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Soil Biology & Biochemistry 39 (2007) 3056–3065

www.elsevier.com/locate/soilbio

Isolation of a selected microbial consortium froma pesticide-contaminated mix-load site soil capable of degrading

the herbicides atrazine and alachlor

Anastasia E.M. Chirnsidea,�, William F. Rittera, Mark Radosevichb

aDepartment of Bioresources Engineering, University of Delaware, 531 South College Avenue, Newark, DE 19716-2140, USAbBiosystems Engineering and Environmental Science, University of Tennessee, 2506 E.J. Chapman Dr., Knoxville, TN 37996, USA

Received 1 February 2007; received in revised form 4 June 2007; accepted 19 June 2007

Available online 25 July 2007

Abstract

Contaminated soil from a 100-year-old mix-load site located in Reading, PA was evaluated for its potential to provide indigenous

microorganisms capable of degrading two widely utilized herbicides, atrazine (2-chloro-4-ethylamino-6-isopropylamino-S-triazine; AT)

and alachlor (2-chloro-20,60-diethyl-N-[methoxymethyl]-acetanilide; AL). Three different locations from the site were chosen for

experimentation based on herbicide handling activities. Standard enrichment techniques were used to isolate a selective microbial

consortium (SCM) with the desirable degrading capabilities. Three enrichment treatment schemes were evaluated; AT and AL, AL alone,

and only AT. Degradative organisms were isolated from only one of the sample locations. Considerable differences in the soil parameters

of the three sample locations were found that might have had an effect on the ability of the indigenous microbial populations within the

soil to degrade AT and AL. In the initial cultures from this location, degradation occurred in the AT and AL treatment only. Because the

AT and AL were the only sources of carbon and nitrogen (N) for the microbes, these results suggest that AL alone was not a sufficient N

source. In general, the ability to degrade AL by the SMC was dependent on AT degradation. Alachlor degradation did not begin until

approximately 15% of the AT was transformed. Once all of the AT was removed very little further AL degradation occurred. The

average half-life (t1/2) of AT was 7.5 d, while average t1/2 for AL degradation was 11 d. Individual colonies from the SMC were identified

by fatty acids methyl ester (FAME) analysis. Five strains were identified with similarity indexes above 70%. These isolates included the

following: Alcaligenes xylosoxydans subsp. denitrificans, Alcaligenes xylosoxydans subsp. xylosoxydans, Pseudomonas putida,

Pseudomonas marginalis, and Providencia rustigianii.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Isolation; Atrazine; Alachlor; Selected microbial consortium; Alcaligenes; Pseudomonas; Providencia

1. Introduction

The two most common herbicides used in the UnitedStates are atrazine (AT) and alachlor (AL). In 1997,approximately 34–37� 106 kg active ingredient (AI) of ATand 6–7� 106 kg AI of AL were used in US agriculture(Aspelin and Grube, 1999). Research indicates that AL isrelatively non-persistent with a reported range of biode-gradative half-lives of about 14–21 d (Barbash et al., 2001;Peter and Weber, 1985). However, AL can persist as a

e front matter r 2007 Elsevier Ltd. All rights reserved.

ilbio.2007.06.018

ing author. Tel.: +1302 831 8871; fax: +1 302 831 8871.

ess: aemc@udel.edu (A.E.M. Chirnside).

dissolved compound in surface and groundwater formonths or years due to its high water solubility(242mg l�1), relatively low soil adsorption coefficient(KOC ¼ 170), and lack of microbial degraders (Battaglinand Hay, 1996). Conversely, AT is considered moderatelypersistent with a reported range of biodegradative half-lives of 21 d to 1 year (Barbash et al., 2001). The t1/2 datafor AT and AL were compiled from a general review ofpublished aerobic soil degradation studies (Barbash et al.,2001). Despite moderate water solubility (33mg l�1), AThas been labeled as highly leachable and has been detectedin the ground and surface water of most of the ruralagricultural areas of the US (US Environmental Protection

ARTICLE IN PRESSA.E.M. Chirnside et al. / Soil Biology & Biochemistry 39 (2007) 3056–3065 3057

Agency (EPA), 1990; Masses et al., 1994; Ritter, 1990).Bulk pesticide mixing and loading (mix-load) sites havebeen identified as major contributors of pesticide groundand surface water contamination due to the nature of thepesticide handling activities occurring upon the soil surface(Ames and Hoyle, 1999; Habecker, 1989). For example, amaximum concentration of 270 and 70mg l�1 of AL andAT, respectively, was detected in surface water locatedspecifically in the loading and mixing areas of these sites.Evaluation of 49 mix-load sites and more than 822 soilsamples depicted AT and AL as the most frequentlydetected pesticides at concentrations ranging from 18,000to 16,291� 106mg kg�1 and from 17,000 to 405,940�106mg kg�1 for AL and AT, respectively (Krapac et al.,1995). The high total mass of pesticides in the mix-load sitesoils could limit bioremediation techniques due to thetoxicity effects of these high concentrations on thedegrading microorganisms (Gan et al., 1996; Roy et al.,1995). The costs of chemical treatment procedures havebeen exorbitant (Preslo et al., 1989). While bioremediation,as a low-cost alternative, has often been unsuccessful dueto complexity of the interactions between the microbialdegraders, the soil, the high concentration of pesticides andthe wide array of compounds present (Maloney, 2001).

