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Analysis of the effect of adding Bacillus on the physicochemical factors and bacterial community structure in ponds

  • Corresponding author: Guangjun WANG, gjwang@prfri.ac.cn
  • Received Date: 2018-11-02
    Accepted Date: 2019-03-11
  • In order to study the effect of adding Bacillus in the pond on the physicochemical factors and bacterial community structure, high throughput sequencing technology was used to analyze the bacterial community structure in water and sediment of the experimental group (adding Bacillus in pond) and the control group (without adding in pond). At the same time, the physicochemical parameters in water and sediment of the two groups were collected. The results indicated that the contents of TN, ${\rm{NH}}_4^ + $-N, NO3—N in water were significantly lower than those of the control group (P<0.05), but the contents of ${\rm{NO}}_3^ - $-N, ${\rm{NH}}_4^ + $-N, TN and TP in sediment were significantly higher than those of the control group (P<0.05). The abundance of Chlamydomonas debaryana, Zymomonas, Roseomonas, Dechloromonas and Chitinophaga in water of the experimental group was significantly higher than that in the control group (P<0.05). Among of these bacterial communities, Chlamydomonas debaryana have the function of removing ${\rm{NH}}_4^ + $-N, Dechloromonas and Chitinophaga have the function of removing ${\rm{NO}}_3^ - $-N, Zymomonas and Roseomonas have the function of denitrification. The Chao1 and Shannon index in water of the experimental group were significantly higher than those in the control group (P<0.05). Hydrogenophaga, Geothermobacter and Haliscomenobacter were negatively correlated with ${\rm{NO}}_3^ - $-N and TP, and positively correlated with ${\rm{NH}}_4^ + $-N and TN. Above all, these results showed that adding Bacillus to the pond would lead to the variations of bacterial community structure, thus realizing the regulation of physicochemical factors in the pond. And the results are meaningful for reducing the pollution of aquaculture tail water and water environment.
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  • [1] 陈学洲, 李苗, 张溢卓. 中国水产养殖保险的现状分析及政策建议——以广东、浙江、宁波、上海等地为例[J]. 中国水产, 2016(5): 38-40. doi: 10.3969/j.issn.1002-6681.2016.05.016Chen X Z, Li M, Zhang Y Z. Analysis of current situation of aquaculture insurance in China and policy suggestions - Taking Guangdong, Zhejiang, Ningbo, Shanghai and other places as examples[J]. China Fisheries, 2016(5): 38-40(in Chinese). doi: 10.3969/j.issn.1002-6681.2016.05.016
    [2] Yang P, Lai D Y F, Jin B S, et al. Dynamics of dissolved nutrients in the aquaculture shrimp ponds of the Min River estuary, China: Concentrations, fluxes and environmental loads[J]. Science of the Total Environment, 2017, 603-604: 256-267. doi: 10.1016/j.scitotenv.2017.06.074
    [3] 张峰峰, 谢凤行, 周可, 等. 利用复合微生物降解养殖水体中亚硝酸盐的初步研究[J]. 水产科学, 2012, 31(10): 593-596. doi: 10.3969/j.issn.1003-1111.2012.10.005Zhang F F, Xie F X, Zhou K, et al. Removal of nitrite from aquaculture water by a mixture of microorganisms[J]. Fisheries Science, 2012, 31(10): 593-596(in Chinese). doi: 10.3969/j.issn.1003-1111.2012.10.005
    [4] 周涛. 草鱼养殖池塘内细菌群落及益生菌对水质和细菌群落的影响[D]. 杭州: 浙江大学, 2012.Zhou T. Bacterial community in grass carp farming ponds and the effects of probiotics on water quality and bacterial community[D]. Hangzhou: Zhejiang University, 2012(in Chinese).
    [5] Qin Y, Hou J, Deng M, et al. Bacterial abundance and diversity in pond water supplied with different feeds[J]. Scientific Reports, 2016, 6: 35232. doi: 10.1038/srep35232
    [6] Islam M S. Nitrogen and phosphorus budget in coastal and marine cage aquaculture and impacts of effluent loading on ecosystem: Review and analysis towards model development[J]. Marine Pollution Bulletin, 2005, 50(1): 48-61. doi: 10.1016/j.marpolbul.2004.08.008
    [7] Guo L G, Li Z J. Effects of nitrogen and phosphorus from fish cage-culture on the communities of a shallow lake in middle Yangtze River basin of China[J]. Aquaculture, 2003, 226(1-4): 201-212. doi: 10.1016/S0044-8486(03)00478-2
    [8] Páez-Osuna F, Guerrero-Galván S R, Ruiz-Fernández A C. Discharge of nutrients from shrimp farming to coastal waters of the gulf of California[J]. Marine Pollution Bulletin, 1999, 38(7): 585-592.
