| Rice paddy is a special land use type in argriculture, which plays an important role in maintaining food safty and soil carbon in China. Carbon sequentation and greenhouse gases (GHGs) mitigation in paddy soils is an important strategy in addressing climatic change. However, rice paddies in China have been increasingly subject to heavy metal pollution since1950s’, expecially in the area of Pearl River delta, Xiang river basin and lower Yangtze region. Heavy metal pollution in paddy soils not only threats the food safty, but probably affects soil biochemical processes, which may further influence soil carbon and nitrogen cycling as well as GHGs production and emission. Thus, study in changes of microbial groups and activities in heavy metal polluted rice paddies, especially in variation of soil microbial community structure, abundance and activity related to GHGs production, could be helpful to understand the carbon sequestration and mitigation in heavy metal polluted rice paddies; and could provide the scientific basis for maintaining and enhancing carbon stock in paddy soils.In the present study, topsoils were collected from heavy metal polluted rice paddies and their counterparts in four sites (Yixing [YX], Dexing [DX], Dayu [DY] and Dabaoshan [DBS]) across South China to conduct a cross-site study, to investigate soil microbial community structure and activity with several methods. Heavy metal contents were determined and the pollution degree was analyzed. Soil basic physicochemical properties and microbial biomass carbon and nitrogen were examined. Culturable colonies of bacteria and fungi were measured with plate counting method; active microbial biomass and community were detected by phospholipids fatty acids (PLFAs) analysis; while the abundance and community structure diversity of bacteria, fungi, methanogens, methanotrophs, ammonia oxidizing bacteria (AOB) and archaea (AOA) as well as denitrifying bacteria were detected with real time PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE) respectively, and some important bands retrieved from DGGE gel were sequenced for phylogenetic analysis. Moreover, the parallel studies of four enzymes (Invertase, Amylase, β-Glucosidase and Polyphenol oxidase) relating to carbon turnover in soil, of soil basal respiration, substrate induced respiration, methanogenic activity, methane oxidation activity, nitrifying activity and denitrifying activity were also conducted. Compared to the corresponding background soil, changes under pollution in the microbial communities, abundances and activities, as well as the relationship among them were investigated. The main results were as follows:1. Changes in soil microbial biomass, community structure, enzyme activity and soil respiration under heavy metal pollution1) Compared with the corresponding background soils, soil microbial biomass carbon (SMBC) and nitrogen (SMBN) had a decreased trendy in all the polluted soils, and microbial quotient reduced significantly. Cultural population size, PLFA content and gene copies of fungi simultaneously showed a consistent decreace in all the polluted soils at different degrees. However, bacteria did not reveal so consistent change in the different polluted soils. Moreover, the ratio of fungal-to-bacterial PLFA contents decreased significantly in all the polluted soils, at a degree of6%to50%. Thus, fungi appeared to be more sensitive than bacteria in heavy metal polluted paddy soils, and fungal dominance decreased. And the decrease of microbial quotient under pollution was significantly connected to the decrease of fungal-to-bacterial ratio. Furthermore, there did occur a significantly increase in metabolic quotient (qCO2)(at a degree of7%to70%) under pollution across the sites. And the increase of qCO2was sharply related to the decrease of microbial quotient; and slightly related (p=0.10) to the decrease of fungal-to-bacterial PLFAs ratio. These observations supported a shift of microbial community with decreased fungal dominance and thus C utilization efficiency under pollution in rice paddies, which may caused the decrease of microbial quotient and increase in qCO2.2) Fatty acid18:4w3c was detected in all polluted soils, while only in the background soils of DY; whereas, the18:1w5c was not detected in the polluted soils, except in that of YX where its content decreased. The concentration of fatty acid18:1w9t (one of fungal biomarkers) reduced in all the polluted soils. Moreover, the ratio of G+to G-bacteria and of saturated to monounsaturated fatty acids (indexes of environmental stress) increased significantly in all the polluted soils compared with their counterparts. Comparison of microbial community structure by principal component analysis (PCA) of PLFA composition and bacterial and fungal DGGE profiles revealed difference between the polluted soils and their counterparts.3) The four studied enzymes (Invertase, Amylase, β-Glucosidase and Polyphenol oxidase) responded differently to heavy metal pollution across the studied sites. Compared with their counterparts, the activities of all the four enzymes did not change significantly in the polluted soils of YX and DBS. In the polluted soils of DY, the activities of invertase and amylase decreased significantly,β-Glucosidase activity did not change, while polyphenol oxidase activity increased. In the polluted soils of DX, β-Glucosidase and polyphenol oxidase activities reduced significantly, yet the activities of other two enzymes did not change clearly.4) Lag time before the priming CO2flush induced by glucosamine, cellobiose or4-hydroxybenzoate in all the polluted soils was longer than that in their counterparts, indicating decreased catabolic versatilities in heavy metal polluted soils. Furthermore, when cellobiose was decomposed actively, total, bacterial and fungal PLFA contents as well as fungal-to-bacterial ratio decreased significantly under pollution; difference of microbial community between polluted and background soils was smaller than that in soils without substrate amended, and the active community of bacteria shifted, yet that of fungi did not change under pollution.2. Changes in soil microbial community structure, abundance and activity with special reference to CH4production and oxidation under heavy metal pollution1) Comparison of the community structures of both methanogens and methanotrophs by PCA of DGGE profiles revealed difference along PC1or PC2between the polluted soils and their counterparts.Phylogenetic analysis