SHORT RESEARCH AND DISCUSSION ARTICLE

Simazine biodegradation and community structuresof ammonia-oxidizing microorganisms in bioaugmented soil:impact of ammonia and nitrate nitrogen sources

Rui Wan & Yuyin Yang & Weimin Sun & Zhao Wang & Shuguang Xie

Received: 21 August 2013 /Accepted: 21 October 2013 /Published online: 6 November 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract The objective of the present study was to investi-gate the impact of ammonia and nitrate nitrogen sources onsimazine biodegradation by Arthrobacter sp. strain SD1 andthe community structures of ammonia-oxidizing archaea(AOA) and bacteria (AOB) in non-agricultural soil. Soil mi-crocosms with different treatments were constructed for her-bicide biodegradation test. The relative abundance of thestrain SD1 and the structures of AOA and AOB communitieswere assessed using quantitative PCR (q-PCR) and terminalrestriction fragment length polymorphism (TRFLP), respec-tively. The co-existence of two inorganic nitrogen sources(ammonia and nitrate) had certain impact on simazine dissi-pation by the strain SD1. Bioaugmentation could induce ashift in the community structures of both AOA and AOB, butAOAwere more responsive. Nitrogen application had signif-icant impacts on AOA and AOB communities inbioaugmented soils. Moreover, in non-bioaugmented soil,the community structure of AOA, instead of AOB, could bequickly recovered after herbicide application. This studycould add some new insights towards the impacts of nitrogensources on s -triazine bioremediation and ammonia-oxidizingmicroorganisms in soil ecosystem.

Keywords Ammonia-oxidizing archaea (AOA) . Ammoniaoxidizingbacteria(AOB) .Bioremediation .Bioaugmentation .

Nitrogen . Pesticide

Introduction

Simazine and other s -triazine herbicides have been commonlyused for the control of broad-leaved weeds in agricultural soilin many regions of the world (Hernández et al. 2011). Toxicityand persistence of these herbicides in soil and aquatic envi-ronments have aroused increasing eco-environmental con-cerns (Hernández et al. 2011; Jablonowski et al. 2011; Guoet al. 2013). Besides intensive agricultural application, acci-dental spillage or intentional disposal can also pose a seriousthreat to the environment (Strong et al. 2002; Silva et al. 2004;Udiković-Kolić et al. 2010). Both chemical and biologicalprocesses can be involved in dissipation of herbicides incontaminated soil, but the latter is mainly responsible for s -triazine removal (Hernández et al. 2008; Udiković-Kolić et al.2010; Zhou et al. 2013). Bioaugmentation is known as aneffective and low-cost remediation approach to clean up pes-ticides in contaminated soil (Morgante et al. 2010; Barreiroset al. 2012). For the purpose of bioremediation practice,s -triazine-degrading bacteria from various genera have beenisolated, and some of them have been used for bioremediationtrial (Silva et al. 2004; Morgante et al. 2010; Guo et al. 2013;Wang et al. 2013a). However, effort of bioaugmentation maybe frustrated by various abiotic and biotic factors (Strutherset al. 1998; Newcombe and Crowley 1999; Silva et al. 2004;Zhou et al. 2013).

Most of the cultivated s -triazine-degrading bacteria can usethe herbicides as nitrogen sources (Yang et al. 2010). Organicand inorganic nitrogen sources in soil might be major con-straints in s -triazine remediation. It has been well documentedthat nitrogen sources can negatively affect biodegradation of

Responsible editor: Robert Duran

Rui Wan and Yuyin Yang contributed equally to this study.

Electronic supplementary material The online version of this article(doi:10.1007/s11356-013-2268-7) contains supplementary material,which is available to authorized users.