    [9] 张大弟, 张晓红, 章家骐, 等. 上海市郊区非点源污染综合调查评价[J]. 上海农业学报, 1997, 13(1): 31-36.Zhang D D, Zhang X H, Zhang J Q, et al. Integrated research and evaluation on nonpoint source pollution in shanghai suburbs[J]. Acta Agriculturae Shanghai, 1997, 13(1): 31-36(in Chinese).
    [10] 戴修赢. 苏州地区七种养殖池塘水质及其氮、磷收支研究[D]. 苏州: 苏州大学, 2010.Dai X Y. Studies on the water quality and nitrogen, phosphorus budgets of seven types of aquaculture ponds in Suzhou region[D]. Suzhou: Soochow University, 2010(in Chinese).
    [11] 李卓佳, 林亮, 杨莺莺, 等. 芽孢杆菌制剂对虾池环境微生物群落的影响[J]. 农业环境科学学报, 2007, 26(3): 1183-1189. doi: 10.3321/j.issn:1672-2043.2007.03.075Li Z J, Lin L, Yang Y Y, et al. Influence of bacillus on the microbial communities in shrimp ponds[J]. Journal of Agro-Environment Science, 2007, 26(3): 1183-1189(in Chinese). doi: 10.3321/j.issn:1672-2043.2007.03.075
    [12] 林亮. 芽孢杆菌制剂对虾池微生物群落以及对虾肠道菌群的影响[D]. 广州: 暨南大学, 2005.Lin L. The effect of Bacillus probiotics on microbial communities and shrimp intestinal microflora[D]. Guangzhou: Jinan University, 2005(in Chinese).
    [13] Park H I, Choi Y J, Pak D. Autohydrogenotrophic denitrifying microbial community in a glass beads biofilm reactor[J]. Biotechnology Letters, 2005, 27(13): 949-953. doi: 10.1007/s10529-005-7654-x
    [14] 杨苗青. 自清洗式活性生物滤池的启动运行和生物特性研究[D]. 郑州: 郑州大学, 2016.Yang M Q. Study on start-up performance and biological characteristics of a self-cleaning activated bio-filter[D]. Zhengzhou: Zhengzhou University, 2016(in Chinese).
    [15] Zhang B, Wang L J, Hasan R, et al. Characterization of a native algae species Chlamydomonas debaryana: Strain selection, bioremediation ability, and lipid characterization[J]. BioResources, 2014, 9(4): 6130-6140.
    [16] 王海燕, 周岳溪, 戴欣, 等. 16S rDNA克隆文库方法分析MDAT-IAT同步脱氮除磷系统细菌多样性研究[J]. 环境科学学报, 2006, 26(6): 903-911. doi: 10.3321/j.issn:0253-2468.2006.06.006Wang H Y, Zhou Y X, Dai X, et al. Bacterial diversity study for the simultaneous nitrogen and phosphorus removal system (MDAT-IAT) by 16S rDNA cloning method[J]. Acta Scientiae Circumstantiae, 2006, 26(6): 903-911(in Chinese). doi: 10.3321/j.issn:0253-2468.2006.06.006
    [17] Shu D T, He Y L, Yue H, et al. Metagenomic and quantitative insights into microbial communities and functional genes of nitrogen and iron cycling in twelve wastewater treatment systems[J]. Chemical Engineering Journal, 2016, 290: 21-30.
    [18] 董媛媛, 乔森, 周集体. 电极改性对电极生物膜反硝化反应器硝酸盐氮去除性能的影响[J]. 环境工程学报, 2016, 10(7): 3565-3572. doi: 10.12030/j.cjee.201501200Dong Y Y, Qiao S, Zhou J T. Effects of electrode modification on nitrate removal of hydrogenotrophic denitrification in biofilm electrode reactor[J]. Chinese Journal of Environmental Engineering, 2016, 10(7): 3565-3572(in Chinese). doi: 10.12030/j.cjee.201501200
    [19] Coates J D, Chakraborty R, Lack J G, et al. Anaerobic benzene oxidation coupled to nitrate reduction in pure culture by two strains of Dechloromonas[J]. Nature, 2001, 411(6841): 1039-1043. doi: 10.1038/35082545
    [20] Lim J H, Baek S H, Lee S T. Ferruginibacter alkalilentus gen. nov., sp. nov. and Ferruginibacter lapsinanis sp. nov., novel members of the family ‘Chitinophagaceae’ in the phylum Bacteroidetes, isolated from freshwater sediment[J]. International Journal of Systematic and Evolutionary Microbiology, 2009, 59(10): 2394-2399. doi: 10.1099/ijs.0.009480-0
    [21] 张睿. 枯草芽孢杆菌对铜绿微囊藻抑制效果的研究[D]. 上海: 上海海洋大学, 2015.Zhang R. Inhibiting Effects of Bacillus subtilis on Microcystis aeruginosa[D]. Shanghai: Shanghai Ocean University, 2015(in Chinese).
    [22] Tilman D, Reich P B, Knops J M H. Biodiversity and ecosystem stability in a decade-long grassland experiment[J]. Nature, 2006, 441(7093): 629-632. doi: 10.1038/nature04742
    [23] Flöder S, Jaschinski S, Wells G, et al. Dominance and compensatory growth in phytoplankton communities under salinity stress[J]. Journal of Experimental Marine Biology and Ecology, 2010, 395(1-2): 223-231. doi: 10.1016/j.jembe.2010.09.006
    [24] 王武. 鱼类增养殖学[M]. 北京: 中国农业出版社, 2000.Wang W. Culture and Enhancement of Fishes[M]. Beijing: China Agriculture Press, 2000(in Chinese).
    [25] 张敬旺. 草鱼养殖池塘溶氧收支平衡及关键影响因子的研究[D]. 上海: 上海海洋大学, 2012.Zhang J W. Study on the oxygen budgets of grass carp ponds and its critical impact factors[D] Shanghai: Shanghai Ocean University, 2012(in Chinese).
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Analysis of the effect of adding Bacillus on the physicochemical factors and bacterial community structure in ponds

