identiﬁcation of bacteria isolated from an oligotrophic...

Identification of Bacteria Isolated from an Oligotrophic Lake with Pesticide

Removal Capacities

L. LOPEZ,1 C. POZO,1 B. RODELAS,2 C. CALVO,2 B. JUAREZ,1 M.V. MARTINEZ-TOLEDO1

AND J. GONZALEZ-LOPEZ1

1Group of Environmental Microbiology, Institute of Water Research, Faculty of Pharmacy, University ofGranada, 18071, Granada, Spain

2Group of Environmental Microbiology, Department of Microbiology, Faculty of Pharmacy, Universityof Granada, Granada, Spain

Accepted 10 December 2003

Abstract. We studied the growth and capacities for pesticides removal of bacterial strains isolated from theLaguna Grande, an oligotrophic lake at the South of Spain (Archidona, Malaga). Strains were isolatedfrom water samples amended with 10 and 50 lg/ml of nine pesticides: organochlorinated insecticides(aldrin and lindane), organophosphorous insecticides (dimetoate, methyl-parathion and methidation),s-triazine herbicides (simazine and atrazine), fungicide (captan) and diflubenzuron (1-(-4-chlorophenyl)-3-(2,6-difluorobenzoyl urea), a chitinase inhibitor. The majority of the strains belonged to the generaPseudomonas and Aeromonas and only 9% of the total of strains were Gram positive. From all the strainsisolated, only 22 showed a wide growth range in all the pesticides tested and 4 of them were chosen forpesticide removal studies. The genetic identification of these strains showed their affiliation to Pseudomonaspseudoalcaligenes, Micrococcus luteus, Bacillus sp. and Exiguobacterium aurantiacum. These last two strainswere those that showed the highest pesticide removal capacities and a high bacterial growth.

Keywords: aquatic microbiota; pesticides; removal; Pseudomonas pseudoalcaligenes; Micrococcus luteus;Bacillus sp; Exiguobacterium aurantiacum

Introduction

The use of pesticides for the control of crop pestsand weeds, plus the discharges from manufactur-ing plants, accidental spills, and natural processessuch as dilution, surface runoff and leaching, arethe causes of the occurrence of these xenobioticcompounds in surface waters (Readman et al.,1997; Dua et al., 2002). The effects of the pesticideson aquatic environments are also due to their

degradation products, which can be more toxicthan the original substances (Thurman et al.,1992). The persistence of pesticides and their deg-radation products in an aquatic system depends onseveral abiotic factors, such as adsorption to sed-iment particles, photochemical degradation, andabsorption by aquatic plants and animals, as wellas on biotic processes in which the microorganismsplay an important role (Aislabe and Lloyd-Jones,1995; Ellis, 2000; Janssen et al., 2001).

The selection of microorganisms with the abilityto degrade or biotransform pesticides from aparticular ecosystem has been an important field

*To whom correspondence should be addressed:

Tel.: +34958244170/+34958249966; Fax: +34958246235;

E-mail: [email protected]

Ecotoxicology, 14, 299–312, 2005

� 2005 Springer Science+Business Media, Inc. Manufactured in The Netherlands.

of research in the last decades. The scientific term‘‘biodegradation’’ has been used to define theprocess of using microorganisms either to immo-bilize or transform environmental contaminants toinnocuous end products (Glazer and Nikaido,1995). Numerous studies have been conductedabout the degradation of pesticides by microor-ganisms, both in pure and mixed culture systems.Microbial strains belonging to the generaPseudomonas, Bacillus, Xanthomonas and Rhodo-coccus, as well as fungi (especially white rot fungi),have demonstrated high degradation capacitiesover a wide range of xenobiotics, including pesti-cides (Bekhi et al., 1993; Mougin et al., 1994;Saagua et al., 1998; Doddamani and Ninnekar,2001; Kazt et al., 2001 ; Abou-Arab, 2002). Thebiodegradation is a very specific process and whenit takes place, it is likely to result from enzymaticactivity; this response either happen immediatelyor only after a period of adaptation to pesticide.

