Bioaugmentation of a 4-chloronitrobenzene contaminated soil with Pseudomonas putida ZWL73

Bioaugmentation of a 4-chloronitrobenzene contaminated soil with Pseudomonas putida ZWL73Gui-Lan Niu a ,b ,Jun-Jie Zhang a ,Shuo Zhao a ,b ,Hong Liu a ,Nico Boon c ,Ning-Yi Zhou a ,*aState Key Laboratory of Virology,Wuhan Institute of Virology,Chinese Academy of Sciences,Wuhan 430071,China bGraduate School,Chinese Academy of Sciences,Beijing 100049,China cLaboratory of Microbial Ecology and Technology (LabMET),Ghent University,Coupure Links 653,B-9000Gent,BelgiumPseudomonas putida ZWL73can accelerate 4CNB removal in lab-scale soils,causing dynamic changes within intrinsic Actinobacteria and Acidobacteria.a r t i c l e i n f oArticle history:Received 9June 2008Received in revised form 17November 2008Accepted 18November 2008Keywords:4-ChloronitrobenzeneAmmonia-oxidizing bacteria BioaugmentationDenaturing gradient gel electrophoresis Pseudomonas putida ZWL73a b s t r a c tThe strain Pseudomonas putida ZWL73,which metabolizes 4-chloronitrobenzene (4CNB)by a partial-reductive pathway,was inoculated into lab-scale 4CNB-contaminated soil for bioaugmentation purposes in this study.The degradation of 4CNB was clearly stimulated,as indicated with the gradual accumu-lation of ammonium and chloride.Simultaneously,the diversity and quantity of cultivable heterotrophic bacteria decreased due to 4CNB contamination,while the quantity of 4CNB-resistant bacteria increased.During the bioaugmentation,denaturing gradient gel electrophoresis analysis showed the changes of diversity in dominant populations of intrinsic soil microbiota.The results showed that Alphaproteobac-teria and Betaproteobacteria were not distinctly affected,but Actinobacteria were apparently stimulated.In addition,an interesting dynamic within Acidobacteria was observed,as well as an influence on ammonia-oxidizing bacteria population.These combined findings demonstrate that the removal of 4CNB in soils by inoculating strain ZWL73is feasible,and that specific populations in soils rapidly changed in response to 4CNB contamination and subsequent bioaugmentation.Ó2008Elsevier Ltd.All rights reserved.1.IntroductionIncreased industrial production of chemicals,as well as their inappropriate use and accidental leakage is increasing the number of polluted areas.In soil bioremediation,bioaugmentation is considered to be a promising approach to clean up contaminations in specific places (Dechesne et al.,2005).There have been both successful and failed attempts to demonstrate the potential of bioaugmentation in soils (Vogel,1996).The key to success is the survival of introduced bacteria in complex ecosystems (Cunliffe and Kertesz,2006;van Veen et al.,1997).In addition,bioaugmentation by inoculated microbial degraders usually releases free ions or immediate products into soil environment (van Veen et al.,1997),probably influencing indigenous soil microbiota.Knowledge about the fate and activity of introduced bacteria in soil,and their impact on the indigenous microbial populations during bioaugmentation processes,is vital but still poorly understood (Dechesne et al.,2005;Gomes et al.,2005).One important group of soil pollutants,the chlorinated nitro-arenes such as 4-chloronitrobenzene (4CNB),are used asintermediates in the synthesis of drugs,dyes,pesticides and other substances in commerce (Katsivela et al.,1999).In particular,4CNB and its derivatives have been detected as serious environmental pollutants in water and soils (Katsivela et al.,1999),and are known to be toxic and mutagenic (Linch,1974).Furthermore,they are considered as more recalcitrant than other aromatics,since the electron-withdrawing character of chloro-and nitro-substitutes makes the aromatic ring more resistant to enzymatic attacks.Pseudomonas putida ZWL73was isolated from 4CNB-contaminated soil for its ability to grow on 4CNB as a sole source of carbon,nitrogen and energy (Zhen et al.,2006)and shown to degrade 4CNB via a partial-reductive pathway with the release of free ammonium (Xiao et al.,2006;Zhen et al.,2006).Soils typically contain abundant bacteria,representing more than a million bacterial species per gram (dry weight)soil sample (Gans et al.,2005).Some of the most dominant phyla in soils,according to 16S rRNA gene libraries,are Proteobacteria ,Acid-obacteria and Actinobacteria (Janssen,2006).Various methods have been developed to evaluate their structure and diversity shifts correlated to changing environmental factors.Molecular ecological techniques,especially those based on analyses of 16S rRNA and genes involved in metabolic activities after PCR amplification,have allowed cultivation-independent investigations of the microbial*Corresponding author.Tel./fax:þ862787197655.E-mail address:n.zhou@ (N.