R. Wan :Y. Yang : Z. Wang : S. Xie (*)College of Environmental Sciences and Engineering, The KeyLaboratory of Water and Sediment Sciences (Ministry of Education),Peking University, Beijing 100871, Chinae-mail: xiesg@pku.edu.cn

W. SunDepartment of Environmental Science, Rutgers, The State Universityof New Jersey, New Brunswick, NJ 08901, USA

Environ Sci Pollut Res (2014) 21:3175–3181DOI 10.1007/s11356-013-2268-7

s -triazine herbicides, either by indigenous microbiota in soilor by selected degraders in liquid culture, but only limitedinformation is available on the effects of nitrogen sources ons -triazine degradation by added degraders in soil environment(Guo et al. 2013; Zhou et al. 2013). Multiple nitrogen sources(e.g., ammonia and nitrate) can coexist with pesticides in soilenvironment. Previous studies usually focused on the effect ofa single nitrogen compound on s-triazine degradation. Infor-mation on the impacts of two or more nitrogen sources ons -triazine degradation in soil by added degraders is still lacking.

Microbial oxidation of ammonia, as the first step in nitrifi-cation process, is an important process for global nitrogencycling. Either ammonia-oxidizing archaea (AOA) or bacteria(AOB) can be the important participants in nitrification pro-cess in soil ecosystems, although their relative contribution tosoil ammonia oxidation remains controversial (Wang et al.2013b). Previous studies indicated that the community struc-tures of AOA and AOB can be affected by nitrogen fertiliza-tion (Wang et al. 2009; Glaser et al. 2010; Hu et al. 2012).However, information on the impacts of s -triazine herbicideson soil AOA and AOB community structures is still verylimited (Chang et al. 2001; Hernández et al. 2011; Guo et al.2013). In addition, few studies have reported the effect ofbioaugmentation on soil AOB community structure (Niuet al. 2009; Zhao et al. 2009; Guo et al. 2013), while theimpact of bioaugmentation on AOA community structureremains unknown. Moreover, no information is available onthe effects of multiple nitrogen sources on AOA and AOBcommunity structures in bioaugmented soil.

The previously isolated Arthrobacter sp. strain SD1 couldquickly dissipate the simazine in soil even at a high dosage ofurea nitrogen (Guo et al. 2013). The strain SD1 harborss -triazine-metabolic genes trzN , atzB , and atzC . The aim ofthe current study was to investigate the impact of two inor-ganic nitrogen sources (ammonia and nitrate) on simazinebiodegradation and the structures of soil AOA and AOBcommunities inoculated with the strain SD1. In this study,the relative abundance of the strain SD1 was assessed usingquantitative PCR (q-PCR) assay, while the structures of AOAand AOB communities were characterized using terminalrestriction fragment length polymorphism (TRFLP).

Materials and methods

Microcosm setup

Soil used for microcosm construction was collected from anon-agricultural site without any link to previous s -triazineexposure. The soil was slightly alkaline (pH 8.2) and loam,with ammonia nitrogen of 6.17 mg kg−1 soil and nitratenitrogen of 1.48 mg kg−1 soil. Following collection, soil washomogenized, sieved through a 4-mm screen, and then stored

at 4 °C until use. In this study, the inoculum was prepared aspreviously described (Guo et al. 2013). Soil microcosms wereprepared in 250-mL jars with 50 g soil (dry weight) and thenincubated at 25 °C in the dark. Soil moisture was maintainedat 10–15 % of the water-holding capacity. Six sets of treat-ments in triplicate were carried out as follows: (A) soil+100 mg kg−1 simazine+3.3×106 CFU g−1 strain SD1, (B)soil+100 mg kg−1 simazine+3.3×106 CFU g−1 strain SD1+100 mg kg−1 nitrate-N+100 mg kg−1 ammonia-N, (C) soil+100 mg kg−1 simazine+3.3×106 CFU g−1 strain SD1+500 mg kg−1 nitrate-N+500 mg kg−1 ammonia-N, (D) soil+100 mg kg−1 simazine, (E) soil+100 mg kg−1 simazine +500 mg kg−1 nitrate-N+500 mg kg−1 ammonia-N, and (F)sterilized soil+100 mg kg−1 simazine.