    Corresponding author: Guangjun WANG, gjwang@prfri.ac.cn
  • 1. Pearl River Fishery Research Institute, Chinese Academy of Fisheries Sciences, Key Laboratory of Tropical & Subtropical Fishery Resource Application and Cultivation, Ministry of Agriculture, Guangzhou    510380, China

Abstract: In order to study the effect of adding Bacillus in the pond on the physicochemical factors and bacterial community structure, high throughput sequencing technology was used to analyze the bacterial community structure in water and sediment of the experimental group (adding Bacillus in pond) and the control group (without adding in pond). At the same time, the physicochemical parameters in water and sediment of the two groups were collected. The results indicated that the contents of TN, ${\rm{NH}}_4^ + $-N, NO3—N in water were significantly lower than those of the control group (P<0.05), but the contents of ${\rm{NO}}_3^ - $-N, ${\rm{NH}}_4^ + $-N, TN and TP in sediment were significantly higher than those of the control group (P<0.05). The abundance of Chlamydomonas debaryana, Zymomonas, Roseomonas, Dechloromonas and Chitinophaga in water of the experimental group was significantly higher than that in the control group (P<0.05). Among of these bacterial communities, Chlamydomonas debaryana have the function of removing ${\rm{NH}}_4^ + $-N, Dechloromonas and Chitinophaga have the function of removing ${\rm{NO}}_3^ - $-N, Zymomonas and Roseomonas have the function of denitrification. The Chao1 and Shannon index in water of the experimental group were significantly higher than those in the control group (P<0.05). Hydrogenophaga, Geothermobacter and Haliscomenobacter were negatively correlated with ${\rm{NO}}_3^ - $-N and TP, and positively correlated with ${\rm{NH}}_4^ + $-N and TN. Above all, these results showed that adding Bacillus to the pond would lead to the variations of bacterial community structure, thus realizing the regulation of physicochemical factors in the pond. And the results are meaningful for reducing the pollution of aquaculture tail water and water environment.

  • 我国是世界渔业生产大国,水产品总产量连续27年位居世界第一,其中73%以上的水产品来自水产养殖[1]。池塘养殖是我国水产养殖的主要生产方式。但随着集约化养殖的发展,池塘水体的污染越来越严重,大量养殖尾水的随意排放造成了环境污染。研究表明,我国每年约有4.77×104吨含氮和3.75×103吨含磷的养殖尾水直接排放到临近水域中[2]。新修订的《水污染防治法》在全国推行之后,养殖尾水的处理已成为被广泛关注的热点问题。为了研究新的池塘养殖模式,减少养殖尾水对水域环境的污染,本团队前期在中山市民众镇开展池塘生态高效养殖技术研究,每天向池塘中添加芽孢杆菌一次,结合水质指标、鱼类摄食情况和养殖周期等参数确定合理的池塘养殖容量。目前已实现养殖全程尾水“零排放”、年产量达到96 000 kg/hm2的效果,有效地减少了养殖尾水的排放。但是对池塘尾水“零排放”的形成原因尚不明确,对其中的微生物群落结构和水体理化因子的关系以及与对照组池塘的差异尚不清楚。

    有研究表明微生物群落结构与池塘水体理化因子之间是相互影响的。水体中微生物的代谢分解显著影响着水体和底泥中营养物质的成分和含量。张峰峰等研究表明在池塘水体中添加复合益生菌制剂可以大幅度降低养殖池塘的硝酸盐和亚硝酸盐的含量[3]。周涛等的研究表明添加外源益生菌可以改变养殖池塘微生物群落结构,进而改善养殖水质[4]。反之,水体中理化因子的变化也可以改变水体中微生物群落的结构,Qin等人的研究表明养殖池塘的水质的变化也在一定程度上引起微生物群落结构的变化,使其中的某些种类增加或减少[5]