In this study, we describe the isolation andcharacterization of microbial strains with highcapacities to remove 9 different pesticides(organochloride, organophosphorous insecticides,s-triazine herbicides, one fungicide and dif-lubenzuron, a chitinase inhibitor) from watersamples of an oligotrophic lake. The growthcharacteristics of these bacterial strains, as well astheir identification by analysis of the sequence ofthe gene encoding 16 S rRNA (16 S rDNA), arereported.

Material and methods

Water samples

The water samples were picked from LagunaGrande, a natural lake located in the region ofAntequera (Malaga, Southern Spain). This lake isconsidered as an oligotrophic system. The sam-pling period was scheduled during 2 years(September 1999–September 2001) and sampleswere taken every 3 months. Previous studies onthis natural lake (Benavente et al., 1998) showedthat the pesticides studied in this report were notdetected in this oligotrophic system.

The water samples were picked near the lakeborder and maintained at 4 �C until their analysisin the laboratory. The mean values of several

chemical and physical characteristics of the sam-ples during the sampling period were the follow-ing: Ca2+, 793 ± 99 mg/l; Mg2+, 142 ± 28 mg/l;Na+, 177 ± 49 mg/l; K+, 7 ± 1 mg/l; Cl),298 ± 54 mg/l; HCO3

), 61 ± 15 mg/l; SO43),

2072 ± 320 mg/l and pH 7.2.

Effect of selected pesticides on platableheterotrophic bacteria

The pesticides used (technical purity grade) werepurchased from Sugerlabor (Barcelona, Spain).The pesticides tested belonged to the followingchemical groups: organochlorinated insecticides(aldrin and lindane), organophosphorous insecti-cides (dimetoate, methyl-parathion and methida-tion), s-triazine herbicides (simazine and atrazine),fungicide (captan) and the chitinase inhibitor dif-lubenzuron (1-(-4-chlorophenyl)-3-(2,6-difluorobe-nzoyl urea).

The nine pesticides were dissolved in acetoneand sterilized by filtration prior to being added tothe water samples, using a sterile filter (0.22 lmpore size, FG type Millipore�). The experimentswere initiated dispensing 350 ml of water samplesin sterile Erlenmeyer flasks (1000 ml) and thenamended with appropriate concentrations of thesterile pesticide stock solutions, to give final con-centrations of 10 and 50 lg/ml. Control sam-ples received the same acetone volumes forcomparison.

All flasks were incubated with continuous agi-tation (150 rpm) at 20 �C in the dark. After 1, 7,14 and 28 days of incubation, total platable hete-rotrophic aerobic bacteria were counted by thedilution plate technique, according to Lopez et al.(2002). The inoculated agar plates (three repli-cates) were incubated at 28 �C for 3 days beforecolonies were counted.

Isolation of potential pesticide-degradingmicroorganisms

Fifty milliliter of water samples were placed in250 ml Erlenmeyer flasks and amended with50 lg/ml of pesticides previously sterilized by fil-tration using a sterile syringe as above described,and then incubated at 28 �C under gentle agitation(150 rpm) for 28 days. Aliquots (0.1 ml) of thesewater samples were spreaded on tripticase soy agar

300 Lopez et al.

plates (TSA, Difco. Detroit, MS, USA). Approx-imately 10 randomly selected bacterial colonieswere sub-cultured from each TSA agar plate. Dishcolony isolates were streaked twice on TSA platesto ensure purity of the cultures.

Characterization of isolated bacteria

Pure isolates were identified using the criteria ofBergey’s Manual of Systematic Bacteriology(1986). The following morphological and bio-chemical tests were used: cell morphology, motil-ity, flagella, spores, Gram stain, pigments, glucosefermentation, utilization of carbon substrates(glucose, arabinose, mannose, mannitol, N-acet-ylglucosamine, maltose, gluconate, caproate, adi-pate, malate, citrate and phenylacetate), oxidase,catalase, NO3

) reduction, O/F test, Indol, ONPG,arginine dihidrolase, Voges-Proskauer test, phen-ylalanine deaminase, urease activity, hydrolysis ofstarch, gelatinase, casein, and tyrosine degrada-tion. Most of the tests of carbon substrate utili-zation were performed using the API20 NEidentification system (bioMerieux, SA, France).