-Y.Zhou).Contents lists available at ScienceDirectEnvironmental Pollutionjournal homepage:/locate/envpol0269-7491/$–see front matter Ó2008Elsevier Ltd.All rights reserved.doi:10.1016/j.envpol.2008.11.024Environmental Pollution 157(2009)763–771populations in many habitats (Boon et al.,2003;Janssen,2006).Although these PCR-based methods are all susceptible to PCR bias and may not represent a complete or accurate picture of the bacterial populations,they still can provide useful information about shifts in soil microbial populations (Jernberg and Jansson,2002).For example,molecular techniques have been proved valu-able in analyzing the diversity and population structure of ecologically important ammonia-oxidizing bacteria (AOB)in soils (Mahmood et al.,2006),which represent important but relatively slow-growing constituents of soil microbial populations.The aim of this study was to assess the bioaugmentation effects of the inoculated strain ZWL73in soils artificially contaminated with 4CNB.Simultaneously,the changes of the structural compo-sition of dominant indigenous bacterial populations including AOB either in the presence or absence of strain ZWL73were also analyzed by denaturing gradient gel electrophoresis (DGGE).2.Materials and methods2.1.Soil characteristics and preparationThe soil studied was an agricultural loam soil from Wuhan Botanical Garden,Chinese Academy of Sciences,without a history of 4CNB contamination.The soil was taken from the top layer (0–10cm)and sieved in a 2-mm mesh before being stored moist at 4 C until use.The soil had a total carbon content of 1.16%,and a total nitrogen content of 0.092%;its pH was 6.77,and its moisture content was 21%.2.2.Preparation of soil microcosmsMicrocosms were set up in 250-ml glass bottles with tightened screw cap tops.Each microcosm consisted of 73g of native or sterile soil (sterilized by autoclaving for 20min at 121 C,every other day during treatments),corresponding to 60g soil dry weight (dw).Four different sets of microcosms were prepared,all in triplicate,with following treatments:(T1)native soil;(T2)native soil with 4CNB added;(T3)native soil with 4CNB added and with strain ZWL73inoculated;(T4)sterilized soil with 4CNB added and with strain ZWL73inoculated.The microcosms were pre-incubated at 30 C for 24h before they were established.The soilspiking procedure was done by thoroughly mixing 4CNB crystals into soil (1mg g À1dw).Strain ZWL73was grown in Luria–Bertani (LB)medium at 30 C to late exponential phase,and collected by centrifugation at 6000Âg for 10min.The pellet obtained was washed twice with sterile saline (0.85%)and finally resuspended in saline.Both microcosms T3and T4were inoculated to a final concentration of approx.1.5Â108colony-forming units (CFU)g À1dw.The same volume of sterile saline was added to non-inoculated soils (T1and T2)to standardize the final moisture content (w 23%)in each microcosm.After vigorous stirring,bottles containing these microcosms were closed and maintained in the dark at 30 C and then sampled for analysis.The bottles were opened during sampling to ensure that 4CNB mineralization was not impeded by lack of oxygen.2.3.Analytical methodsThe concentration of 4CNB in the soil microcosms was determined by high-performance liquid chromatography (HPLC).This was performed at 30 C with a Gilson 715system equipped with a C18reversed-phase column (25cm Â4.6mm,5m m particle size;Supelco).The mobile phase consisted of methanol (80%)and water (20%)with a flow rate of 0.8ml/min.A Gilson 119UV/vis detector was used at 280nm;retention time was 6.6min for the authentic 4CNB under those conditions.The soil samples (0.50g each)were mixed thoroughly with 1ml methanol by shaking at 200rpm for 30min and filtered before HPLC analysis.Ammonium-N content was determined by colorimetry (Nkonge and Ballance,1982),in which soil samples (0.50g each)were extracted with 2.5ml of 2M KCl and shaken on a rotary shaker at 200rpm for 30min followed by centrifugation at 10,000Âg for 5min.Chloride content was detected by Thermo Orion Model 868Cl Àdetector,in which 4ml MilliQ water was added to soil samples (0.50g each),and shaken on a rotary shaker at 200rpm for 30min followed by centrifugation at 10,000Âg for 5min.2.4.Bacterial enumerationThe cultivable heterotrophic bacteria of the treatments were counted on both non-selective nutrient agar (NA)and selective NA with 4CNB,both containing the fungicide nystatin (25m g ml À1).Simultaneously,the variation in cell numbers of strain ZWL73during 