showed that the retrieved species of methanogens from the polluted soils of YX, DX and DBS affiliated with Methanobacteriacea, Methanocellales (Rice cluster) and Methanospirillaceae, respectively. The methanotrophs’clones from the polluted and background soils in DX affiliated with type I methanotrophs, while they grouped into two different clusters. Two clones from DY polluted soils, belonging to type I and typeⅡ methanotrophs respectively, did not grouped into the same cluster with that from their counterparts. These indicated that, to an extent, the methanothrophs in the polluted soils of DX or DY differed in phylogenesis from that in their counterparts.2) In the polluted soils, the abundances of methanogens decreased significantly in DY, yet did not change clearly in other sites; while its diversities reduced in DY, did not change in DBS, increased significantly in YX and DX. However, the abundances of methanotrophs reduced significantly in the polluted soils, at a degree of16%to74%, except in DX, while its diversities decreased clearly in the polluted soils of YX and DX, did not change in that of DY, but increased significantly in that of DBS.3) The methanogenic activity decreased significantly in the polluted soils of DY, yet did not change clearly in those of other sites; while the methane oxidation activity appeared to be more sensitive to metal pollution, the activity decreased significantly in the polluted soils, at a degree of37%to56%, except in DX.3. Changes in soil microbial community structure, abundance and activity with special reference to N2O production under heavy metal pollution(1) Ammonia oxidizers’community structure and abundance as well as nitrifying activity1) Comparison of the community structures of AOB and AOA by PC A of DGGE profiles revealed that the difference of AOB community between the polluted and background soils was only found in YX and DBS, while the AOA community differed along PC1or PC2in all the polluted soils from their counterparts.Phylogenetic analysis showed that all the retrieved AOB sequences in this study fell into different clusters in the genus Nitrosospira. Clones retrived from the background soils in YX and DBS respectively grouped into Cluster3a.1and Cluster4, which almost can not be detected in the polluted soils. These indicated some species in Cluster3a.1and Cluster4were more sensitive to heavy metal pollution. Two AOA sequences from the polluted soils in YX and DY distributed in soil/sediment cluster of phylum Crenarchaeote, while other three clones fall outside of all of the known three clusters; whereas, clones from the background soils in DY affiliated with water column cluster.2) AOB abundances decreased significantly in the heavy metal polluted soils of DX and DBS, did not change clearly in other two sites; while it diversities did not change in DX polluted soils, decreased clearly in DBS, increased in other two sites. While AOA abundances and diversities did not change significantly in the polluted soils of YX, decreased in that of DX, but changed inconsistent in that of DY or DBS. 3) Nitrifying activity was inhibited significantly in the polluted soils of DX and DBS, at a degree more than60%, but did not change clearly in other two sites.(2) Denitrifers’community structure and abundance as well as denitrifying activity1) Comparison of the community structures of nirK-and nosZ-denitrifiers by PCA of DGGE profiles revealed difference along PC1or PC2between the polluted soils and their counterparts, except the nirK community in DBS.All the retrived nirK clones did not belong to either α-or β-proteobacteria, while most of the nosZ species affiliated with a-proteobacteria. All the nirK and nosZ clones were not closely related to known cultural species in phylogenesis, and were widespread among different bacterial genera. While in a single site, the nirK-or nosZ-denitrifers from the polluted and background soils were different in phylogenesis to an extent.2) Abundances of nirK and nosZ decreased significantly in the metal polluted soils in YX and DY, did not change clearly in other sites. Diversities of nirK reduced significantly in DY polluted soils, did not change in other soils; while that of nosZ increased clearly in the polluted soils in DX and DBS, yet not change significantly in other sites.3) Denitrifying activity reduced significantly in polluted soils in YX, DX and DY, and N2O production rate also reduced more or less under pollution in the three sites, yet N2O reduction rate decreased significantly in DX site only. These suggested that comparing with N2O reduction activity, N2O production activity may be more inhibited in the relatively seriously polluted paddy soils.In a word, in the four heavy metal polluted rice paddies, there did occur a decline in fungal dominance, in microbial quotient and an increase in metabolic quotient, which were the common change and may exert an impact on soil biogeochemical process related to C metabolism and CO2evolution in polluted rice paddies. Compared with methanogens and methanogenic activity, methanotrophs and methane oxidation activity seemed to be more sensitive to heavy metal pollution in paddy soils, which probably enhance CH4emission from paddy soils and deserve more field studies to examine. However, the changes of ammonia oxidizers and nitrifying activity, as well as denitrifers and denitrifying activity varied in different soils or due to different heavy metal elements or contents.The changes in the several microbial activities detemined in this study had ambiguous relationships with the changes in community structures or diversities of the related functional microorganisms. However, under heavy metal pollution, increase in qCO2was related to decrease in fungal-to-bacterial PLFAs ratio (p=0.10); methanogenic activity and methanogenes’abundance, methane oxidation activity and methanotrophs’abundance, as well as nitrifying activity and AOB abundance reduced significantly and consistently or unchanged simultaneously. These indicated that, when soil microbial diversities maintained stability in a certain extent, microbial activities appeared to be more related to their abundances. However, no significant relationship between the change in microbial activity and in its abundance were observed in this study, which indicated that other aspects except for microorganisms probably should be concerned to explore the changes in GHGs emission under heavy metal pollution. |
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