Microbial growth in presence of pesticide

For construction of growth curves, pure isolateswere cultured at 28 �C for 1, 2 and 3 days underaerobic conditions (rotary shaker, 150 rpm) inmodified liquid Banat medium (Banat et al., 1993)amended with 0 or 50 lg/ml of a pesticide (aldrin,lindane, methidation, methylparation, atrazine,simazine, dimetoate, diflubenzuron or captan).The composition of the liquid medium was (g/ldistilled water): Na2HPO4, 2.2 g; KH2PO4, 1.4 g,MgSO4 Æ 7H2O, 0.6 g; (NH4)2SO4, 0.3 g; NaCl,0.05 g; CaCl2, 0.02 g; FeSO4 Æ 7H2, 0.01 g, andyeast extract 0.1 g. The pH was adjusted at 7.0using NaOH (0.1 N). The yeast extract was addedin the mineral medium as a growth factor. Platablecell counts were estimated by the dilution platetechnique (three replicates from each dilution),using TSA amended with 50 lg/ml of the corre-sponding pesticide.

Genetic identification of pesticide-degrading strains

Selected bacterial isolates were identified by anal-ysis of the sequence of the gene encoding 16 S

rRNA (16 S rDNA). Primers fD1 and rD1(Weisburg et al. 1991) were synthesized by Amer-sham Pharmacia (Sweden) and used to amplifyalmost the full length of 16 S rRNA gene from thestrains. The sequences of the primers are follow-ing: fD1, 5¢ccgaattcgtcgacaacAGAGTTTGATCCTGGCTCAG3¢, rD1, 5¢ccgggatccaagctt AAGGAGGTGATCCAGCC3¢. Linker sequencescontaining restriction sites for cloning are indi-cated in lower case letter. Upper case letters spanpositions 8–27 (fD1) and 1541–1525 (rD1) of E.coli strain K12 16 S rDNA sequence.

A fresh cultured colony of each strain was lysedby the addition of 20 ll of a mixture of NaOH(0.05 M)-SDS (0.25%, w/v) and then boiled for15 min. The lysate was adjusted to 200 ll withsterile water and centrifuged at 10,000 · g for5 min. The cleared lysate (4 ll) was used as tem-plate for amplification. PCR was done adding tothe lysate 1· PCR buffer (GeneCraft, Germany),1.5 mM MgCl2(GeneCraft, Germany), 200 lMdNTPs (Roche Molecular Biochemicals,Germany), 20 pmol of each primer, and 1 U ofTaq polymerase (GeneCraft, Germany). Finalvolume of the reaction tubes was adjusted to 50 ll.Reactions were run in a Perkin Elmer GeneAmpPCR system 2400 (Perkin Elmer, Norwalk, USA).The temperature profile was the one previouslydescribed by Vinuesa et al. (1998). PCR productswere run on 1% agarose gels and the bands werepurified using the Quiaex II kit (Quiagen,Germany). The nucleotide sequence of the purifiedbands was determined by the dideoxy chain ter-minator method, using the ABI-PRISM Big DyeTerminator Cycle Sequencing Ready Reaction kit,(Perkin Elmer, USA) and automated sequencerApplied Biosystems ABI 373 (Perkin-Elmer,USA). Sequence data were analyzed using theGCG Wisconsin Package v. 10.1. programs(Genetics Computing Group, Madison, Wiscon-sin, USA), and were compared to sequences inEMBL bank using FASTA v. 3.3t07 (Pearson andLipman, 1988).