4CNB removal was also monitored by plating the sample on minimum medium (MM)(Liu et al.,2005)agar plate containing nystatin (25m g ml À1)and chloramphenicol (34m g ml À1,for selecting strain ZWL73).The 4CNB crystals (about 50mg)were spread on the lids of inverted plates.Soil dilution series were prepared from 0.20g of wet soil in 0.8ml sterile extracting solution (Na 4P 2O 7,2g l À1)(Cavalca et al.,2002).The suspension was mixed by vortexing for 1min and then shaking at 200rpm for 30min.After a 10-min sedimentation period,appropriate 10-fold dilutions in sterile saline were plated.2.5.DNA extraction and PCR amplification of 16S rRNA gene fragmentsThe UltraClean Soil DNA kit (Mo Bio Labs)was used for extracting DNA from soil according to the manufacturer’s instruction.The primers used in this study are listed in Table 1.Universal primers F984-GC and R1378(V6–V8variable regions of the 16S rRNA gene)were used for amplification of bacterial 16S rRNA gene fragments,according to a published method (Gomes et al.,2005).For the amplification of actinobacterial,alpha-and betaproteobacterial 16S rRNA gene fragments,a nested PCR approach was performed with a procedure described previously (Costa et al.,2006).In the first PCR round,the forward primer was the group-specific primer and the reverse primer was R1492,and in the second PCR round,primers F984-GC and R1378were used.The same nested PCR strategy was performed for the amplification of acidobacterial populations with the following program:an initial denaturation step of 5min at 94 C,followed by 20cycles of denaturation at 94 C for 1min,annealing at 64 C for 1min and elongation at 72 C for 1min,then a final extension step of 10min at 72 C.To amplify the 16S rRNA gene fragments of AOB,a published procedure was followed (Mahmood et al.,2006).The universal primers F27and R1492were used in the first PCR round and the CTO189AB/C with CTO653R pairs were used in the second round.2.6.DGGE analysesDGGE analyses were performed with the Dcode System (Bio-Rad).For Alphap-roteobacteria ,Betaproteobacteria ,Actinobacteria and Acidobacteria analyses,a double gradient gel consisting of 6–9%polyacrylamide with a gradient of 45–60%of denaturant was used.For AOB analysis,6%polyacrylamide gel with a gradient of 45–60%of denaturant was used.The electrophoresis was carried out with 1ÂTAE buffer at 60 C with a constant voltage of 80V for 14h.The gels were silver-stained according to a published method (McCaig et al.,2001).2.7.Cluster analysisA dendrogram was constructed by the Pearson correlation index for each pair of lanes within the gels and cluster analysis with arithmetic mean (UPGMA)algorithms in the Quantity One software version 4.6(Bio-Rad).2.8.Cloning and sequencingSelected bands were excised from silver-stained DGGE gels and then extracted according to the protocol described previously (Schwieger and Tebbe,1998).TheTable 1G.-L.Niu et al./Environmental Pollution 157(2009)763–771764recovered DNA was re-amplified with primers F984-GC and R1378.After confirming the correct electrophoretic mobility of the excised bands by DGGE,the PCR products were ligated into a pGEM-T vector(Promega)and transformed into competent cellsof Escherichia coli DH5a for sequencing.2.9.Nucleotide sequence accession numbersThe sequences of the DGGE bands determined in this study were deposited in the GenBank database under accession numbers EU486825–EU486846.The sequences of clone libraries of PCR-amplified16S rRNA gene fragments of treatment T3were deposited under accession numbers EU520346–EU520391.3.Results3.1.4CNB removal and accumulation of ammonium(NH4þ)and chloride(ClÀ)The variations of4CNB concentration in four different treat-ments were examined(Fig.1a).After30days,the4CNB concen-tration of the control treatment T2(soilþ4CNB)decreased by20% (149Æ104m g gÀ1dw)comparing with the initial concentration.In the bioaugmentation treatments T3(non-sterile soilþ4CNBþZWL73)and T4(sterile soilþ4CNBþZWL73),98% (922Æ136m g gÀ1dw and892Æ35m g gÀ1dw)of the added4CNB in microcosms were removed due to the inoculated strain ZWL73 after6days.After8days the4CNB concentration had decreased from1000to4.33Æ0.47m g gÀ1dw soil in treatment T3.But the removal was slightly slower in treatment T3of the non-sterile soil than that in treatment T4of sterile soil during thefirst4days (Fig.1a).The4CNB concentration of treatment T3decreased by35% (329Æ53m g gÀ1dw)on day2and86%(801Æ236m g gÀ1dw)on day4,in contrast to the76%(685Æ253m g gÀ1dw)and96% (856Æ22m g gÀ1dw)decrease of treatment T4.Concomitant with the gradual removal of4CNB,the accumula-tion of ammonium(Fig.1b)and chloride(Fig.1c)was also observed