Bioaugmentation remediation techniques have ofteninvolved the addition of acclimated indigenous microbesthat can degrade the target compounds at accelerated rates.The microbes are isolated from the contaminated soil in thelaboratory through a selection and enrichment process(Cookson, 1995). The enrichment culture technique devel-ops a culture that has increased numbers of a consortiumof microorganisms able to degrade the target compounds(Alexander, 1994). These microbes often exhibit a remedialadvantage over the existing non-specific heterogeneouspopulations in that they can metabolize the recalcitranttarget compounds (Nestler, 2001). Utilizing a SMC isolatedfrom mix-load sites may overcome many of the difficultiesassociated with these sites. Therefore, the objective of thisresearch was to isolate a SMC from the pesticide-contaminated mix-load site soil capable of degrading ATand AL so that future in situ bioremediation strategiescould be developed.

2. Materials and methods

2.1. Study site description and sampling technique

Soil was acquired from a 100-year-old contaminatedmix-load site located at the intersection of Routes 176 and724 in Reading, Pennsylvania. Previous environmentalinvestigations of this 11.7 acre site detected triazine andacetanilide herbicides, as well as organo-chlorine andorgano-phosphate pesticides with most contaminantsfound in the areas where pesticides were stored orloaded/unloaded. Concentrations of AT (52mgkg�1,RBC ¼ 13.0mg kg�1); simazine (33.0mg kg�1, RBC ¼24.0mg kg�1); AL (200mgkg�1, RBC ¼ 36.0mg kg�1);

dieldrin (0.31mg kg�1, RBC ¼ 0.18mg kg�1); and hepta-chlor (0.93mg kg�1, RBC ¼ 0.64mg kg�1) in soil samplestaken in the vicinity of the mix-load area were above theEPA risk-based concentrations (RBC) for commercialareas (Xin et al., 1995). Due to the gravelly matrix, soilsamples were taken to a depth of 46 cm utilizing both apick and a shovel that were scrubbed clean betweensamples. The locations of the three sample sites wereselected based on previous data indicating high concentra-tions of AT and AL. The sample sites S-5A (east side ofloading dock) and S-5B (south side of loading dock) werelocated within 1m of the bulk pesticide storage buildingand loading dock, while S-6 was on the west side betweenthe railroad spurs that ran several feet behind the loadingdock where product was unloaded. The three samples wereplaced in clean plastic 113.6 l garbage cans, covered withfitted lids and immediately transported to the laboratory.The samples were stored for 7–14 d at 0 1C until analysis.

2.2. Soil characterization

Soils were air dried, ground and passed through a 4-mmsieve. Twenty grams of the dried, ground soil wereextracted with 2M potassium chloride (KCl) and analyzedfor ammonia-nitrogen (NH4-N) and nitrate/nitrite-nitro-gen (NO3-N) utilizing steam distillation/acid titration(Standard Methods for the Examination of Water andWastewater 17th Edition, Method 350.1/417D [Clesceriet al., 1989]) and Devardo’s Alloy distillation/acid titration(Method 350.2/417D), respectively. Table 1 lists thestandard physical and chemical properties of the soil thatwere determined by the University of Delaware SoilTesting Laboratory (Sims and Heckendorn, 1991). Waterholding capacity of the three soil samples was determinedusing a standard pressure membrane apparatus. Fifty-gramaliquots of the dried, ground soil were extracted for ATand AL using a solid phase extraction method previouslydescribed (Chirnside and Ritter, 1992) followed by analysisby HPLC (Table 1). The concentrations of AT and ALwere quantified utilizing the HPLC system (ThermalSeparation Products, West Palm Beach, FL) under thefollowing conditions: (a) mobile phase—65% acetonitrilein deionized water; (b) flow rate—1.3mlmin�1; (c) column-RP-18 reverse phase, 25mm� 4.6 cm, 5 mm (E. MerkSeparations Technology, Darmstadt, Germany); and(d) detector-UV, 220 nm.