    为了研究添加芽孢杆菌对池塘水体中微生物群落结构和理化因子影响,探明在草鱼养殖中微生物群落结构与池塘水体理化因子的关系,本文比较分析了中山市民众镇的三个实验组(添加芽孢杆菌池塘)和三个对照组(普通池塘)表层和底层水体及底泥理化指标,并采用高通量测序技术对表层和底层水体及底泥中细菌群落进行了研究,以期了解添加芽孢杆菌池塘与普通池塘细菌群落构成以及理化因素的差异,为分析评价添加芽孢杆菌池塘细菌对养殖环境的影响提供理论支撑。

    • 采样池塘位于中山市民众镇的三个实验组池塘(面积1公顷,平均水深1.80 m)(定期添加芽孢杆菌),并将与其临近且管理模式相同、放养密度接近的三个池塘作为对照池塘(面积0.97公顷,平均水深1.85 m)。采样时间为2017年8月和9月各一次。在每个池塘中使用5点采样法采集表层水(深度为0.2 m)和底层水(深度为1.2 m)各200 ml。一部分样品被用来立即测量物理化学指标,另一部分样品采集后置于含冰袋的恒温箱中,运回实验室中进一步处理,并在水样采集后12小时内将这些水样通过0.22 μm核孔过滤器(直径:47 mm)过滤,然后保持在−80 °C直到DNA提取。在每个池塘中使用5点采样法采集底泥600 g,置于保温箱中用冰块保存。这些底泥样品中的一部分被用来立即测量物理化学指标,另一部分样品保存在−80 °C直到DNA提取。

    • 用YSI仪器(ProPlus,USA)对每个池塘的温度(T)、溶解氧(DO)、酸碱度(pH)、电导率(Specific Conductance,SPC)、总溶解固体(Total Dissolved Solids,TDS)进行原位测量。采用国标法测定水体中的氨氮(${\rm{NH}}_4^ + $-N)、亚硝酸盐(${\rm{NO}}_2^ - $-N)、硝酸盐(${\rm{NO}}_3^ - $-N)、磷酸盐(${\rm{PO}}_4^ - $-P)、总氮(TN)和总磷(TP)的含量,以及底泥中的氨氮、硝酸盐、总氮和总磷的含量。

    • 高通量测序文库的构建和基于Illumina MiSeq平台的测序由GENEWIZ公司(苏州,中国)完成。本研究使用Qubit 2.0 Fluorometer(Invitrogen,Carlsbad,CA)检测DNA样品的浓度,使用MetaVxTM文库构建试剂盒(GENEWIZ,Inc.,South Plainfield,NJ,USA)构建测序文库。

      用水体DNA提取试剂盒(Omega,USA)提取水体微生物总DNA。用天根土壤提取试剂盒提取底泥微生物总DNA。将提取的水体和底泥总DNA送至苏州金唯智生物科技有限公司,以30-50ng DNA为模板,使用金唯智设计的一系列PCR引物扩增原核生物16SrDNA上包括V3及V4的2个高度可变区。采用包含“CCTACGGRRBGCASCAGKVRVGAAT”序列的上游引物和包含“GGACTACNVGGGTWTCTAATCC”序列的下游引物扩增V3和V4区。另外,通过PCR向16S rDNA的PCR产物末端加上带有Index的接头,以便进行NGS测序。

      使用Agilent 2100生物分析仪(Agilent Technologies,Palo Alto,CA,USA)检测文库质量,并且通过Qubit2.0 Fluorometer(Invitrogen,Carlsbad,CA)检测文库浓度。DNA文库混合后,按Illumina MiSeq(Illumina,San Diego,CA,USA)仪器使用说明书进行2×300 bp双端测序(PE),由MiSeq自带的MiSeq Control Software (MCS)读取序列信息。

    • 双端测序得到的正反向reads首先进行两两组装连接,过滤拼接结果中含有N的序列,保留序列长度大于200 bp的序列。经过质量过滤,去除嵌合体序列,最终得到的序列用于OTU分析,使用VSEARCH(1.9.6)进行序列聚类(序列相似性设为97%),比对的16S rRNA参考数据库是Silva 123。然后用RDP classifier (Ribosomal Database Program)贝叶斯算法对OTU的代表性序列进行物种分类学分析,并在不同物种分类水平下统计每个样本的群落组成。基于OTU的分析结果,采用对样本序列进行随机抽样的方法,分别计算Shannon、Chao1等α多样性指数。

      所有数据均以平均值±标准差(mean±SD)表示。采用单向方差分析方法,对两种池塘样品中水体、底泥理化指标以及水体中微生物属、种的丰度差异进行比较分析。采用RDA(冗余分析)图,运用CANOCO 4.5程序分析池塘水群落组成与环境因子之间的关系。