in treatments T3and T4apparently due to4CNB degradation.The amounts of ammonium and chloride in treatment T4reached 82.5Æ1.9m g gÀ1and231.5Æ28.7m g gÀ1dw,respectively.The molar ratio of4CNB removal to the accumulation of ammonium and chloride was about1:0.70:0.75(treatment T4)on day30.A moderate increase of ammonium concentration was detected in treatment T2after14days.Fig.1c shows that the chloride accu-mulation in treatment T3was relatively slower than that in treat-ment T4during thefirst4days,which was correlated with the degradation rate of4CNB between these two treatments(Fig.1a).3.2.Impact of soil treatments on relative abundance of specific bacterial populations in soil3.2.1.Impact on cultivable bacterial populations in soil microcosmsThe cell numbers of cultivable heterotrophic bacteria of different treatments were determined by plate counts on both NA and4CNB-containing NA plates(Fig.2).The diversity was evaluated by classification on colony shape,size and color.The results showed the growth of cultivable heterotrophic bacteria was apparently repressed in treatment T2in comparison with untreated control soil microcosms(T1)(Fig.2a).The cell numbers of cultivable heterotrophic bacteria decreased from7.7Â107on day0to 9.5Â106CFU gÀ1dw soil on day18in treatment T2.The diversity observation based on colony morphology indicated an obvious decrease from original8types to no more than2types after30days in treatment T2(data not shown).Furthermore,the4CNB-resistant cultivable heterotrophic bacteria gradually increased to become the dominant population in treatment T2after18days(Fig.2b).A significant increase of strain ZWL73was observed in the 4CNB-contaminated treatments T3and T4,2days after inoculation (Fig.2c).However,after6days,cell numbers of strain ZWL73insoil Fig.1.Removal of4CNB in soil microcosms inoculated with Pseudomonas putida ZWL73and non-inoculated controls,together with variations of ammonium and chloride.(a)Removal of4CNB in soil microcosms;(b)variation of ammonium concentration;and(c)variation of chloride concentration.Samples were collected at different intervals,and lasted for30days.Data are averages of triplicates.Error bars represent the standard deviation of the mean of triplicates;in some cases,the error bars were too small to illustrate.G.-L.Niu et al./Environmental Pollution157(2009)763–771765Fig.2.Variation of heterotrophic cultivable bacteria during the bioaugmentation against 4CNB in soil microcosms inoculated with strain ZWL73and non-inoculated controls.The cell numbers of heterotrophic cultivable bacteria of different treatments were counted on both non-selective NA (a)and NA with 4CNB plates (b)to monitor the variation of total heterotrophic bacteria and 4CNB-resistant bacteria.The MM agar plate (c)with 4CNB-containing chloramphenicol (34m g ml À1)was designed for the variation of strain ZWL73in treatments T3and T4during 4CNB removal.Data are averages of triplicates.Error bars represent the standard deviation of the mean of triplicates;in some cases,the error bars were too small to illustrate.G.-L.Niu et al./Environmental Pollution 157(2009)763–771766microcosms of treatment T3decreased due to the removal of4CNB. The growth rate of strain ZWL73in treatment T3was also appar-ently slower than that in treatment T4.3.2.2.DGGE analysis of the dominant groups of indigenous bacteria in soil microcosmsDGGE targeted16S rRNA gene analyses were performed to examine the effects of the bioaugmentation on dominant microbial groups in soils.The bacterial DGGE profiles of total bacteria revealed significantly reduced microbial complexity due to the numerical dominance of the inoculant in treatment T3(data not shown).Therefore,in order to efficiently evaluate changes of a limited diversity,a group-specific nested PCR approach was used for the Alphaproteobacteria,Betaproteobacteria,Actinobacteria and Acidobacteria.The PCR products of16S rRNA genes sampled at days0,4and18 of treatments T2and T3were analyzed by DGGE in detail.Day 0represents the start of the incubations,day4represents disrup-tion of the microcosm in the case of treatment T2and the active phase of bioaugmentation in the case of treatment T3,and day18 represents recovery phase.The group-specific profiles for the 4CNB-contaminated soils with or without inoculation of strain ZWL73,showed rather similar patterns for the Alphaproteobacteria, Betaproteobacteria and Actinobacteria(Fig.3a–c,respectively). Several additional bands could be observed on day18of treatment T3in all these patterns.However,the bands were relatively more distinct in the profile of Actinobacteria,in which an additional band appeared as