2.3. Enrichment design and development

Standard enrichment techniques were used to obtainindigenous microbial cultures capable of degrading AT andAL following the methods of Radosevich et al. (1993). Thesoil samples, S-5A, S-5B and S-6, were assessed individu-ally for the presence of microbial degradation of AT andAL. The following three herbicide treatment schemes wereutilized for the development of the SMC: TMT (1) AT andAL as the only source of carbon (C) and nitrogen (N),

ARTICLE IN PRESS

Table 1

Physical and chemical properties of soil samples (dw basis)

Sample S-5A S-5B S-6

Location East side of loading dock South side of loading dock West side between RR spurs

Textural class Loamy sand Clay loam Sandy loam

SOM (%) 1.1 (0)a 1.4 (0) 2.8 (0.07)

pH 7.33 7.60 6.13

NH4-N (mgkg�1) 10.49 (0.31) 0.27 (0.36) 1.02 (0.41)

NO3-N (mgkg�1) 50.24 (3.12) 22.75 (0.67) 26.69 (4.08)

Sol salts (mmho cm�1) 1.34 (0.11) 0.83 (0.03) 1.20 (0.05)

Acidity (meq 100 g�1) 0.033 (0.029) 0.003 (0.006) 0.023 (0.006)

Moisture content, field capacity (%) 8.61 20.04 19.54

Atrazine (mgkg�1) 205.1 (10.2) 0.7 (0.1) 1.1 (0.1)

Alachlor (mg kg�1) 108.5 (8.3) 3.6 (0.2) 1.6 (0.1)

aNumber in parentheses represents standard deviation of three values.

A.E.M. Chirnside et al. / Soil Biology & Biochemistry 39 (2007) 3056–30653058

TMT (2) AL as the sole source of C and N, and TMT(3) AT as the sole source of C and N. The herbicidetreatments were made by dissolving 27.0mg AL and/or21.6mg of AT in one liter of deionized distilled water(DDW) containing 10ml of concentrated phosphate buffer(0.24M KH2PO4 and 0.20M K2HPO4, pH ¼ 6.8). Thebuffered solutions were stirred overnight to ensurecomplete dissolution of the herbicides and then autoclavedat 120 1C for 20min. A concentrated salt solution (0.20MMgSO4 � 7H2O) and a concentrated trace element solution(3.7� 10�3M FeCl3, 6.8� 10�3M CaCl2, 8.0� 10�5MMnCl2, and 6.2� 10�7M ZnSO4; from Radosevich et al.,1993) were made individually and filter sterilized using a0.20 mm filter system. Under aseptic conditions, 39ml ofthe appropriate buffered herbicide solution and 0.5ml ofeach of the concentrated filter sterilized solutions wereadded to 250ml Erlenmeyer incubation flasks for a finalpesticide concentration of 100 mM. Two flasks of eachtreatment were inoculated with 10 g of dried, ground soilfor each of the three sites sampled. A control flask for eachsoil site containing the combined herbicide treatment (ATand AL) and 10 g of autoclaved dried, ground soil weremonitored to account for any non-microbial loss of ATand AL during experimentation. The flasks were sealedwith foam plugs to allow for aerobic conditions andincubated for 6 weeks at 25 1C on an orbital shaker at150 revmin�1 and sampled periodically for loss of parentcompound as described in the succeeding SMC analysis.

After 6 weeks, all flasks were subcultured by asepticallytransferring 10ml of the well-mixed original culture intonew flasks with fresh media containing the identicalherbicide treatments. Flasks were subcultured again after22 weeks. Selected treatment flasks from these initialcultures that exhibited a reduction in herbicide concentra-tion were subcultured into fresh media and monitored for22 weeks as outlined above. At this point only culturesexhibiting herbicide degradation were maintained bytransferring to flasks containing media consisting of thethree original herbicide treatment schemes. Based on thelack of AL degradation in the treatment flasks containing

only AL, the most successful subcultures from the finaldevelopment study were maintained on aqueous mediacontaining the original Treatment 1, i.e., AT and AL as theonly source of C and N.

2.4. SMC analysis

Prior to sampling, the flasks were weighed and correctedfor evaporative water loss by addition of sterile water. A0.5ml aliquot was removed and mixed with 0.5ml ofacetonitrile in a microcentrifuge tube and centrifuged atapproximately 12,000g for 10min to remove any suspendedsoil particles. The supernatant was transferred to HPLCvials for analysis of AT and AL utilizing the methoddescribed above for the soil extractions. Percent recoveriesof AT and AL were 10175.8% and 9778.7% (*Po0.05),respectively. Percent recovery was based on the analysis ofseven concentrations of spiked soil and water samples runseven times.