Chemical and molecular analyses

Soil samples (1 g, dry weight) from each treatment werecollected at days 0, 2, 4, and 6. Residual simazine in soilwas extracted and analyzed according to the literature (Guoet al. 2013). Soil DNA was extracted using the PowersoilDNA extraction kit (Mobio Laboratories). DNA concentrationwas quantified using a Bio-Photometer (Eppendorf, Hamburg,Germany). For q-PCR assays, the primers for amplification ofthe 16S rRNA and s -triazine-degrading trzN genes were 16Sf(5′-CTGGTAGTCCACGCCGTAAA-3′), 16Sr (5′-CGAATTAAACCACATGCTCCAC-3′), trzNf (5′-CACCAGCACCTGTACGAAGG-3′), and trzNr (5′-GATTCGAACCATTCCAAACG-3′), using the same PCR conditions as previouslydescribed (Guo et al. 2013; Wang et al. 2013a; Xie et al.2013). The amplification efficiency and coefficient (r2) foramplification of the 16S rRNA and trzN genes were 101 %and 99 %, 0.997 and 0.998, respectively. Ratio of trzN genecopies to 16S rRNA gene copies was used to estimate theproportion of s-triazine degrader in soil. Analysis of variance(two-way analysis of variance) was used to determine thesignificant differences (P <0.05) in residual herbicide concen-tration and the relative abundance of trzN gene among thedifferent treatments during the incubation period, using thestatistical software SPSS 20.

TRFLP analyses of AOA and AOB communities werecarried out according to the literature (Feng et al. 2012). Theprimers for amplification of the ammonia monooxygenaseA (amoA ) genes of AOA and AOB were Arch-amoAF(5′-STAATGGTCTGGCTTAGACG-3′; 5′ end-labeled withcarboxyfluorescine), Arch-amoAR (5′-GCGGCCATCCATCTGTATGT-3′), amoA -1F (5′-GGGGTTTCTACTGGTGGT-3′; 5′ end-labeled with carboxyfluorescine), and amoA-2R (5′-CCCCTCKGSAAAGCCTTCTTC-3′). Hha I wasused to digest purified PCR products and TRFLP fragmentswere determined using an ABI 3730 DNA Analyzer (AppliedBiosystems) and Genemapper v3.5 software (AppliedBiosystems). Terminal fragments under 200 fluorescent units

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or smaller than 50 bp were excluded from further analysis.Fragments that differed by less than 1 bp were clustered.Bray–Curtis similarity index was calculated using PRIMER5.0 software to evaluate the similarities among different sam-ples (Clarke andWarwick 2001). Clustering of samples, basedon Bray–Curtis similarity index, was performed using thesoftware PRIMER 5.0 and the UPGMA (unweighted pairgroup mean average) method (Clarke and Warwick 2001).

Results and discussion

Simazine dissipation and the relative abundance of trzN gene

In the present study, soil microcosms with a total of sixtreatments were constructed for herbicide degradation test.Applied simazine concentration (100 mg kg−1) in these soilmicrocosms was much higher than that used in others’ previ-ous microcosm biodegradation experiments (4.5–20 mg kg−1;Liao and Xie 2008; Jose et al. 2010; Morgante et al. 2012).Figure 1 shows a very limited decline of simazine in non-bioaugmented microcosms (with treatments D–F) during the6-day incubation period. However, at days 2, 4, and 6, residualherbicide concentration was significantly lower (P <0.05) inbioaugmented soils than in non-bioaugmented ones. On day4, in the bioaugmented microcosms with treatments A–C, theresidual ratios of simazine were 24 %, 27.4 %, and 53.8 %,respectively. Moreover, on day 6, a nearly complete removalof simazine occurred in the microcosms with treatments A and

B, but a residual rate of 10.5 % still existed in the microcosmwith treatment C. At days 4 and 6, residual herbicide concen-tration in each microcosm was also significantly differentfrom that in the other ones (P <0.05). For treatmentwith C, residual herbicide concentration was significantlyhigh (P <0.05) compared to treatments with A and B. Theseresults indicated a negative impact of two inorganic nitrogensources (ammonia and nitrate) on simazine dissipation.