early as day4.Thus the bioaugmentation seemed to have a more significant effect on Actinobacteria than either Alphaproteobacteria or Betaproteobacteria.No obvious differences were observed for the dominant bands,even from patterns of samples on day18of treatment T2.The appearance of additional bands in the patterns for treatment T3taken at day18made it distinguishable from other samples in the dendrograms,in which samples on day18of treatment T3formed a separate group (Fig.4a–c).Subsequent sequencing results from these recovered bands confirmed their phylogenetic affiliation as shown in Table2.Fig.3.DGGE population profiles of16S rRNA gene fragments of soil bacterial populations in samples contaminated with4CNB,with and without inoculation of strain ZWL73(0,4 and18days).(a)Population of Alphaproteobacteria;(b)population of Betaproteobacteria;(c)population of Actinobacteria;(d)population of Acidobacteria;(e)population of ammonia-oxidizing bacteria.Bands indicated with arrows were excised from the gel and sequenced.G.-L.Niu et al./Environmental Pollution157(2009)763–771767On days 0and 4,in treatment T3,an intense band of acidobacterial DGGE fingerprints was distinctly present,but was hardly visible on day 18(band Acido_04in Fig.3d).This may indicate that special populations of Acidobacteria were stimulated in the primary phase of bioaugmentation just 12h (when sampling was taking place at day 0)after inoculating strain ZWL73.When the 4CNB removal was completed,the growth of this population was then suppressed.The sequencing data revealed this band was most similar to Gp1of Acid-obacteriaceae (Table 2).In addition,some bands of treatment T2were visible in DGGE fingerprints of Acidobacteria on day 0but faint on day 18,which also suggested that 4CNB contamination could inhibit growth of some Acidobacteria .Nevertheless,the bioaugmentation seemed to protect the Acidobacteria populations in treatment T3from the samples taken at day 18.Cluster analysis indicated samples taken at days 0and 4from treatment T3formed a separate cluster and shared less than 45%similarity to other samples’profiles,while the T3samples from day 18formed a group that was more similar to T2samples from day 0in the dendrograms (Fig.4d).Free ammonium was released during 4CNB bioaugmentation by strain ZWL73in treatment T3(Fig.1b).A more detailed analysis was performed to evaluate its effects on the structural composition of the AOB population.When the DGGE patterns of the AOB pop-ulation for different treatments were compared,all samples were highly similar and shared at least 87%similarity by cluster analysis (Fig.4e).However,the samples obtained from treatment T3taken at day 18were shown clustered separately from the other samples (Fig.4e).In addition,the signal intensities of bands in the DGGE patterns of treatment T2on day 18had obviously decreased compared to other samples.The assignment of the rRNA sequences from these dominant bands on DGGE is shown in Table 2.3.3.Libraries construction and analysis of 16S rRNA gene fragments of the AOB populationSince non-AOB sequences were also obtained in the bands affiliation analysis,separate libraries were constructedusingFig.4.Dendrograms constructed with the corresponding DGGE profiles in Fig.3.The differences between the profiles are shown by percentage of similarity.(a)Population of Alphaproteobacteria ;(b)population of Betaproteobacteria ;(c)population of Actinobacteria ;(d)population of Acidobacteria ;(e)population of ammonia-oxidizing bacteria.G.-L.Niu et al./Environmental Pollution 157(2009)763–771768PCR-amplified 16S rRNA gene fragments of treatment T3sampled at days 0and 18;50clones were randomly selected for sequencing.The sequence analysis showed the clone library of day 0contained a high proportion (80%)of sequences that fell outside the target clade,while the proportion decreased to 20%in the clone library of day 18.Furthermore,all AOB sequences obtained from either DGGE profiles or clone library were classified as Nitrosospira spp.These sequences can be assigned to cluster 3of the AOB population (Fig.5),proposed by Stephen et al.(1996)and Purkhold et al.