2.5. Isolation and identification of the bacteria in the SMC

Isolation of the bacteria present in the subcultures wasaccomplished by streaking 100 ml of culture onto agar Petridishes. Four different types of agar plates were used for theisolations representing the three treatment schemes as wellas a nutrient agar. The treatment solutions described abovethat were used for the SMC development were mixed withagar to form plates representing TMT 1 (100 mM of ATand AL), TMT 2 (100 mM of AL), and TMT 3 (100 mM ofAT). Nutrient agar was commercially available dextroseagar prepared according to the manufacturer (Difco:Becton Dickinson Microbial Systems, Sparks, MD). Foreach treatment, typical serial dilutions of the SMC flaskwith the greatest amount of pesticide degradation weremade. Dilutions from the TMT 1 SMC flask weretransferred to both the TMT 1 agar and the nutrient agar.Dilutions from the TMT 2 SMC flask were transferred toboth the TMT 2 agar and the nutrient agar. Dilutions fromthe TMT 3 SMC flask were transferred to both the TMT 3

ARTICLE IN PRESS

Table 2

Parent pesticide concentrations measured over time (years) for the three

soil samples taken from the mix-load site

Atrazine

(mg kg�1)

Alachlor

(mg kg�1)

NH4-N

(mgkg�1)

NO3-N

(mgkg�1)

S-5 1988 51.72 2.05 NDa 1150

S-5 1989 23 2.05 ND 112

S-5 1994 33.0 2.10 ND ND

S-5 1997 205.1 108.5 10.5 50.2

S-5B 1988 6.65 o0.4 ND 105

S-5B 1989 0.2 9.06 ND 91

S-5B 1994 o0.1 o0.6 ND ND

S-5B 1997 0.723 3.61 0.27 22.8

S-6 1988 19.54 31.67 ND 1150

S-6 1989 2.0 9.06 ND ND

S-6 1994 o0.1 o0.61 ND ND

S-6 1997 1.09 1.61 1.02 26.7

aND, not determined.

A.E.M. Chirnside et al. / Soil Biology & Biochemistry 39 (2007) 3056–3065 3059

agar and the nutrient agar. The Petri plates were incubatedat 27 1C for 1 week. Five distinctive, isolated colonies fromthe plates were transferred to trypticase soy agar plates(TSP) and incubated at 27 1C for 24 h in preparation forFatty Acid Methyl Ester (FAME) profile analysis that hasbeen developed for the taxonomic identification of puremicrobial isolates (MIDI, Newark, DE).

For FAME analysis, one loop of bacterial cells from theisolated colonies on each plate was added to a 20ml testtube and closed with a Teflon-lined screw cap. Each tubereceived 1ml of saponification reagent and was mixed(vortex mixer) and heated at 100 1C for 30min to liberatefatty acids from the cellular lipids. Once cooled, each tubereceived 2ml of methylation reagent, was mixed again andheated at 80 1C for 10min to form methyl esters of the fattyacids. The FAMEs were extracted from this solution byadding 1.25ml of one part methyl-tert-butyl ether in onepart hexane (v:v) (ultrapure grade, VWR ScientificProducts, Bridgeport, NJ) and placing the closed tubeson an end-over-end mixer for 10min. Finally, each tubewas centrifuged for 10min at 10,000g, the bottom aqueouslayer removed with a pastuer pipet and the supernatantwashed with 3ml of dilute NaOH (0.27M NaOH). Theextracted fatty acids were analyzed by a Hewlett Packard(HP) gas chromatograph (Wilmington, DE) equipped witha HP Ultra 2 Cross-linked 5% phenyl methyl Siliconecolumn (250mm� 0.2mm� 0.33 mm) and a flame ioniza-tion detector (FID) using H2 as the carrier gas. Theinjection and FID temperatures were 250 and 300 1C,respectively. The oven temperature was increased linearlyfrom 170 to 300 1C at 5 1Cmin�1. The FAME profile wasanalyzed utilizing the MIDI, Inc. eukaryote program thatcovers the fatty acids with a larger range of chain lengthcompared to the aerobe program. Fatty acids with chainlengths exceeding twenty carbons (characteristic of higherorganisms, e.g., plants) were not considered in the analysisin an effort to focus strictly on microbial organisms.

3. Results

3.1. Soil characterization

The physical and chemical properties of the three soilsamples are presented in Table 1. High concentrations ofboth NH3-N (10.5mg kg�1) and NO3-N (50.2mg kg�1)were found in S-5A (Table 1). Only 0.3 and 1.0mg kg�1 ofNH3-N was found in S-5B and in S-6, respectively. Similarconcentrations of NO3-N were found in S-5B and in S-6(22.8 and 26.7mg kg�1, respectively). S-5A was character-ized as a loamy sand with 1.1% soil organic matter (SOM),1.34mmho cm�1 soluble salts, an exchangeable acidity of0.033 cmol kg�1, and a neutral pH of 7.33. Similarly, S-5Bwas characterized as a clay sand with 1.4% SOM,0.83mmho cm�1 soluble salts, an exchangeable acidity of0.0033 cmol kg�1, and a neutral pH of 7.60. S-6 wascharacterized as a sandy loam with 2.75% SOM,1.20mmho cm�1 soluble salts, an exchangeable acidity of