Biodegradation studies

Inoculated and control (lacking bacteria) modifiedBanat medium amended with 50 lg/ml of selectedpesticides were cleared by centrifugation at10,000 · g for 10 min and filtered thought

Identification of Bacteria Isolated from an Oligotrophic Lake 301

LOG

CFU/mL

0

1

2

3

4

5

6

7

0 1 7 14 28

ALDRIN

ATRAZINE

0

1

2

3

4

5

6

7

0 1 7 14 28

CAPTAN

0

1

2

3

4

5

6

7

0 1 7 14 28

DIFLUBENZURON

0

1

2

3

4

5

6

7

DIMETOATE

0

1

2

3

4

5

6

7

0 1 7 14 28

LINDANE

0

1

2

3

4

5

6

7

0 1 7 14 28

METHIDATI0N

0

1

2

3

4

5

6

7

0 1 7 14 28

METHYLPARATHION

0

12

3

4

56

7

0 1 7 14 28

SIMAZINE

0

1

2

3

4

5

6

7

0 1 7 14 28

INCUBATION TIME (days)

Figure 1. Number of platable heterotrophic bacteria in water samples amended with 0: n, 10: h, and 50: j – l g/ml of aldrin, lin-

dane, dimetoate, methyl-parathion, methidation, atrazine, simazine, captan, and diflubenzuron. The values area median of three

replicates (p £ 0.05).

302 Lopez et al.

0.22 lm membrane filters. Samples (50 ml) wereextracted 3 times with chloroform (1:1); the sol-vent was removed under vacuum. Dried extractswere re-suspended in 1 ml chloroform, re-evapo-rated to dryness, re-dissolved in 50 l l acetone andtransferred to a vial, ready for gas chromatogra-phy determination. The solution was analyzed forpesticide by injecting 0.1 l l into a GC–MS spec-trometer, HP-5973 A-Model (Hewlett Packard,Palo Alto, CA, USA). The operating conditionswere as follows: injector temperature: 250 �C,detector temperature: 300 �C, oven temperature:100 �C, carrier flow rate: He 1 ml/min. Standardcurves for pesticides were calculated from10 concentrations (1–100 lg/ml) in modifiedBanat medium. The correlation coefficient was>0.99 and the sensitivity of the method was in therange of 0.2 lg/ml of pesticide.

Statistical analysis

Two-way analysis of variance (ANOVA) using thesoftware package STATGRAPHICS 3.0 Plus ver-sion (STSC Inc., Rockville, MD, USA) was per-formed to identify significant differences betweenthe treatments. A significance level of 95%(p < 0.05) was selected.

Results

The plate-count data indicated that the numbers oftotal viable bacteria in the water samples amendedwith 10 and 50 lg/ml pesticides were higher thanunamended controls. Our results indicate (Fig. 1)

that after the treatment with aldrin, lindane,dimetoate, captan and atrazine at concentrationsof 10 and 50 lg/ml, bacterial populations increasesignificantly after 1 day, whereas when watersamples were amended with methidation, meth-ylparation and diflubenzuron amended samplesdid not exceed the control until day seven. Afterthe treatment with simazine at concentrations of10 and 50 lg/ml, bacterial microbiota did notincrease until 14 days of incubation.

The results of the identification into broadgroups of the randomly selected bacteria isolatedfrom a second set of water samples amended with50 lg/ml of pesticides and incubated at 28 �C for28 days are given in Table 1. Most (90%) of the1330 isolates which were sub-culturable were Gramnegative rods and among these, Pseudomonas andAeromonas isolates were numerically dominant.

The group which accounted for the greatestproportion of the isolates (41%) consisted of Pseu-domonas spp. Aeromonas group was the secondlargest component of the heterotrophic microbiota(32%) and Agrobacterium group (14%) was also animportant fraction of the total number of strainsidentified. Only 9% of the isolated bacteria wereGram positive bacteria (5% Bacillus spp. and 4%Micrococcus spp.) and the other 4% of the totalcould not to be classified by biochemical and mor-phological methods. Of 1330 strains isolated, only22 were able to grow in modified liquid Banatmedium amended with 50 lg/ml of selected pesti-cides. Three strains were isolated from mediaamended with aldrin, 1 from atrazine, 1 from cap-tan, 4 from diflubenzuron, 4 from media amendedwith dimetoate, 1 from lindane, 3 frommethidation,

Table 1. The identification into broad groups of randomly selected aerobic bacterial isolates from oligotrophic lake water samples

amended with 50 lg/ml of different pesticides: aldrin (1), atrazine (2), captan (3), diflubenzuron (4), dimetoate (5), lindane (6)

methidation (7), methylparathion (8), and simazine (9)

Pesticide 1 2 3 4 5 6 7 8 9 Total

Group

Pseudomonas 56 71 60 47 102 30 74 45 60 545

Aeromonas 45 50 35 58 50 30 45 69 44 426

Agrobacterium 19 20 15 25 38 10 25 18 16 186

Bacillus 20 0 0 0 20 0 20 0 7 67

Micrococcus 0 9 0 0 10 0 11 10 13 53

N.I.a 0 10 10 10 0 0 5 8 10 53

Total 140 160 120 140 220 70 180 150 150 1330

aNot identified.