    2.   结果
    • 表1可知,8月和9月实验组池塘水体中的SPC、TDS显著低于对照组(P<0.05),而T、DO、pH无明显差异(P>0.05)。由图1ab可知,8月实验组池塘水体中${\rm{NO}}_2^ - $-N含量显著高于对照组(P<0.05),而实验组池塘水体中TN、${\rm{NH}}_4^ + $-N、${\rm{NO}}_3^ - $-N含量显著低于对照组(P<0.05),TP、${\rm{PO}}_4^ - $-P含量无明显差异(P>0.05)。由图1cd可知,9月实验组池塘水体中TN、TP、${\rm{NH}}_4^ + $-N、${\rm{NO}}_3^ - $-N含量显著低于对照组(P<0.05),而${\rm{PO}}_4^ - $-P、${\rm{NO}}_2^ - $-N含量无明显差异(P>0.05)。8、9月份两种池塘表层与底层水体中T、DO、pH、SPC、TDS、TN、TP、${\rm{NH}}_4^ + $-N、${\rm{NO}}_3^ - $-N、${\rm{NO}}_2^ - $-N、${\rm{PO}}_4^ - $-P含量无明显差异(P>0.05)。

      样品 sampleT(°C)DO(mg/L)SPC(ms/cm)TDS(mg/L)pH
      z1 30.38±1.6 5.47±1.44 691.20±60.86* 499.80±39.64* 7.53±0.44
      n1 29.53±0.15 6.39±0.24 2775±880.56 1804.83±574.37 7.69±0.085
      z2 31.18±0.27 6.62±1.65 660.00±61.95* 430.30±40.96* 7.29±0.18
      n2 31.03±0.06 8.38±1.22 3113±120.20 1746.33±546.05 7.46±0.43
      注:z1代表8月实验组池塘水样;n1代表8月对照组池塘水样;z2代表9月实验组池塘水样;n2代表9月对照组池塘水样。同列数据上标加*表示与同月份的数据间存在显著差异(P<0.05)。
      Note: z1 represents experimental group ponds water sample in August; n1 represents the August control ponds water sample; z2 represents the September experimental group ponds water sample; n2 represents the September control ponds water sample. The superscript plus * indicates that there is significant difference in the same month (P<0.05).

      Table 1.  Routine water quality indicators of experimental group and          control group

      Figure 1.  Experimental group and control group water physical and chemical indicators (mean±SD)

      表2可知,8月实验组池塘底泥pH、${\rm{NH}}_4^ + $-N、TN和TC含量显著高于对照组(P<0.05),而${\rm{NO}}_3^ - $-N和TP含量无明显差异(P>0.05)。9月实验组池塘底泥${\rm{NO}}_3^ - $-N、${\rm{NH}}_4^ + $-N、TN和TP含量显著高于对照组(P<0.05),而pH和TC含量无明显差异(P>0.05)。

      样品 samplepHn=3;${\rm{NH}}_4^ + {\rm -N(mg/kg)}$n=3;${\rm{N0}}_3^ - {\rm -N (mg/kg)}$TN(mg/kg)TC(mg/kg)TP(mg/kg)
      z1 7.64±0.09 52.23±20.28* 3.17±1.93 2 360±114.02* 4 972 000±663 716.81* 2 024±220.41
      n1 7.61±0.13 11.02±3.19 0.75±0.37 1 500±435.89 3 826 666.67±283 607.71 1 750±111.36
      z2 7.74±1.24 96.88±11.73* 0.97±0.03* 2 360±270.19* 3 763 333.33±852 310.59 1 680±44.72*
      n2 7.72±0.9 49.53±9.61 0.49±0.14 1 666.67±404.15 3 986 666.67±680 612.47 1 170±315.75
      注:z1代表8月实验组池塘底泥样品;n1代表8月对照组池塘底泥样品;z2代表9月实验组池塘底泥样品;n2代表9月对照组池塘底泥样品。同列数据上标加*表示同月份的数据间存在显著差异(P<0.05)。
      Note: z1 represents experimental group ponds sediment sample in August; n1 represents the August control ponds sediment sample; z2 represents the September experimental group ponds sediment sample; n2 represents the September control ponds sediment sample. The superscript plus * indicates that there is significant difference in the same month (P<0.05).

      Table 2.  Physical and chemical indicators of sediment in two types of ponds in          August and September

    • 图2可得实验组池塘水体Chao1指数和Shannon指数显著高于对照组(P<0.05),实验组池塘底泥Chao1指数和Shannon指数显著高于对照组(P<0.05)。

      Figure 2.  Bacterial community diversity index in water and sediment of two kinds of ponds (mean±SD)