0.023 cmol kg�1, and a slightly acidic pH of 6.13. Con-centrations of AT and AL found in the three soil samplesare also presented in Table 1. AT and AL soil concentra-tions (dry weight (dw) basis) were highest in S-5A, with205mgkg�1 of AT and 108mgkg�1 of AL. S-5B and S-6had AT concentrations of 0.72 and 1.09mg kg�1 and ALconcentrations of 3.61 and 1.61mg kg�1, respectively.The mix-load site had been monitored for pesticide

contaminants three other times when the property changedownership (Table 2; Xin et al., 1995). Despite these changesin ownership, the site has continued as an active pesticideand fertilizer mix-load site. No degradation of AT and ALwas found at the S-5A site where nutrient concentrationsand AT and AL concentrations were high suggesting thatthere was a lack of microorganism capable of degradingAT and AL in the soil or the availability of the nutrients tothe microbes inhibited degradation of the more recalcitrantherbicides. Conversely, previous analysis of S-5B (Table 2)revealed that the herbicides had been present at highconcentrations indicating that degradation did occur at thissampling location over time, thus suggesting the possibilitythat microbial degraders with the ability to degrade ATand AL are present in the soil at S-5B.

3.2. SMC enrichment

3.2.1. Results from initial cultures

In the initial SMC cultures, degradation of the parentcompounds occurred in TMT 1 only, which contained bothAT and AL. Table 3 lists the disappearance of AT and ALfrom the SMC flasks. S-5A had not exhibited anydegradation in all treatments (Fig. 1a). S-5B exhibiteddegradation of both compounds in the combined treatment(TMT 1) demonstrating a time lag in the degradation of theAL (Fig. 1b). Finally, S-6 exhibited AT degradation butonly in the combined treatment, TMT 1 (Fig. 1c).

ARTICLE IN PRESS

Table 3

Disappearance of AT and AL from SMC flask solution (Treatment 1)

Site 5A Site 5B Site 6 Control

Week AT AL AT AL AT AL AT AL

0 114.0 (1.23)a 130.2 (4.57) 115.4 (8.26) 109.8 (3.59) 112.3 (0.361) 115.9 (3.02) 117.5 (8.39) 121.3 (4.11)

2 117.5 (3.51) 139.0 (4.36) 108.7 (0.79) 57.2 (19.3) 117.2 (15.7) 116.0 (8.47) 117.6 (3.47) 126.9 (12.4)

5 107.6 (0.30) 129.2 (5.87) 0.891 (0) 52.2 (11.1) 79.6 (2.67) 95.4 (5.16) 108.5 (3.29) 110.0 (11.1)

7 109.0 (4.01) 129.1 (0.45) 0.891 (0) 51.3 (10.6) 39.8 (19.2) 95.9 (6.10) 110.1 (1.67) 109.3 (15.6)

8 105.4 (0.90) 124.4 (8.13) 0.891 (0) 49.7 (6.88) 0.891 (0) 94.7 (2.54) 107.6 (4.82) 114.3 (9.41)

12 123.8 (7.93) 131.9 (15.1) 0.891 (0) 37.4 (17.1) 0.891 (0) 92.7 (5.24) 120.6 (4.99) 115.9 (6.46)

Concentration (mM) is the average of two replications.aNumber in parentheses represents standard deviation of three values.

A.E.M. Chirnside et al. / Soil Biology & Biochemistry 39 (2007) 3056–30653060

3.2.2. Results from the subcultures of flasks with pesticide

degradation

Subsequent subcultures of the flasks with the greatestpesticide removal resulted in inconsistent degradationof AT and AL (Fig. 2). Replicated subcultures from thesame flask showed high variability in the amount ofpesticide degraded. In general, the results were similar tothe first cultures in that the subcultures from S-5Bexhibited AT and AL degradation in TMT 1. The lengthof time for complete disappearance of AT differedamong replications (Fig. 2), while it took an average of7.5 d until half of the concentration of AT was degraded;i.e., experimental half-life (t1/2) ¼ 7.5 d. AL degradationwas more varied among the replicates of TMT 1. Fig. 2shows no change in AL concentration for one replicate(Flask ]5) while AL was completely degraded in Flask ]6(Fig. 2) with an AL t1/2 of approximately 11 d. ALdegradation occurred in one of the AL only flasks (TMT2) from S-5B, which differed from the initial cultures (Fig.4). Degradation did proceed slowly for the first 2 weeks butdisappeared by week 4 with a t1/2 of 9.5 d. Subcultures ofTMT 3 (AT only) exhibited a 2-week lag period followedby rapid disappearance of AT (Fig. 5) with an averaget1/2 ¼ 16 d.