LOG

CFU/mL

Strain12Al

3

4

5

6

7

8

9

10

0 1 2 3 4

Control50 µg/ml aldrin

Strain 13Al

3

4

5

6

7

8

9

10

0 1 2 3 4

Control50 µg/mlaldrin

Strain 12 Cap

3

4

5

6

7

8

9

10

0 1 2 3 4

Control50 µg/mlcaptan

Strain 14Al

3

4

5

6

7

8

9

10

0 1 2 3 4

Control50 µg/mlaldrin

Strain 14Atrz

3

4

5

6

7

8

9

10

0 1 2 3 4

4

Control50 µg/mlatrazine

Strain 5Df

3

4

5

6

7

8

9

10

0 1 2 3

Control50 µg/ml diflubenzuron

Strain 6Df

3

4

5

6

7

8

9

10

0 1 2 3 4


Strain 7Df

3

4

5

6

7

8

9

10

0 1 2 3 4

Control50 µg/mldiflubenzuron

Strain 8Df

3

4

5

6

7

8

9

10

0 1 2 3 4


INCUBATION TIME (days)

Figure 2. Growth of several bacterial strains isolated from an oligotrophic lake able to grow in modified liquid Banat medium

amended with 50 lg/ml of aldrin, atrazine, captan and diflubenzuron. The values are a median of three replicates (p £ 0.05).

304 Lopez et al.

2 frommethylparathion and 3 frommedia amendedwith simazine. These isolates were studied in detailwith regard to their growth (Figs. 2, 3 and 4) andpesticides spectrum (Table 2).

Our results indicate that four pure isolates,named as 14 Atrz, 8M, 9D and 2Sz, gave the highestgrowth rates in modified Banat medium amendedwith 50 lg/ml of aldrin, lindane, methidation,

INCUBATION TIME (hours)

Strain 4D

3

4

5

6

7

8

9

10

0 1 2 3 4

Control

50 µg/ml dimetoate

Strain 9D

3

4

5

6

7

8

9

10

0 1 2 3 4

Control

50 µg/ml dimetoate

Strain 18D

3

4

5

6

7

8

9

10

0 1 2 3 4

Control50 µg/ml dimetoate

Strain 19D

3

4

5

6

7

8

9

10

0 1 2 3 4

Control50 µg/ml dimetoate

Strain 3Li

3

4

5

6

7

8

9

10

0 1 2 3 4

Control50 µg/ml lindane

Strain 8M

3

4

5

6

7

8

9

10

0 1 2

Control

50 µg/ml methidation

Strain 10M

3

4

5

6

7

8

9

10

0 1 2 3

Control


Strain 12M

3

4

5

6

7

8

9

10

0 1

Control


L

O

G

C

F

U

/

m

L

3 4

4 2 3 4


amended with 50 lg/ml of dimetoate, lindane and methidation. The values are a median of three replicates (p £ 0.05).


dimetoate, methylparathion, atrazine, simazine,captan or diflubenzuron. Consequently, thesestrains were selected for further biodegradationstudies and were identified at the genetic level.

The strategy used to sequence the 16 S rDNAgene of the 4 strains above mentioned produced acontinuous stretch of ca. 1.4 kb, representing>95% of the primary 16 S rDNA sequence. Per-centage similarity values obtained after pairwisealignment of the sequence of 16 S rDNA of strains8M, 2Sz, 9D and 14Atraz, versus EMBL databasesequences are shown in Table 3. The gene sequencecomparison demonstrated the affiliation of strain

14Atraz to Pseudomonas pseudoalcaligenes (98.12–98.37% identity), 9D strain to Bacillus sp. (99.03–99.6% identity), 8M to the Micrococcus luteusspecies (99.51–99.79% identity) and 2Sz strain toExiguobacterium aurantiacum (98.15% identity).