The known s -triazine-degrading Arthrobacter species usu-ally carry three degrading genes trzN , atzB and atzC (Wangand Xie 2012). trzN gene is responsible for transformingatrazine to hydroxyatrazine. In this study, ratio of trzN genecopies to 16S rRNA gene copies was used to assess theproportion of simazine degraders in soil. In the non-bioaugmented microcosms (with treatments D and E), trzNgene always remained below q-PCR detection limit during the6-day incubation period (data not shown). A significant in-crease (P <0.05) in the proportion of trzN gene was found ineach bioaugmented soil in 2 days after inoculation of the strainSD1 (Fig. 2). In the microcosms with treatments A and B,the proportion of trzN gene reached its peak value on day4 and followed by a significant decline (P <0.05) insubsequent incubation. For treatment C, a significant dif-ference (P <0.05) in the proportion of trzN gene wasobserved during the incubation period. A continuous in-crease in the proportion of trzN gene occurred in themicrocosm with treatment C. In addition, the density oftrzN gene in the bioaugmented soils shows a similarchange pattern (Fig. S1).

Several previous studies have been performed to investi-gate the impacts of ammonia or nitrate nitrogen on the degra-dation activity of s -triazine-degrading bacteria in liquid cul-ture. Sajjaphan et al. (2010) found that supplementation ofnitrate nitrogen completely inhibited the atrazine degradation

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Fig. 1 Percentage of residual simazine in microcosms with treatment A–F. Treatment A soil+100 mg kg−1 simazine+3.3×106 CFU g−1

Arthrobacter strain SD1; treatment B: soil+100 mg kg−1 simazine+3.3×106 CFU g−1 Arthrobacter strain SD1+ 100 mg kg−1 nitrate-N+100 mg kg−1 ammonia-N; treatment C: soil+100 mg kg−1 simazine+3.3×106 CFU g−1 Arthrobacter strain SD1+ 500 mg kg−1 nitrate-N+500 mg kg−1 ammonia-N; treatment D: soil+100 mg kg−1 simazine;treatment E: soil+100 mg kg−1 simazine +500 mg kg−1 nitrate-N+500 mg kg−1 ammonia-N; and treatment F: sterilized soil+100 mg kg−1

simazine. Values are the average of three independent experiments.Vertical bars indicate standard deviations

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Fig. 2 Change of the relative abundance of trzN gene in thebioaugmented microcosm during the incubation period. Treatment A:soil+100 mg kg−1 simazine+3.3×106 CFU g−1 Arthrobacter strainSD1; treatment B: soil+100 mg kg−1 simazine+3.3×106 CFU g−1

Arthrobacter strain SD1+ 100 mg kg−1 nitrate-N+100 mg kg−1 ammo-nia-N; and treatment C: soil+100 mg kg−1 simazine+3.3×106 CFU g−1

Arthrobacter strain SD1+ 500 mg kg−1 nitrate-N+500 mg kg−1 ammo-nia-N. Values are the average of three independent experiments. Verticalbars indicate standard deviations

