什么是作文提纲R E S E A R C H A R T I C L E
Resistance and resilience of Cu-polluted soil after Cu perturbation,tested bya wide range of soil microbial parameters
端午节要干什么Huan Deng 1,Xiao-Fang Li 2,Wang-Da Cheng 3&Yong-Guan Zhu 1,2
1
Rearch Center for Eco-Environmental Sciences,Chine Academy of Sciences,Beijing,China;2Institute of Urban Environment,Chine Academy of Sciences,Xiamen,China;and 3Jiaxing Academy of Agricultural Sciences,Jiaxing,China
Correspondence:Yong-Guan Zhu,Rearch Center for Eco-Environmental Science,
Chine Academy of Science,PO Box 2871,Beijing 100085,China.Tel.:1861062936940;fax:1861062923563;e-mail:ygzhu@rcees.ac
Received 3February 2009;revid 16May 2009;accepted 24May 2009.
Final version published online 3August 2009.DOI:10.1111/j.1574-6941.2009.00741.x Editor:Max H ¨aggblom
Keywords
ammonia oxidation;free copper activity;general parameters;specific parameters;resilience.
Abstract
Copper (Cu)-polluted and unpolluted soils were ud to study the effect of initial pollution on soil biological resistance and resilience by measuring the respons to perturbation using different parameters.Microbial biomass carbon,substrate-induced respiration and copy numbers of 16S rRNA gene were grouped as general parameters,while potential ammonia oxidation rate and copy numbers of amo A gene were grouped as specific functions.In addition,to illustrate how initial pollution affects soil biological resistance and resilience following condary perturbation,the microbial community structure,together with free Cu 21activities ([Cu 21])in soil pore water and soil pH were also measured after condary perturbation.Results showed that general parameters were
more stable than specific ones.High [Cu 21]and low pH in soil pore water induced by Cu addition may lead to apparently low resistance and resilience,whereas the formation of a tolerant community after Cu pollution,condary perturbation and Cu aging may contribute to resistance and resilience.Analysis of the phospholipid fatty acids profile showed that microbial community structure shifted along with the [Cu 21]gradient.The microbial community structure of the control soil was both resistant and resilient to 400mg kg À1Cu perturbation,whereas other treatments were neither resistant nor resilient.
Introduction
Heavy metal pollution in soil ecosystems has been of major environmental concern.Copper (Cu)contamination is common,mainly due to mining and smelting activities,and also the application of Cu-containing fungicides (Loland &Singh,2004;Karbassi et al .,2008;Wightwick et al .,2008).It has been demonstrated that Cu contamination can perturb the soil microbial community,leading to a decrea of micro-bial biomass and enzyme activities,as well as changes in the microbial community composition (Chander &Brookes,1991;Wang et al .,2007).Conquently,the stability of the microbial community following perturbation directly influ-ences soil functions,and therefore the sustainability of soil quality (Griffiths et al .,2005).Stability compris resistance and resilience,of which the ability of t
he soil to withstand the immediate effects of perturbation is defined as resistance and the ability to recover from perturbation is known as resilience (Pimm,1984).The resistance and resilience of the
soil microbial community following heavy metal perturba-tions have been widely studied in recent years,including pollution by Cu (Griffiths et al .,2001,2004),lead (Pb)(Lazzaro et al .,2006)and mercury (Hg)(Harris-Hellal et al .,2009).However,the influence of initial pollution on soil resistance and resilience to condary perturbation has been less studied and is still being debated (Philippot et al .,2008).There are contrasting theories and results regarding the effect of initial pollution on ecological stability.It has been predicted that nonstresd systems are more stable becau survival in a polluted environment is metabolically costly and thus there is less resistance to additional stressors (Stone et al .,2001).Another theory predicts that stresd systems are more stable becau exposure to an initial stress may cau a development of community tolerance towards this particular stress and such tolerance is positively correlated with the pollutant concentration (Odum,1981;Demoling
&B ˚a
˚a th,2008).Tobor-Kaplon et al .(2005)showed Cu-contaminated soil was less resistant to Pb perturbation.