The capacity to remove or degrade selectedpesticides from a chemically defined media by thefour selected strains, was assayed in modifiedBanat media amended with 50 lg/ml of the pesti-cides used previously which allowed the highestgrowth rates for each particular strain. Accordingto this criteria, the hypothetical pesticide removalcapacity of Pseudomonas pseudoalcaligenes was

INCUBATION TIME (hours)

Strain 8MP

3

4

5

6

7

8

9

10

0 1 2 3 4

Control50 µg/ml methyl parathion

Strain 10MP

3

4

5

6

7

8

9

10

0 1 2 3 4

Control50 µg/ml methil parathion

Strain 2Sz

3

4

5

6

7

8

9

10

0 1 2 3 4 0 1 2

Control

50 µg/mlsimazine

Strain 8Sz

3

4

5

6

7

8

9

10

Control50 µg/mlsimazine

Strain 9Sz

3

4

5

6

7

8

9

10

0 1 2 3 4

Control

50 µg/mlsimazinaa

L

O

G

C

F

U

/

m

L

3 4


amended with 50 lg/ml of methylparathion and simazine. The values are a median of three replicates (p £ 0.05).

306 Lopez et al.

Table 2. Growth screening (as growth abundance criteria) of the 22 strains isolated from water samples amended with the pesti-

cides assayed (50 l g ml): aldrin (1), atrazine (2), captan (3), diflubenzuron (4), dimetoate (5), lindane (6) methidation (7), methyl-

parathion (8), and simazine (9)

Strain 1 2 3 4 5 6 7 8 9

12 Al + + + + + + + + +

13 Al + + + + + + + + +

14 Al ++ + + + + ++ + + +

14 Atrz ++ +++++ + ++++ ++ +++ +++ +

12 Cap + ++ + + + + + + ++

5 Df + + + + + + + + +

6 Df + + + ++ + + + + +

7 Df ++ + + ++ + ++ + + +

8 Df ++ ++ ++ ++ ++ ++ ++ ++ ++

4 D + ++ + ++ ++ + ++ ++ ++

9 D +++ ++++ +++ ++++ +++++ +++++ +++++ +++++ +++++

18 D + + + ++ ++ + + + +

19 D ++ + ++ ++ + ++ + + +

3 Li ++ ++ ++ ++ ++ ++ ++ ++ ++

8 M ++ ++ ++ ++ ++++ ++ ++ ++++ ++

10M ++ ++ ++ ++ ++ ++ + + +

12M ++ ++ ++ ++ ++ ++ ++ ++ ++

8 MP + ++ ++ ++ + + + ++ +

10MP + ++ + ++ ++ ++ + ++ +

2 Sz +++ ++ ++ +++ +++ ++ +++ +++ +++++

8 Sz ++ ++ + ++ ++ ++ + + ++

9 Sz + + + + + + + + +

+,++,+++,++++. Growth abundance.

Table 3. Identity of strains 8M, 2Sz, 9D and 14 Atrz 16S rDNA sequence with sequences in the EMBL database (FASTA search

output)

Strain EMBL sequence of 16S rDNA from strain % identity Nucleotides overlap

8M AJ276811 Micrococcus sp. 99.44% 1434

AJ409096 Micrococcus luteus 99.58% 1420

AF057289 Micrococcus luteus 99.51% 1429

AJ313024 Micrococcus sp. 99.23% 1420

AJ409095 Micrococcus luteus 99.79% 1418

2Sz X70316 Exiguobacterium aurantiacum 98.15% 1414

AJ344151 Exiguobacterium undae 94.08% 1423

D55730 Exiguobacterium acetylicum 94.07% 1392

AJ297437 Exiguobacterium acetylicum 94.00% 1370

X86064 Exiguobacterium sp. 94.74% 1356

9D AJ315062 Bacillus sp. 99.66% 1476

AJ315059 Bacillus sp. 99.59% 1476

AJ315057 Bacillus sp. 99.53% 1476

AF441729 Bacillus sp. 99.45% 1486

AY030319 Bacillus macroides 99.03% 1449

14Atr Z76666 Pseudomonas pseudoalcaligenes 98.36% 1461

AB030583 Pseudomonas alcaliphila 98.21% 1458

AB021379 Pseudomonas pseudoalcaligenes 98.35% 1461

AF238494 Pseudomonas pseudoalcaligenes 98.12% 1441

AF181570 Pseudomonas pseudoalcaligenes 98.27% 1445

.