Subcultures from S-6 flasks exhibited the same degrada-tion pattern as seen in the initial cultures. No ALdegradation was seen in any of the treatments (Figs. 3and 4). AT degradation occurred after 4 weeks in TMT 1(Fig. 3) and TMT 3 (Fig. 5).

3.2.3. Serial dilution plate count results

Agar plate counts of the enrichment culture resulted intoo numerous to count (TNTC) tiny colonies from serialdilution 10�6 and lesser for TMT 1, 2, and 3. Nutrient agarplates were similar but the numbers of colonies appearedgreater than on the treatment plates for equivalentdilutions. The AT and AL selective agar (TMT 1) producedmore colonies than TMT 2 and 3 (Table 4).

There were five morphologically distinctive types foundon both the selective agars and the nutrient agar. However,the colonies appeared darker in color when growing on the

nutrient agar. The five morphologically distinct coloniescan be described as follows:

A.

Tiny in size; clear to white in color; round to elliptical inshape with irregular edges; with a flat growth patternon the plate.

B.

Small to medium in size; red in color with darker centersand a shiny appearance; round in shape with smoothedges; with a raised growth pattern on the plate.

C.

Medium to large in size; creamy white to beige in colorwith an opaque appearance; round in shape with smoothedges; with a raised growth pattern on the plate.

D.

Large in size; creamy white in color with yellow to orangecolor in the center; round in shape with somewhatsmooth edges; with a raised growth pattern on the plate.

E.

Small to medium in size; red to reddish brown in color;round in shape with smooth edges; with a raised growthpattern on the plate.

3.2.4. Results of the FAME analysis

FAME analysis of the five isolated colony typesdescribed above and chosen for FAME analysis were fromthe TMT 1 agar plates (AT and AL) containing serialdilutions of the active SMC. Results from the analysis arelisted in Table 5. As seen from the table, some of thematches had similarity indexes greater than 70%. Therewas an 86.4% similarity to the bacteria Alcaligenes. Thespecies A. xylosoxydans and the subspecies A. x. denitri-

ficans also had similarity indices of 86.4%. Anotherbacterium in the culture, Pseudomonas putida, had asimilarity index of over 80% as well.

4. Discussion

4.1. SMC enrichment procedure

A SMC capable of degrading AT and AL wassuccessfully isolated from only one of the three sitessampled, S-5B. Degradation occurred in the combined ATand AL treatments only. Alachlor alone was not asufficient N source for the microbes. Site 5A soil exhibited

ARTICLE IN PRESS

S-5A -SMC FLASKSAT and AL Degradation

10

15

20

25

30

35

40

Incubation time (weeks)

Con

cent

ratio

n (m

g l-1

sol

utio

n)

AT

Control AT

AL

Control AL

0 7 1252 8

S-6 SMC FLASKSAT and AL Degradation

Con

cent

ratio

n (m

g l-1

sol

utio

n)

Incubation time (weeks)

0

5

10

15

20

25

30

35

0 7 1252 8

AT

Control AT

AL

Control AL

S-5B -SMC FLASKSAT and AL Degradation

Incubation time (weeks)

Con

cent

ratio

n (m

g l-1

sol

utio

n)

AT

Control AT

AL

Control AL

0

5

10

15

20

25

30

35

5 8 120 2 7

Fig. 1. Disappearance of AT and AL form SMC cultures for Treatment I;

(a) Site 5A, (b) Site 5B, and (c) Site 6.

S-5B -FLASK 5 Atrazine & Alachlor

-10

10

30

50

70

90

110

0 12108642

Incubation time (weeks)

Con

cent

ratio

n <

mu>

M

AT

Control AT

AL

Control AL

S-5B -FLASK 6 Atrazine & Alachlor

Con

cent

ratio

n <

mu>

M

-2

0

20

40

60

80

100

120

0 12108642

Incubation time (weeks)

AT

Control AT

AL

Control AL

Fig. 2. Disappearance of AL and AT from selective flasks from

subcultures of the initial SMC (Treatment 1).