(2000)based on phylogenetic analysis.Other products attributing to non-AOB amplification all belong to Methylophilus ,another Betaproteo-bacteria genus (Table 2).However,the AOB genus of Nitrosomonas spp.was not detected in this case.4.DiscussionIn this study,the bioaugmentation of a 4CNB-contaminated model soil was examined.The strain ZWL73could accelerate the 4CNB removal from soil microcosms,which was different from a previous report on rhizoremediation using plant–microbe system (Liu et al.,2007).Furthermore,the effects of bioaugmentation with the inoculant strain ZWL73,and of 4CNB contamination,on the dominant populations of soil microbial populations were also evaluated in detail,and obviously dynamic changes within intrinsic Actinobacteria and Acidobacteria populations were observed.An effect on the AOB population had also been noticed.Generally,a key factor of successful bioaugmentation is the density and activity of introduced microorganisms (van Veen et al.,1997).As a result of bioaugmentation in treatments T3and T4of this study,contaminated 4CNB was effectively degraded concomi-tant with the accumulation of ammonium and chloride (Fig.1a–c),confirming strain ZWL73s role in the degradation.Moreover,the active growth phase of strain ZWL73corresponded to the rapid loss phase of 4CNB,as well as the rapid accumulation phase of ammo-nium and chloride,indicating a close correlation among them.However,all the rates,including 4CNB removal and the accumu-lation of ammonium and chloride,were apparently slower in treatment T3than those in treatment T4.These combined data suggested that there may be a process of the inoculated strain that competes with indigenous microorganisms and predatory protozoa present in non-sterile soil,causing lower countable cell numbers of strain ZWL73from treatment T3than those from treatment T4(Fig.2c)and the delay in the 4CNB degradation.These biotic factors were thought to play a major role in controlling the population dynamics of introduced bacteria in soils (Cunliffe and Kertesz,2006;van Veen et al.,1997),which even could cause a bio-augmentation failure because of the poor survival of introduced bacteria (Bouchez et al.,2000).Therefore,the successful bio-augmentation in treatment T3demonstrated that the strain ZWL73could compete well with soil indigenous microorganisms for utilization of 4CNB as carbon and nitrogen sources.In general,it is considered that introduced microorganisms capable of degrading a particular contaminant survive well when this contaminant is present,but are not able to persist in its absence (Cunliffe and Kertesz,2006;van Veen et al.,1997).In this study,after the 4CNB removal was completed,strain ZWL73lost the competitive advantage with indigenous microorganisms in treatment T3,as demonstrated by the decrease of its cell numbers,but there was not a distinct decrease in treatment T4(Fig.2c).Simultaneously,the ammonium concentration in non-sterile soil of treatment T3could not reach the same high levels as the sterile soil of treatment T4,since some indigenous fungi and bacteria in soil microcosms of treatment T3could transform free ammonium during the bio-augmentation.But a slight increase of ammonium concentration was observed in treatment T2probably because the inhibition of nitrification by 4CNB,similar to a study in which the inhibition of nitrification by 3-chloroaniline caused the accumulation of ammonium (Boon et al.,2003).It is likely the accumulation of ammonium in both studies has the same cause.Comparisons of treatment T3with treatment T4indicate that indigenous microbial populations in soils affected the survival of the inoculated strain ZWL73.Moreover,the addition of strain ZWL73and 4CNB into soils influenced the indigenous microbes.Based on cultivation,an obvious decrease in both diversity and the cell numbers of cultivable heterotrophic bacteria was observed,as well as increased cell numbers of 4CNB-resistant bacteria,apparently due to 4CNB selective stress.However,the changes demonstrated by the cultivation-independent approach (DGGE)were not as obvious.This may be attributed to the complexity of soil as a biological system and the rather low proportion of cultivable heterotrophic bacteria in the overall soil bacterial populations.In addition,PCR bias and the drawbacks of DGGE method may also affect results (Nannipieri et al.,2003).Thus,the cultured isolates are still very important in developing our understanding of bacterial diversity.Nevertheless,the approach of determining microbial diversity from the number of isolates has become less popular,not only because a limited number of microorganisms can be cultured but also because the procedures are laborious (Nannipieri et al.,2003).Ammonia-oxidizing bacteria oxidize ammonia to nitrite,the first and most critical step of the nitrification process,playinganTable 2*Closest matches to band sequences were obtained with the sequence match tool of Ribosomal Database Project II release 9(Cole et al.,2007).Similarities between the band sequence and database entries were determined by use of the RDP-II tool.G.-L.Niu et al./Environmental Pollution 157(2009)763–771769。