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Mertens et al.(2007),on the other hand,demonstrated that zinc contamination did not influence soil resistance and resilience to pesticides,freeze–thaw and dry–wet.Griffiths et al.(2005)even found that initial contamination with heavy metal incread soil functional stability to Cu pertur-bation.There are many factors that lead to such disagree-ments and make it impossible to compare results from different experiments.First,the studies ud different microbial parameters as indicators of resistance and resi-lience;however,it has been shown that different soil func-tions or communities may respond differently to a particular stress(Kuan et al.,2007;Carreira et al.,2008). Secondly,various types of perturbation were ud in differ-ent studies;however,both the type and the level of the perturbation will also affect the respon of microbial para-meters(T obor-Kaplon et al.,2005,2006;Banning&Murphy, 2008);Thirdly,the initial pollutant and perturbed pollutant can form mixture contaminants in soil.Previous studies have shown noninteractive and interactive actions for
mixture contaminants.For noninteractive actions,the chemicals act independently,and the toxicity of a mixture can be predicted statistically from the respons to the individual chemicals, and when chemicals act similarly,the toxicity can be additive (Mumtaz et al.,1994).For interactive actions,interaction between pollutants could result in a stronger(synergistic, potentiating,supra-additive)or a weaker(antagonistic,in-hibitive,subadditive,infra-additive)toxicological effect than expected on the basis of additivity(Feron&Groten,2002). Therefore,to make an overall evaluation of resistance and resilience,it is necessary to consider the parameters and perturbations,as well as the interaction between perturbations and contaminants.
It has been demonstrated that pollutants such as heavy metals or organic compounds can alter the microbial com-munity structure,and that this alteration is mainly due to the replacement of nsitive species by more resistant species (B˚a˚a th et al.,2005;Mertens et al.,2006;Ogilvie&Grant, 2008).However,how altered community structure responds to the condary perturbation is hardly known.Phospho-lipid fatty acids(PLFAs)analysis has been widely ud as a reliable tool to evaluate the effect of heavy metals on microbial community structure(B˚a˚a th et al.,1998;Hinojosa et al.,2005).Changes in PLFA profiles are indicative of changes in the overall structure of microbial communities (Frosteg˚a rd et al.,1996)and‘signature’PLFAs can provide information on specific grou
ps of microorganisms prent in a community(Frosteg˚a rd et al.,1993);in addition,PLFA analysis offers an advantage over isolation-bad techniques becau it avoids the lectivity inherent in the isolation of microorganisms(Cavigelli et al.,1995).
T o characterize metal stress,most previous studies ud total metal concentration as an indicator of potential toxicity, but they did not distinguish among different forms of metals in soil.In recent years,the bioavailable fraction of metals in soil has been widely ud to indicate the level of stress.Free metal ion concentration has been considered a better indicator of toxicity and availability(Sunda&Huntsman,2000;Ma et al.,2006a),and free Cu21activity([Cu21])is the most bioavailable species of Cu(Rachou et al.,2007).
In this paper,we evaluated the resistance and resilience of soil microbial communities to Cu perturbation that have been initially expod to Cu pollution for1year.The initial pollution is long,which allows a tolerant community to develop.The objectives of this study were to illustrate(1) how initial pollution affects soil resistance and resilience to additional perturbation,and(2)the characterization of resistance and resilience with different microbial para-meters.For the purpos,Cu-polluted soil was perturbed with different Cu concentrations.Parameters of microbial biomass carbon(C mic),substrate-induced respiration(SIR), potential ammonia oxidation(PAO)rate,w
hich is a repre-ntative key soil function in nitrogen(N)cycling(Gremion et al.,2004),copy numbers of16S rRNA and amo A genes, microbial community structure and[Cu21]and soil pH were investigated before and after perturbation. Materials and methods
Site description
The sampling sites were located in the Experimental Station of Jiaxing Academy of Agricultural Sciences in Zhejiang Pro-vince,Southeast China(30135.30N,120137.60E).The climate is subtropical and wet with an average annual precipitation of 1200mm and mean annual temperature of15.71C.
The sampling sites were in a paddyfield before the year 2000;maize was planted in the sites from2000to2006,then fallowed in2007.Chemical fertilizers[N(as urea)20.0g mÀ2, P2O5(as calcium superphosphate)3.1g mÀ2,K2O(as potas-sium chloride)18.7g mÀ2]were applied annually from2000 to2007.One plot was artificially polluted in May2007with 800mg kgÀ1Cu(in CuSO4)and the other plot was left unpolluted.A mulchfilm was placed1m down in the soil to prevent leakage of soil solution between the two sites.After Cu addition,thefield experienced aflooding period until October and maintained water content clo to its maximum water-holding capacity(MWHC)for rice cultivation.
Soil sampling
公文写作范文Sampling was carried out in May2008.Soil samples were collected from polluted and unpolluted plots(6m2per plot, 3mÂ2m).In each plot,three surface soil samples(0–20cm in depth)were randomly collected in equal amounts. Samples from the same plot were mixed thoroughly to form a reprentative sample for that plot.The fresh soil samples were pasd through a2-mm sieve.After thorough mixing,
294H.Deng et al.
part of the sieved soil was immediately adjusted to 40%of the MWHC and preincubated at 251C for 14days before soil biological stability testing.The remaining sieved soil was air-dried for chemical analysis.