    • 图3可知,在纲水平下,实验组池塘水体细菌结构按照相关丰富度由高到低排序依次为β-变形菌(Betaproteobacteria)、放线菌(Actinobacteria)、α-变形杆菌纲(Alphaproteobacteria)、Chloroplast、蓝藻细菌(Cyanobacteria)、鞘脂杆菌纲(Sphingobacteriia)、丙型变形菌纲(Gammaproteobacteria)、酸微菌纲(Acidimicrobiia)、绿菌纲(Chlorobia)、变形菌纲(Deltaproteobacteria)。对照组池塘水体细菌按照相关丰富度由高到低排序依次为β-变形菌、α-变形杆菌纲、放线菌、蓝藻细菌、鞘脂杆菌纲、丙型变形菌纲、Flavobacteriia、酸微菌纲、绿菌纲。实验组池塘表层水体细菌群落结构与底层水体细菌群落结构无显著差异(P>0.05),对照组池塘表层水体细菌群落结构与底层水体细菌群落结构也无显著差异(P>0.05)。实验组池塘底泥细菌按照相关丰富度由高到低排序依次为Unclassified、δ变形菌纲、β-变形菌、拟杆菌属(Bacteroidia)、绿菌纲、Bacteroidetes vadinHA17、丙型变形菌纲、厌氧绳菌纲(Anaerolineae)、螺旋体属(Spirochaetes)、Ignavibacteria。对照组池塘底泥细菌按照相关丰富度由高到低排序依次为Unclassified、δ变形菌纲、丙型变形菌纲、β-变形菌、Sphingobacterii、拟杆菌属、Bacteroidetes vadinHA17、厌氧绳菌纲、Ignavibacteria、Flavobacteriia。两组池塘水体和底泥中优势菌的种类有些差异,均在第二位上的菌落开始出现不同,特别在水体中位于对照组第四位的蓝藻细菌,在实验组数量更少,两者差异显著(P<0.05)。

      Figure 3.  Composition of bacteria communities in two types of ponds water bodies and sediments at the class level

      图4可知,在属水平下,实验组池塘水体细菌按照相关丰富度由高到低排序依次为UnclassifiedhgcI cladeAmbiguous taxaCL500-29 marine groupMWH-UniP1 aquatic groupPolynucleobacter、浮丝藻属(Planktothrix)、LimnobacterAlpinimonasFluviicola。对照组池塘水体细菌按照相关丰富度由高到低排序依次为UnclassifiedFluviicolaAmbiguous taxaMWH-UniP1 aquatic grouphgcI cladeCL500-29 marine groupLimnobacterDinghuibacterAcidibacterHaliscomenobacter。实验组池塘表层水体细菌群落结构与底层水体细菌群落结构无显著差异(P>0.05),对照组池塘表层水体细菌群落结构与底层水体细菌群落结构也无显著差异(P>0.05)。在属水平下,实验组池塘水体细菌中的Polynucleobacter、浮丝藻属、Alsobacter、噬氢菌属(Hydrogenophaga)、脱氯单胞菌(Dechloromonas)、发酵单胞菌属(zymomonas)、玫瑰单胞菌(Roseomonas)、噬几丁质菌属(Chitinophaga)的绝对丰富度显著高于对照组池塘(P<0.05),实验组池塘水体细菌中的GeothermobacterHaliscomenobacterKomvophoron kgarii str. Wanggoolba creekIgnavibacteriumThiobacillusLewinellaSulfuritaleaGuillardia theta的绝对丰富度显著低于对照组池塘(P<0.05)。实验组池塘底泥细菌按照相关丰富度由高到低排序依次为UnclassifiedAmbiguous taxaGeobacterHydrogenispora、脱氯单胞菌、DesulfatiglansSyntrophorhabdusGeothermobacterSpirochaeta 2Smithella。对照组池塘底泥细菌按照相关丰富度由高到低排序依次为UnclassifiedAmbiguous taxaGeothermobacterFluviicolaDesulfatiglansBD1-7 cladeGeobacterSyntrophorhabdusSva0081 sediment groupCaldithrix。在属水平下,实验组池塘底泥细菌中的GeobacterHydrogenisporaPolynucleobacter,脱氯单胞菌,Spirochaeta 2CetobacteriumErcella,噬几丁质菌属的绝对丰富度显著高于对照组池塘(P<0.05),实验组池塘底泥细菌中的FluviicolaGeothermobacterCaldithrixActibacterThioalkalispiraThaueraDesulfuromonasDehalococcoidia bacterium SCGC AB-540-C11的绝对丰富度显著低于对照组池塘(P<0.05)。

      Figure 4.  Composition of bacteria communities in two types of ponds water bodies and sediments at the genus level

      表3可知,在种水平下,实验组池塘水体细菌中的Ambiguous taxaEuglenaria caudataEuglenaria anabaenaPhacus limnophilaLepocinclis spirogyroidesChlamydomonas debaryanaTerrimonas sp. 16-45a的绝对丰富度显著高于对照组池塘(P<0.05)。由表4可知,在种水平下,实验组池塘底泥细菌中的Iron-reducing bacterium enrichment culture clone fea 2 a6Candidate division wor 1 bacterium dg 54 3Candidatus peribacter riflensis的绝对丰富度显著高于对照组池塘(P<0.05)。