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activity of Arthrobacter sp strain KU001, whereas ammoni-um nitrogen showed no effect. Bichat et al. (1999) indicatedthat atrazine degradation by Pseudomonas strain sp. ADP andAgrobacterium radiobacter J14a was unaffected by the pres-ence of ammonia or nitrate nitrogen, whereas ammonia nitro-gen completely inhabited herbicide degradation by bacteriumM91-3. Interestingly, atrazine biodegradation byArthrobactersp. strain DAT1 could even be enhanced by addition ofammonia or nitrate nitrogen (Wang and Xie 2012). Yanget al. (2010) found that NH4NO3 addition could suppressatrazine degradation by a consortium of Klebsiella sp. A1and Comamonas sp. A2. Moreover, there are also severalreports on the impacts of ammonia or nitrate nitrogen on s -triazine degradation by soil indigenous microbiota. Atrazinemineralization was significantly inhibited in soil amendedwith nitrate nitrogen of 133–280 mg kg−1 (Garces et al.2007). Abdelhafid et al. (2000) indicated that either ammonianitrogen (211.9 mg kg−1) or nitrate nitrogen (170.6 mg kg−1)could significantly decrease atrazine degradation in adaptedagricultural soil. Zablotowicz et al. (2008) reported that max-imum daily rates of atrazine mineralization in adapted agri-cultural soil were reduced from 55% to 41% by application of1 to 8 g kg−1 of ammonium sulfate. Unfortunately, little isknown about the impact of two inorganic nitrogen sources ons -triazine degradation by soil indigenous microbiota. Entryet al. (1993) and Entry (1999) found that addition of 200–500 kg N ha–1as NH4NO3 suppressed atrazine mineralizationin grassland and redwater wetland soils but not blackwaterwetland soil. Therefore, the impacts of nitrogen sources on s -triazine biodegradation seem to be dependent on type ofdegrader and type of inorganic nitrogen compound, as wellas culture condition.

Information on the impacts of inorganic nitrogen sourceson s -triazine biodegradation in bioaugmented soil is stillscanty. Garcia-Gonzalez et al. (2003) showed that nitrateamendment (500 mg kg−1) could markedly repress bioreme-diation of atrazine-contaminated soil using Pseudomonas sp.ADP. Atrazine biodegradation by Arthrobacter strain DAT1in soil could be almost completely inhibited by addition of 1,000 mg kg−1 nitrate nitrogen, but unaffected by 200 mg kg−1

nitrate nitrogen (Zhou et al. 2013).Our previous study indicat-ed simazine biodegradation by the strain SD1 was not signif-icantly inhibited even at a high level of ammonia nitrogen(1,000 mg kg−1; data not shown). Different types andlevels of amended nitrogen sources might have variousimpacts on soil s -triazine remediation using bioaugmenta-tion process (Guo et al. 2013). To the authors’ knowledge,this was the first report on the impact of two inorganicnitrogen sources on the selected degrader for s -triazinedissipation in soil. In this study, simazine biodegradationby the strain SD1 was only slightly affected by a mediumlevel of inorganic nitrogen (100 mg kg−1 nitrate-N+100 mg kg−1 ammonia-N), but more evidently by a high

level of inorganic nitrogen (500 mg kg−1 nitrate-N+500 mg kg−1 ammonia-N).

In this study, the quick herbicide dissipation in bioaugmentedsoils could be attributed to a significant increase in the propor-tion of s-triazine-degrading genes. However, the impacts ofnitrogen sources on the degradation activities of added bacteriamight not be accurately estimated by the abundance of meta-bolic genes (Guo et al. 2013). Our previous study found that theproportion of s-triazine-degrading genes could not be correlatedwith the dissipation rate of simazine in inoculated soil amendedwith different levels of urea-N (Guo et al. 2013). The resultobtained in this studywas in agreement with that in our previousstudy. In this study, on day 6, although residual herbicide in themicrocosms with treatments A and B was much lower than thatin the microcosm with treatment C, a higher proportion of themetabolic gene was observed in the latter. The reason was notfully understood. One possible hypothesis was that the expres-sion of the s -triazine catabolic or transport gene could berepressed at high nitrogen levels (Garcia-Gonzalez et al.2003), so a redundancy of the metabolic gene might be neededin order to effectively utilize the herbicide. In addition, thedecrease of selective pressure from herbicide might also reducethe proportion of the s-triazine-degrading gene.

