研究样品的固氮微生物群落分布[48-53]。另外,本研究三月份水体样品的总氮和氨氮含量均高于六月份,这与前人对三峡库区消落带土壤氮素吸附释放规律的研究结果相符,其研究表明三峡水库消落带土壤在低水位的干期大量吸纳来自农田和地表径流的氮素,在高水位的淹水期向相对稳定的水体中释放大量的氮素[54]。前人研究表明氨氮的增加会抑制固氮微生物的生长[55],故铵根离子会显著影响本研究样品固氮微生物的组成,同时它也可能是导致三月份样品固氮微生物α-多样性较六月份低的原因之一。

此外,值得我们注意的是本研究发现三峡库区水体存在大量(相对丰度 > 25%)的Firmiutes固氮微生物。前人研究表明,Firmicutes固氮微生物主要分布在沉积物和土壤中[57-58],也有少数分布在陆地热泉、海草和盐沼生态系统中[59]。在本研究中,Firmicutes在每个水体样品中均有较高的占比,且在三月的样品中比例最高,我们推测这与三峡库区蓄清排浑的水利调节方式导致的消落带有关。三月份水位由最低水位145 m上涨达到175 m的最高水位,高程差达到30 m,致使消落带土壤完全淹没在水下,大量土壤中的固氮微生物进入三峡库区水体中[60],从而导致三月研究样品的固氮微生物主要以土壤来源的Firmicutes固氮微生物为主。

4 结论

本文以三峡水库不同时间(三月和六月)和不同空间(干流和支流)的水体样品中固氮微生物作为研究对象,通过对水体中固氮功能基因建立克隆文库并结合水体地球化学参数进行综合分析,对三峡库区固氮微生物多样性、群落构成及其环境影响因素进行了探究,结论如下:(1)三峡库区水体存在大量的固氮微生物群落,主要是Proteobacteria、Firmicutes和Cyanobacteria为主。该结果暗示内源固氮作用产生的氨态氮可能是库区水体氮营养元素的又一重要来源;(2)环境因素的时间(三月和六月)和空间(干流和支流)变化,导致了三峡库区水体固氮微生物多样性和群落结构的时空差异性。其中,温度和pH的时空差异是控制三峡库区固氮微生物群落组成差异的主要环境因素。另外,由于克隆文库分析方法本身的技术局限性,即该方法仅能探测到相对丰度 > 0.1%的微生物种群,而对稀有微生物种群(相对丰度 < 0.1%)检测能力有限[61],所以本研究工作结果仅能揭示三峡库区水体中优势固氮微生物群落的多样性及其分布规律。鉴于稀有微生物种群在环境生物地球化学循环中的重要作用[62],因此十分必要在后续工作中采用高通量测序手段深入研究三峡库区水体和沉积物固氮微生物群落构成。综上,本研究结果有助于进一步理解三峡库区水体氮元素的来源和及其循环过程,并可为全面评价库区水体环境变化提供基础研究数据。

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