      样品
      sample
      Ambiguous
      taxa
      Euglenaria caudataEuglenaria anabaenaPhacus
      limnophila
      Lepocinclis spirogyroidesChlamydomonas debaryanaTerrimonas sp.
      16-45a
      z1 8 621.30±1 408.85* 7.4±5.3* 54.90±20.56* 17.8±15.2* 15.90±12.96* 23.10±11.68* 2.25±1.26*
      n1 5 332.83±571.97 0.17±0.41 3.83±3.92 0 0 0 0
      z2 9 323.00±761.55* 87.70±70.76* 33.50±26.93* 40.80±26.12* 15.40±15.41* 3.40±3.13* 2.00±0.82*
      n2 8 330.00±943.25 0 0.17±0.41 0.83±1.60 0 0 0
      注:z1代表8月实验组池塘水样,n1代表8月对照组池塘水样,z2代表9月实验组池塘水样,n2代表9月对照组池塘水样. 同列数据上标加*表示同月份的数据间存在显著差异(P<0.05)。
      Note: z1 represents experimental group ponds water sample in August, n1 represents the control ponds water sample in August, z2 represents experimental group ponds water sample in September, and n2 represents the September control ponds water sample. The superscript plus * indicates that there is significant difference in the same month (P<0.05).

      Table 3.  Absolute abundance of bacteria in the experimental group ponds water and ordinary pond       water at the species level (P<0.05)

      样品 sampleIron-reducing bacterium enrichment culture clone fea 2 a6Candidate division wor 1
      bacterium dg 54 3
      Candidatus peribacter riflensis
      z1 46.00±11.25* 22.40±9.91* 12.40±5.22*
      n1 15.00±6.25 7.00±7.21 1.67±1.15
      z2 33.50±5.20* 30.20±12.91* 9.50±4.36*
      n2 14.67±1.53 9.33±6.81 1.30±0.58
      注:z1代表8月实验组池塘底泥样品,n1代表8月对照组池塘底泥样品,z2代表9月实验组池塘底泥样品,n2代表9月对照组池塘底泥样品。同列数据上标加*表示与同月份的数据间存在显著差异(P<0.05)。
      Note: z1 represents the August experimental group ponds sediment sample, n1 represents the August control ponds sediment sample, z2 represents the September experimental group ponds sediment sample, and n2 represents the September control ponds sediment sample. The superscript plus * indicates that there is a significant difference in the same month (P<0.05).

      Table 4.  the absolute abundance of bacteria with significant differences in experimental group ponds sediments and         common pond sediments at the species level (P<0.05)

    • 图5的RDA图可知,水体细菌群落中的噬氢菌属、GeothermobacterHaliscomenobacter与硝酸盐、总磷呈负相关性,与氨氮、总氮呈正相关性。浮丝藻属、脱氯单胞菌、玫瑰单胞菌、发酵单胞菌属与氨氮、总氮呈负相关性。

      Figure 5.  Reduplication Analysis (RDA) of Bacterial Community and Environmental Variables in Water

    3.   讨论
    • 水产养殖尾水的排放给周围环境造成的污染越来越受到关注。Islam等人的研究表明,在饲料系数为2.5,饲料氮、磷含量分别为6.5%和1.4%,鱼体氮、磷含量分别为3%和1%的情况下,每生产一吨的鱼,将有132.5 kg的氮和25.0 kg的磷被释放到环境中去[6]。Guo等人认为每产出一吨鱼的氮、磷释放量分别为159.7 kg和53.3 kg[7]。Páez-Osuna等人关于墨西哥虾类养殖的研究表明,每公顷虾池通过养殖尾水释放的氮和磷的质量分别为15.2 kg和13.5 kg[8]。张大弟等调查表明精养鱼池塘排放尾水中TN和TP的含量分别是101 kg/hm2和11 kg/hm2[9]。戴修赢等人共调查了432.18 hm2的草鱼主养池塘,得出草鱼主养池塘因换水而造成的氮、磷排放量,分别为86.55 kg/hm2和10.21 kg/hm2[10]。而本文所研究的对象——实验组池塘通过定期向池塘中添加芽孢杆菌,结合水质指标、摄食情况和养殖周期等参数确定池塘养殖容量,已经达到养殖全程尾水零排放、年产量96 000 kg/hm2的效果,有效地降低了养殖池塘水体的排放。说明向水体中定期添加有益微生物,结合其他管理措施,可以减少或者不向外界排放养殖尾水。