Soil chemical analysis
Soil chemical analysis was conducted bad on standard methods.In brief,soil total Cu was determined by atomic absorption spectrophotometer (Page et al .,1982).Soil organic carbon [total organic carbon (TOC)]was deter-mined by wet oxidation with K 2CrO 4,total N (TN)by Kjeldahl digestion (Bremner &Mulvaney,1982)and soil clay content according to Kettler et al .(2001).
Soil biological stability
Soil incubation and the measurement of biological stability followed the procedures described by Griffiths et al .(2001).Each soil sample was divided into three aliquots with three replicates for each aliquot.Various amount of Cu solution (CuSO 4,analytical reagent)were added to two of the three aliquots to 400and 800mg Cu kg À1dry soil,respectively.The third aliquot was treated as unperturbed control.Accordingly,six treatments were generated:unpolluted control soil without perturbation (C-0)or perturbed with 400mg kg À1Cu (C-4)or with 800mg kg À1Cu (C-8);polluted soil without perturbation (P-0)or perturbed with 400mg kg À1Cu (P-4)or with 800mg kg À1Cu (P-8).After thorough mixing,each treatment of soil was placed into 18vials (500mL).All vials were subquently incubated at a constant 251C for 40days.Soil water content was adjusted to 50%of MWHC.At intervals of 1,3,8,20and 40days after Cu addition,the soils from three randomly lected vials for each Cu treatment were measured to determine C mic ,SIR,PAO rate,copy numbers of 16S rRNA and amo A genes,PLFAs profile and free Cu 21activity and soil pH in extracted soil solution.
Free Cu 21activity and pH in soil solution Soil solutions were extracted at each sampling time using the centrifugation method according to Di-Bonito (2005).In brief,a double-chamber ultrafiltration device (Millipore Cor-poration)was ud to obtain soil solution,and the solutions were filtered through a 0.45-m m nitrocellulo membrane filter before analysis.Free Cu 21activity ([Cu 21])in soil solution
s was measured using a cupric ion-lective electrode (Orion 94-29)coupled with a reference electrode (Orion 900200)and a millivolt meter (Orion 720)(Ma et al .,2006b).The pH of soil solutions was also measured by conventional methods (Tan,2005).
Microbial biomass carbon
Microbial biomass carbon (C mic )was extracted using the protocol described by Vance et al .(1987).Soil samples (10g dry weight equivalent)taken at the end of the incubation were fumigated with ethanol-free CHCl 3and extracted with 0.5mol L À1K 2SO 4.C mic was calculated as the difference between fumigated and unfumigated samples divided by the calibration factor (kc =0.45).The organic carbon in the soil extract was measured using an automated TOC analyr (Allison et al .,2006).
SIR
SIR was measured according to Wada &Toyota (2007).Gluco was added to 10g of soil (dry weight equivalent)at a rate of 5mg g À1dry soil.The vials were aled and incubated for 6h at room temperature.CO 2was trapped in a 0.2M NaOH solution in 15-mL flask placed in the 250-mL vial and determined by titration with 0.05M HCl following the addition of 1M BaCl 2(Bekku et al .,1997).
PAO
PAO rate was measured using the chlorate inhibition method described previously (Xia et al .,2007).Briefly,20mL phos-phate-buffered saline solution (g L À1:NaCl,8.0;KCl,0.2;Na 2HPO 4,0.2;NaH 2PO 4,0.2;pH 7.1),containing 1mM (di-)ammonium sulphate and 50mM sodium chlorate,was added to 5g of soil sample (dry weight equivalent)in a 50-mL centrifuge tube.After 24-h incubation in a rotary shaker at
咖啡作用room temperature,samples were subjected to NO 2À_
N extrac-tion and 5mL of 2M KCl was added to the tubes.After
centrifugation,5-mL aliquots were sampled and NO 2À_
N was measured colorimetrically at 545nm using sulphonamide and naphthylethylene diamide as reagents.The standard curve for
NO 2À_N was generated with KNO 2over a range of 0–0.8mg L À1.
DNA extraction
DNA was extracted from 0.5g (dry weight equivalent)of soil using a Bio 101Fast DNA SPIN Kit for soil (Bio 101Inc.,Vista,CA),following the manufacturer’s instructions.The concentration and quality of the extracted DNA was deter-mined by spectroscopic analysis (Nanodrop,Wilmington,DE)and agaro gel electrophoresis.DNA was stored at À201C before u.