A.E.M. Chirnside et al. / Soil Biology & Biochemistry 39 (2007) 3056–3065 3061

no degradation in any of the treatments. The soil had highconcentrations of NH4-N and NO3-N; therefore themicrobes did not need AT and AL as a source of N.Atrazine degradation has been negatively correlated toinorganic nitrogen concentrations suggesting AT-nitrogenutilization by the degrading microorganisms (Alvey andCrowley, 1995; Ames and Hoyle, 1999; Bichat et al., 1999;Gan et al.,1996; Radosevich et al., 1993). Thus, thepresence of high concentrations of inorganic N at S-5A

could have inhibited the selection pressure for N-utilizingAT degraders. Concomitantly, the low levels of inorganicN and the initial high AT concentrations at S- 5B may haveincreased the selection pressure for indigenous microbescapable of degrading AT for its N content resulting in adecrease of AT over time. Addition of AT and AL to theenrichment cultures triggered the induction of the neces-sary enzymes for degradation to occur.In general, initial enrichment cultures demonstrated that

the ability to degrade AL by the SMC was dependent onAT degradation. AL degradation did not begin untilapproximately 15% of the AT was transformed. Researchhas shown that one of the first steps in AT degradation isN-dealkylation (Bichat et al., 1999; Radosevich et al.,

ARTICLE IN PRESS

S-6 -FLASK 9 Atrazine and Alachlor

0

20

40

60

80

100

120

140

Incubation time (weeks)

CO

NC

EN

TR

AT

ION

<

mu>

M

0 4 6 8 10 122

AT

Control AT

AL

Control AL

S-6 -FLASK 10Atrazine and Alachlor

0

20

40

60

80

100

120

140

160

180

CO

NC

EN

TR

AT

ION

<m

u>M

Incubation time (weeks)0 4 6 8 10 122

AT

Control AT

AL

Control AL

Fig. 3. Disappearance of AL and AT from selective flasks from

subcultures of the initial SMC (Treatment I). Flasks are from Site 6 soil.

S-5B -FLASKS 5 & 6 Alachlor

-10

10

30

50

70

90

110

0 2 4 6 8 10 12Incubation time (weeks)

Con

cent

ratio

n <

mu>

M

AL-5 AL-6 Control AL

Con

cent

ratio

n <

mu>

M

S-6 -FLASKS 9 & 10 Alachlor

0 2 4 6 8 10 12Incubation time (weeks)

40

60

80

100

120

140

AL-9 AL-10 Control AL

Fig. 4. Disappearance of AL from selective flasks from subcultures of the

initial SMC (Treatment 2).

A.E.M. Chirnside et al. / Soil Biology & Biochemistry 39 (2007) 3056–30653062

1993). After utilization of the C’s located on the alkyl sidechains on the triazine ring, the microbes must havedegraded AL for the additional C because of the non-zeroreductance degree of the ring C’s, which cannot be amicrobial source of energy or growth (Radosevich et al.,1995; Yassir et al., 1999). Bichat et al. (1999) saw microbialgrowth when AT was the sole source of C and N but littlering cleavage suggesting that the side alkyl chains cansupply the C and energy required for growth. Further

growth and/or ring cleavage would require the energyderived from additional C sources. Sun et al. (1990)isolated an SMC capable of using AL as the sole C andenergy source. Added C (0.01% sucrose) decreasedmineralization from 12% to 5.7% indicating AL as theutilized C source. Thus, the AL in the enrichment culturescould have provided C to the microbes for energy needed inorder to cleave the triazine ring. With the completemineralization of AT, there would be no N for furtherAL degradation. In this study, adding more AT to theenrichment cultures that had had complete degradation ofAT resulted in the removal of more AL (data not shown).The instability of the SMC’s ability to degrade ALsuggested that the enzymes involved in the AL degradationmechanisms were inducible. Another possibility is that themicrobes responsible for the AL degradation may not havebeen as competitive as the other microbes within the SMC;and therefore, their numbers were low within the totalconsortium population.The effect of site characteristics (SOM, water content,

soil texture, and the nature of the contamination) on the

ARTICLE IN PRESS

S-5B -FLASKS 5 & 6 Atrazine

-30

-10

10

30

50

70

90

110

0 2 4 6 8 10 12

Incubation time (weeks)

Con

cent

ratio

n<

mu>

M

AL-5 AL-6 CONTROL AT

AL-9 AL-10 CONTROL AT

S-6 -FLASKS 9 & 10 Atrazine

0 2 4 6 8 10 120

20

40

60

80

100

120

140

Incubation time (weeks)

Con

cent

ratio

n <

mu>

M

Fig. 5. Disappearance of AT from selective flasks from subcultures of the

initial SMC (Treatment 3).

Table 4

Plate counts from initial SMC flasks

Treatment Dilution Selective agar Nutrient agar Transfera

AT and AL 10�6 TNTC TNTC

10�7 30 35 30

10�8 10 7

AL 10�6 25 TNTC

10�7 7 1 20

10�8 3 0

AT 10�6 16 20

10�7 2 5 16

10�8 1 0

aTransfers were from the selective agar plates of the 10�7 dilution to

similar selective agar plate.