      水体中微生物的代谢分解对水体和底泥中营养物质的分解有显著影响,有研究表明向养殖池塘中添加益生菌可以改变微生物群落结构,从而达到养殖水质的改善[11-12],这可能是实验组池塘达到养殖尾水“零排放”的机制。在本研究中,实验组池塘水体中的氨氮和硝酸盐含量明显低于对照组池塘,实验组池塘水体细菌在种水平下的Euglenaria caudataEuglenaria anabaenaPhacus limnophilaLepocinclis spirogyroidesChlamydomonas debaryanaPhacus trimarginatusTerrimonas sp. 16-45a的绝对丰富度显著高于对照组池塘。Park等通过DGGE技术发现生物膜反应器中的优势菌噬氢菌属是新型反硝化细菌,可以利用氢作为电子供体来去除硝酸盐[13]。杨苗青等人的研究也得出了类似的结果,他们使用高通量测序的方法,结果表明以噬氢菌属为优势菌的自清洗式活性生物滤池对硝酸盐和氮有去除作用[14]。由于Terrimonas sp. 16-45a属于噬氢菌属,推测其也可能有去除硝酸盐的功能。Zhang等人的研究表明在高压灭菌尾水培养基中培养Chlamydomonas debaryana,结果得出Chlamydomonas debaryana可以去除污水中的氨[15]。因此可以推断,Terrimonas sp. 16-45aChlamydomonas debaryana在两种不同模式池塘丰度的差异性可能是实验组池塘水体总氮和氨氮含量低于对照组池塘的原因之一。同时,实验组池塘底泥细菌中的Iron-reducing bacterium enrichment culture clone fea 2 a6Candidate division wor 1 bacterium dg 54 3Candidatus peribacter riflensis的绝对丰富度显著高于对照组池塘。而实验组池塘底泥中的硝酸盐、氨氮、总氮、总磷含量显著高于对照组。因此我们推测实验组底泥细菌中的Iron-reducing bacterium enrichment culture clone fea 2 a6Candidate division wor 1 bacterium dg 54 3Candidatus peribacter riflensis可能是与某些可以分解氮磷的菌群有一定的拮抗作用,导致这类细菌的数量减少。

      在属水平下,实验组池塘水体细菌中的Polynucleobacter、浮丝藻属、Alsobacter、噬氢菌属、脱氯单胞菌、发酵单胞菌属、玫瑰单胞菌、噬几丁质菌属的绝对丰富度显著高于对照组池塘。水体细菌群落中的噬氢菌属、Geothermobacter、束缚杆菌属与硝态氮、总磷呈负相关性,浮丝藻属、脱氯单胞菌、玫瑰单胞菌、发酵单胞菌属与氨氮、总氮呈负相关性。有研究表明变形菌门是脱氮的功能性菌门[16-17] 。同时PolynucleobacterAlsobacter、发酵单胞菌属、玫瑰单胞菌属于变形菌门,因此可以推测出PolynucleobacterAlsobacter、发酵单胞菌属、玫瑰单胞菌可能具有脱氮的功能。有研究表明噬氢菌属有除去硝酸盐的作用[15-16]。董媛媛等人用16SrDNA和水杨酸紫外分光光度法,得出以噬氢菌属和脱氯单胞菌为主要优势菌的电极生物膜反应器对硝酸盐有很强的去除作用[18]。Coates等人的研究结果也可以表明脱氯单胞菌可以利用有机物提供电子将硝酸盐还原成氮气[19]。同时也有研究报道表明噬几丁质菌属可以还原硝酸盐作为末端电子受体[20]。因此我们推断出实验组池塘水体中PolynucleobacterAlsobacter、发酵单胞菌属、玫瑰单胞菌、噬氢菌属、脱氯单胞菌和噬几丁质菌属细菌群落丰度高于对照组池塘可能是实验组池塘水体中的总氮和硝酸盐含量显著低于对照组池塘的主要原因。

      在纲水平下,实验组池塘水体中的蓝藻细菌绝对丰富度显著低于对照组池塘。有研究表明在水中添加芽孢杆菌可以有效抑制蓝藻细菌的丰富度[21]。蓝藻细菌是诱发水体蓝藻爆发的重要菌群[21],因此我们推测添加芽孢杆菌可以有效抑制水华的发生。

      物种多样性越丰富,其所在的生态系统越稳定[22-23]。在本实验中,实验组池塘水体和底泥细菌群落多样性显著高于对照组池塘,说明实验组池塘生态系统相对于对照组池塘生态系统更加的稳定。

      同时,本研究表明,实验组和对照组表层水体理化指标和底层水体理化指标、水体中微生物的多样性种类、优势种群没有显著差异。这和传统的养殖池塘相关研究结果有一定的差距,王武等研究表明,在精养池塘中,水体存在明显的分层,溶解氧等理化指标垂直分布明显[24]。究其原因,可能是目前池塘养殖中大量增氧机的使用对水体的搅动有关[25]

    4.   结论
    • 在实验组池塘添加芽孢杆菌丰富了养殖池塘细菌群落多样性,表现为实验组池塘水体和底泥细菌多样性显著高于对照组池塘,其中实验组池塘水体中Terrimonas sp. 16-45aChlamydomonas debaryanaPolynucleobacterAlsobacter、发酵单胞菌属、玫瑰单胞菌、噬氢菌属、脱氯单胞菌和噬几丁质菌属细菌丰度显著高于对照组池塘,且实验组池塘总氮,氨氮和硝酸盐含量显著低于对照组池塘。

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