AOA and AOB community structures

Analysis of AOA community structures in the inoculated andnon- inoculated microcosms was performed by TRFLP. AOAterminal fragment 167 bp (Hha I) predominated in each sam-ple, while its relative abundance varied among samples(Fig. S2). In addition, a marked variation of some otherfragments and their proportions were also observed amongdifferent samples. These results indicated a difference of AOAcommunity structure among samples. Figure 3 shows thedendrogram constructed for the structure of AOA communityin soil with each treatment. For each treatment, the sample onday 0 was not always closely clustered with the ones at othersampling dates, indicating a shift in AOA community struc-ture during the incubation period. For treatment D, the sam-ples at days 0 and 6 were grouped, but distantly separatedfrom those at days 2 and 4. This suggested a strong resilienceof AOA community after herbicide application. Moreover, fortreatment E, clustering of the samples at days 0, 4, and 6indicated that the recovery of AOA community structurecould also be achieved in few days after application of herbi-cide and inorganic nitrogen sources. However, at days 2, 4,and 6, the sample with treatment D was usually distantlyseparated from that with treatment E, showing a strong impactof nitrogen amendment on the AOA community structure inthe non-bioaugmented soil. The sample with treatment Awasalways distantly separated from that with treatment D at days2, 4, and 6, suggesting a large shift in AOA communitystructure induced by the bioaugmentation process. In addition,

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at days 2, 4, and 6, the sample with treatment A was usuallydistantly separated from those with treatments B and C. Dur-ing the incubation, the samples from the microcosm withtreatment C were closely clustered. These results illustrate asignificant impact of inorganic nitrogen sources on AOAcommunity in the bioaugmented soils. Recovery of AOAcommunity structure did not occur in each bioaugmented soilduring the 6-day incubation.

The variation of the compositions of AOB fragments andtheir proportions shows a shift in AOB community structureamong different samples (Fig. S3). Figure 4 illustrates thedendrogram constructed for the structure of AOB communityin soil with each treatment. For each treatment, the sample onday 0 was not clustered with the ones at other sampling dates,indicating a shift in AOB community structure during theincubation period. For treatment D, the samples at days 4and 6 were clustered, but distantly separated from those atdays 0 and 2. This indicated that the recovery of AOB com-munity structure was not achieved after herbicide application.The recovery of AOB community also did not occur in themicrocosm with treatment E during the 6-day incubation. Thesample with treatment D was distantly separated from thatwith treatment E at days 4 and 6, illustrating a strong impact ofinorganic nitrogen on the AOB community structure in thenon-bioaugmented soil. Moreover, the sample with treatmentA was separated from that with treatment D at days 2 and 4,but clustered on day 6. This suggested bioaugmentation

process alone might only result in a transient shift in AOBcommunity structure. In addition, at days 2, 4 and 6, thesample in each bioaugmented microcosm was usually distant-ly separated from that in the other two ones, illustrating astrong impact of inorganic nitrogen onAOB community in theinoculated soils. Recovery of AOB community structure wasnot found in each bioaugmented soil during the 6-dayincubation.

Pesticide application can usually have a strong impact onmicrobial community structure (Mahía et al. 2011; Sura et al.2012; Verdenelli et al. 2012; Marinozzi et al. 2013; Xie et al.2013), yet information on the impacts of pesticides on AOAand AOB community structures is still very limited. Fewprevious studies indicated that pesticide application couldinduce a shift in the structure of AOB community (Changet al. 2001; Li et al. 2008; Hernández et al. 2011; Puglisi et al.2012; Guo et al. 2013) and AOA community (Puglisi et al.2012). Hernández et al. (2011) found that simazine applicationdid not affect AOA community structure in adapted agricul-tural soil. In the current study, in non-adapted soil, a transientshift in AOA community structure was found after simazineapplication. Therefore, the impacts of s -triazine herbicides onsoil AOA community structure might be different. Moreover,there was a recovery of AOA community structure in few daysafter simazine application, but not AOB. This suggested si-mazine application had a more profound impact on AOB thanAOA. However, Puglisi et al. (2012) indicated that AOAweremore responsive to fungicides penconazole and cyprodinil

Fig. 4 Cluster diagram of AOB community similarity values for soilsamples with each treatment. Similarity levels are indicated below thediagram. Upper case letters refer to treatment, and digits indicate sam-pling date. Treatment A: soil+100 mg kg−1 simazine+3.3×106 CFU g−1