Table 5

Results from the FAME analysis of the SMC

Microorganism Similarilty index

Colony type A

Alcaligenes 0.864

Alcaligenes xylosoxydans 0.864

Alcaligenes xylosoxydans denitrificans 0.864

Alcaligenes xylosoxydans xylosoxydans 0.498

Colony types B and E

Pseudomonas 0.839

Pseudomonas putida 0.839

Pseudomonas putida biotype A 0.839

Pseudomonas marginalis 0.691

Pseudomonas chlororaphis 0.381

Colony type C

Providencia

Providencia rustigianii 0.716

Erwina chrysanthemi 0.598

Erwina chrysanthemi biotype V 0.598

Erwina chrysanthemi biotype III 0.558

Erwina chrysanthemi biotype II 0.532

Erwina chrysanthemi biotype V 0.598

Colony type D

Actinobacillus 0.354

Actinobacillus lignieresii 0.354

A.E.M. Chirnside et al. / Soil Biology & Biochemistry 39 (2007) 3056–3065 3063

degradative mechanisms of these indigenous populationscould have resulted in differing AT degrading capabilities.Ames and Hoyle (1999) found AT-degrading populationsin soils collected from a mix-load site but found that theirspatial distribution was not uniform. Certain selected areaswithin the mix-load site saw decreases in AT concentrationover time, while other areas did not. Enrichment culturesfrom these areas of reduced AT concentration exhibitedAT degradation activity. There were no positive enrich-ment cultures from the samples collected from the areaswhere there had not been a reduction in AT. The samephenomenon occurred at the mix-load site of this study.

The indigenous microbes from two of three locationssampled were able to degrade AT and AL in enrichmentcultures but required an extensive lag period of 2 weeks.Reintroduction of AT and AL to the enrichment culturestriggered the induction of the required degradative enzymesby the indigenous microbes present.

4.2. Discussion of FAME results

From the results of the FAME analysis, the best matchwas from the clear white colony (A) and was identified asan Alcaligenes. Certain species of this genus have beenshown to degrade some halogenated aliphatic compounds

ARTICLE IN PRESSA.E.M. Chirnside et al. / Soil Biology & Biochemistry 39 (2007) 3056–30653064

cometabolically under aerobic conditions (Cookson, 1995).These organisms generate oxygenase enzymes of broadsubstrate specificity. They have been shown to mineralizemultiple ring aromatics as well as PCBs (Cookson, 1995).Alcaligenes denitrificans was able to utilize the chlorophe-noxy herbicide (R)-(+)-mecoprop as the sole source of Cand energy through monooxygenase catalyzed reactions(Teft et al., 1997). Ring cleavage of the correspondingcatechol was through the ortho cleavage pathway. Thedegrading enzyme systems of A. denitrificans showed adegree of non-specificity toward methylated catechols andcatechol intermediates. When oxygen concentration wasgreater than stoichiometric amounts, aerobic mineraliza-tion of 2,4-dinitrotoluene (2,4-DNT) was achieved byAlcaligenes sp. JS867. This Alcaligenes species was tenta-tively identified as A. xylosoxidans and was closely relatedto A. x. denitrificans (Smets and Mueller, 2001). Anothergood match from the FAME analysis of the red colonies(B, E) was Pseudomonas and in particular P. putida.

Pseudomonas is a well-studied bacterium with severalspecies known to degrade AT (Alexander, 1994; Cookson,1995). They are labeled as Gram-negative, organotrophicorganisms with approximately 30 recognized species. Theyare known to utilize about 60–100 different organiccompounds as sole C and N sources (Cookson, 1995).Earlier work by Sun et al. (1990) found an SMC able tomineralize AL containing seven distinct bacterial strainswith three of those strains representing 75% of the CFUs.Two of those three strains were Pseudomonas spp. Inanother study (Mandelbaum et al., 1995), characterizationof an AT-degrading SMC (sole C and N) resulted in theisolation of several Pseudomonas species. Colonies weredescribed as circular with an irregular border, opaque andlight brown with darker coloration in the center. Substrateutilization tests found the isolate belong to RNA group 1of genus Pseudomonas, specifically Pseudomonas citronel-

losis (similarity index ¼ 0.544), while FAME analysisidentified Pseudomonas aeruginosa. The isolate was labeledPseudomonas sp. strain ADP (Mandelbaum et al., 1995).

In conclusion, a SMC capable of degrading AT and ALwas isolated from a contaminated mix-load site soil. Theconsortium exhibited a unique degradation pattern in thatAL degradation was dependent on AT degradation.Further work with the isolated SMC is needed to elucidatethe degradative metabolites and to identify the environ-mental factors required for optimal degradation. Manip-ulation of the cometabolic pattern of pesticide degradationcould lead to the development of the SMC for large scale insitu remediation of pesticide-contaminated soils.

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