Real-time PCR
The abundance of the functional ammonia monooxygena gene (amo A)and the 16S rRNA gene was determined by real-time PCR using an iCycler iQ 5thermocycler (Bio-Rad,Hercules,CA)(Li et al .,2009).The 25-m L reaction mixtures included 1m L of template DNA,12.5m L of SYBR Premix Ex
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Taq(TaKaRa Bio Inc.,Shiga,Japan),500nM of the primers amo A-1FÃ(GGGGTTTCTACTGGTGGT)a
nd amo A-2R (CCCCTCKGSAAAGCCTTCTTC)for bacterial amo A(Rot-thauwe et al.,1997);F27(AGAGTTTGATCMTG GCTCAG) (Lane,1991)and R1492(TACGGYTACCTTGTTACGACTT) (Suzuki et al.,2000)for16S rRNA gene.For bacterial amo A amplification the protocol was:941C for3min,40cycles consisting of941C for30s,551C for30s,721C for45s.For 16S rRNA gene amplification the protocol was:941C for 4min,30cycles consisting of941C for60s,551C for60s, 721C for60s;and721C for10min,followed by melting curve analysis at65–981C,0.21C per reading,6-s hold.Fluorescence was read during each cycle at831C.
Standard curve
Standard genes of known copy numbers were prepared as described by He et al.(2007).In brief,PCR products were ligated into pGEM-T Easy(Promega)and then transformed into competent Escherichia coli cells following the manufac-turer’s technical manual.Transformed cells were incubated, followed by plating onto Luria–Bertani plates containing 100m g mLÀ1ampicillin,0.5mM IPTG and80m g mLÀ1 X-Gal.Positive colonies were lected and subjected to quencing and plasmid DNA extraction with a MiniBEST Plasmid Purification Kit(TaKaRa).Tenfold rial dilutions of a known copy number of the plasmid DNA were ud to perform real-time PCR assays in triplicate to generate standard curves.The amplification efficiencies ranged from 93.0%to102.5%and the linear coefficient
correlation(R2) of standard curves from0.986to0.993.
PLFA analysis
The soil phospholipids were extracted according to Bossio& Scow(1998).In brief,3g of freeze-dried soil was added to chloroform–methanol–phosphate buffer solution(1:2:0.8, v/v;pH4).After centrifugation,the supernatants were com-bined andfiltered.The phospholipids were parated from neutral lipids and glycolipids using a silicic acid column (Varian Bond Elut SILICA SI500mg).The parated phos-pholipids were subjected to a mild alkaline methanolysis at 501C and the resulting fatty acid methyl esters were detected using an Agilent6820gas chromatograph equipped with an HP-5MS capillary column(0.32mmÂ30m,0.25-m mfilm thickness)and aflame ionization detector.Helium was ud as carrier gas at aflow rate of0.9mL min1.The temperature of the injector was2801C.The time–temperature program for the oven was as follows:initial temperature901C for2min, incread by301C minÀ1until1601C,incread by31C minÀ1 until2801C,final temperature2801C for10min.The PLFAs were identified using a standard qualitative bacterial acid methyl ester mixture(Supelco,Supelco UK,Poole,Dort, UK)that ranged from C11to C20as an external standard.
The quantitative concentration(nmol PLFA gÀ1dry weight
soil)of single PLFA was calculated according to Paloj¨a rvi
(2006).Bad on the data,relative concentrations of differ-
ent PLFAs in percentage(mol%)were derived.The branched,
saturated PLFAs were chon to reprent gram-positive(G1)
bacteria and the monoenoic and cyclopropane unsaturated
fatty acids and saturated fatty acids containing the OH group
reprented gram-negative(GÀ)bacteria(Chen et al.,2008). Data analysis
All statistical tests were performed using SPSS software(version
14.0).Significant differences between means(n=3)were
determined by one-way ANOV A at a level of P o0.05.Perturbed
treatments that showed no significant difference(P o0.05)
精熟with unperturbed treatments at day1were defined as
‘resistant’and at following days as‘resilient’or‘recovered’.
Significant correlations between quantitative descriptors were
determined by applying the Pearson correlation matrix.
The individual PLFAs(a total of26PLFAs were identi-
天蝎座男
fied)were expresd as percentage of the total amount of
PLFAs detected in a soil sample(mol%).PLFA profiles were
analyd initially by principal component analysis(PCA)to
reduce the dimensionality.The results of PCA along two
principal components PC1and PC2were compared by ANOV A.The scores along PC1were subjected to a stepwi regression analysis with the soil[Cu21]and pH to deter-
mine their effects on PLFA profiles.