Arthrobacter strain SD1; treatment B: soil+100 mg kg−1 simazine+3.3×106 CFU g−1 Arthrobacter strain SD1+ 100 mg kg−1 nitrate-N+100 mg kg−1 ammonia-N; treatment C: soil+100 mg kg−1 simazine+3.3×106 CFU g−1 Arthrobacter strain SD1+ 500 mg kg−1 nitrate-N+500 mg kg−1 ammonia-N; treatment D: soil+100 mg kg−1 simazine; andtreatment E: soil+100 mg kg−1 simazine +500 mg kg−1 nitrate-N+500 mg kg−1 ammonia-N. Replicates illustrated the same trend

Fig. 3 Cluster diagram of AOA community similarity values for soilsamples with each treatment. Similarity levels are indicated below thediagram. Upper case letters refer to treatment, and digits indicate sam-pling date. Treatment A: soil+100 mg kg−1 simazine+3.3×106 CFU g−1

Arthrobacter strain SD1; treatment B: soil+100 mg kg−1 simazine+3.3×106 CFU g−1 Arthrobacter strain SD1+ 100 mg kg−1 nitrate-N+100 mg kg−1 ammonia-N; treatment C: soil+100 mg kg−1 simazine+3.3×106 CFU g−1 Arthrobacter strain SD1+ 500 mg kg−1 nitrate-N+500 mg kg−1 ammonia-N; treatment D: soil+100 mg kg−1 simazine; andtreatment E: soil+100 mg kg−1 simazine +500 mg kg−1 nitrate-N+500 mg kg−1 ammonia-N. Replicates illustrated the same trend

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compared to AOB. It seems that the response of AOA andAOB communities to pesticide application was dependent onpesticide type. In addition, although it is known that thecommunity structures of AOA and AOB can be affected bynitrogen fertilization, the effects of pesticide and nitrogensources on AOA and AOB communities remains unclear. Inthis study, inorganic nitrogen sources (ammonia and nitrate)could significantly affect AOA and AOB communities inherbicide-amended soil.

To date, few studies have shown that bioaugmentationcould alter soil AOB community structure (Niu et al. 2009;Zhao et al. 2009; Guo et al. 2013). However, to the author’sknowledge, this was the first report on the impact of bioaug-mentation on AOA community. In this study, bioaugmenta-tion induced a significant shift in AOA community structure,but only resulted in a transient shift in AOB communitystructure in soil with no nitrogen amendment. Therefore,AOA community was more responsive to bioaugmentationcompared to AOB community. Moreover, this was also firststudy to investigate the effect of multiple nitrogen sources onAOA and AOB community structures in bioaugmented soil.In this study, application of two inorganic nitrogen sources(ammonia and nitrate) had a significant impact on either AOAor AOB community in bioaugmented soil.

DNA-based techniques can not differentiate between liveand dead microorganisms. Extracellular DNA from dead cellsmay degrade quite slowly in the environment (Levy-Boothet al. 2007). By comparison, RNA synthesis rates are higherthan those of DNA. The continual turnover of RNA in theenvironment is a reflection of cellular activity independent ofreplication (Manefield et al. 2002). Therefore, RNA-basedtechniques might provide alternatives to characterization ofmicrobial community structure in pesticide-amended systems.

Concluding remarks

Simazine biodegradation by Arthrobacter strain SD1 wasslightly affected by a moderate level of inorganic nitrogencompounds, but more evidently by a high level. The commu-nity structure of AOA, instead of AOB, could be recovered infew days after simazine application in non-bioaugmented soil.Bioaugmentation could induce a shift in the community struc-tures of both AOA and AOB, but AOAwere more responsive.Application of ammonia and nitrate nitrogen had a significantimpact on both AOA and AOB communities in bioaugmentedsoil.

Acknowledgments This work was financially supported by NationalNatural Science Foundation of China (No. 50979002) and State KeyLaboratory of Ecohydraulic Engineering in Shaanxi.

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