Results
Soil properties
T otal Cu in control and polluted soil was31and816mg kgÀ1,
respectively.There was no significant difference between
control and polluted soil in TOC,TN and clay content;the
average mean value for the two soil treatments was14g kgÀ1
for TOC,1.5g kgÀ1for TN and316g kgÀ1for clay content. Free Cu21activity
Free Cu21activity in soil pore water was significantly higher
(P o0.05)in polluted soil than in the control soil,and
incread significantly(P o0.05)after Cu addition,com-
pared with the unperturbed treatments.One day after Cu
perturbation,the[Cu21]of the C-8treatment incread
from0.04to9.41mg LÀ1,and that of the P-8treatment
incread from0.73to25.44mg LÀ1(Fig.1).[Cu21]of all
treatments decread at all sampling times,to0.77and
4.56mg LÀ1for the C-8and P-8treatments,respectively,40
days after Cu perturbation.[Cu21]of the C-0treatment was
stable during the40-day incubation time.
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Soil pH
Soil pH was significantly higher (P o 0.05)in the control soil than in the polluted soil.One day after Cu perturbation,the soil pH decread from 6.39to 4.88for the C-8treatment,and from 5.42to 4.75for the P-8treatment,compared with unperturbed treatments C-0and P-0,respectively.The soil pH of all tr
eatments was relatively stable (Æ0.35U)with the order unperturbed 4perturbed,with that of soils perturbed with 400mg kg À1greater than that perturbed with 800mg kg À1throughout the incubation.
Microbial biomass carbon
The microbial biomass carbon of the control soil was significantly higher (P o 0.05)than that of the polluted soil.Compared with the unperturbed C-0treatment,C mic of the C-4and C-8treatments decread significantly (P o 0.05)1day after Cu perturbation.The C mic of the C-4treatment recovered at day 8,but that of the C-8treatment did not recover during the incubation period (Fig.2).
The C mic of the P-4treatment did not change significantly (P Z 0.05)following Cu perturbation compared with the unperturbed P-0treatment,whereas C mic of the P-8treat-ment decread significantly (P o 0.05)1day after pertur-bation,and had not recovered 40days after perturbation.
SIR
The SIR was similar to C mic in the respon to Cu pollution and Cu perturbation.SIR was significantly higher (P o 0.05)in the control soil than in the polluted soil.Compared with the unperturbed C-0treatment,the SIR of both the C-4and C-8treatments significantly decread (P o 0.05)1day after
Cu perturbation;the SIR of the C-4treatment recovered at day 8,whereas that of the C-8treatment did not recover during the 40-day incubation time,although it incread dramatically from day 1to day 40(Fig.3).
Compared with the unperturbed P-0treatment,the SIR of the P-4treatment did not change following Cu perturba-tion.P-8treatment decread significantly (P o 0.05)1day after perturbation and recovered 8days after perturbation.
PAO rate
There was no significant difference (P Z 0.05)between the control soil and the polluted soil in PAO rate.A significant hormetic effect on PAO was obrved in both C-4and C-8treatments 1day after Cu perturbation.However,PAO of perturbed soil sharply decread 3days after perturbation and remained significantly lower than the unperturbed C-0treatment from day 8to day 40(Fig.4).
There was no hormetic effect in the polluted soil.PAO of both the P-4and the P-8treatments decread significantly (P o 0.05)1day after Cu perturbation,and neither of them recovered during the 40-day incubation.
Copy numbers of 16S rRNA and amo A genes The copy numbers of 16S rRNA and amo A genes repre-nted the abundance of total bacterial and ammonia-oxidizing bacteria,respectively.The copy number of 16S rRNA gene was two magnitudes higher than that of amo A gene.At days 1,8and 40,the copy numbers of 16S rRNA gene
0.01
0.1
1
10 +400 +800L o g 10 [C u 2+] (m g L –1)
Days after perturbation
0.01
0.1110Fig.1.Free Cu 21activity ([Cu 21])of control soil (C)and polluted soil (P)after Cu addition during the 40-day incubation.Mean ÆSE (n =3).
020*******
+0
+400+800
m g C k g –1 s o i l
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Days after perturbation
200
400600C
安卓系统更新Fig.2.Microbial biomass carbon of control soil (C)and polluted soil (P)after Cu perturbation during the 40-day incubation.The different letters above the bars indicate statistical differences (P o 0.05,Duncan’s test,n =3)among the soils for the 10(without perturbation),1400(perturbed with 400mg kg À1Cu)and 1800(perturbed with 800mg kg À1Cu)treat-ments,at each sampling time respectively.Bars reprent SEs.
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