assayed in modified Banat media amended withatrazine (Fig. 5). Bacillus sp. removal capacity wasassayed with lindane, simazine and dimetoate(Fig. 6); the capacity of Micrococcus luteus wastested in media amended with methylparathion(Fig. 7) and finally, the biodegradation of theherbicide simazine was assayed with Exiguobacte-rium aurantiacum (Fig. 8). Parallel to the evolutionof the concentration of the pesticide available inthe culture medium measured by GC–MS, growthcurves (as log CFU/ml) for each of the microor-ganisms were constructed. Bacillus sp. andE. aurantiacum were the isolated bacteria withhigher capacity for pesticide removal.

Discussion

There have been constant pressures on authoritiescontrolling the registration and approval of agri-cultural chemicals, to include measurements ofside effects on soil and water microbiota in theirrequirements. However, in order to encourageuniformity of data and aid interpretation andcomparison the recommended methods use thenormal conditions of laboratory experiments inenvironmental microbiology.

The addition of pesticides in water samplesfrom an oligotrophic lake studied here, increasedthe number of total bacteria. It has been reportedthat pesticides are metabolized by a variety of soiland water bacteria, indicating that these com-pounds could be utilized as carbon sources (Ellisand Camper, 1982; Spain et al., 1984; Cook, 1987;Howard, 1993; Mougin et al., 1994; Johnsen et al.,2001; Lopez et al., 2002).

Microbial transformation is recognized as acritical factor affecting the fate and behavior ofpesticides in aquatic ecosystems. Aquatic micro-biota comprises a diversity of species and is adynamic community able to degrade a wide varietyof chemically complex compounds (Salmeronet al., 1991). In the absence of data on the min-eralization or biotransformation of pesticidesassayed in water samples, the proliferation ofbacterial microbiota in treated waters may beassociated with the transformation of thesechemicals by the water microbiota.

The major group of bacteria responsible for thepesticide removal isolated from water samplesobtained from an oligotrophic lake, have beenidentified as Gram negative rods classified asmembers of the genus Pseudomonas (41%) andAeromonas (31%). These genus have been reported

Figure 5. Pesticide removal and growth of Pseudomonas pseudoalcaligenes in medium amended with atrazine. Concentration of pes-

ticide in media without bacterial strain: ; Concentration of pesticide in media inoculated with bacterial strain: ; Growth of

bacteria in media without pesticide: (–m–). Growth of bacteria in media amended with pesticide: (–n–). Values are a median of

three replicates (p £ 0.05).

308 Lopez et al.

Figure 6. Pesticide removal and growth of Bacillus sp. in media amended with lindane, simazine and dimetoate. Concentration of

pesticides in media without bacterial strain: ; Concentration of pesticides in media inoculated with bacterial strain: ;

Growth of bacteria in media without pesticide: (–m–). Growth of bacteria in media amended with pesticide: (–n–). Values are a

median of three replicates (p £ 0.05).


too by Edward et al. (2001) as typical Gramnegative bacteria in a eutrophic lake in the Northof England. According to our experiments, fourpure isolates classified as Exiguobacterium auran-tiacum, Pseudomonas pseudoalcaligenes, Micrococ-cus luteus and Bacillus sp. showed a high ability forinitial pesticide removal, and with the onlyexception of Exiguobacterium aurantiacum, which

is a microorganism reported as a facultative alk-alophile with Na+ requirements for its growth(Gee et al., 1980), the other bacterial strains havebeen previously reported as microorganisms com-monly showing xenobiotic biodegradation capaci-ties (Saagua et al., 1998; Doddamani andNinnekar, 2001; Kazt et al., 2001; Abou-Arab,2002). In particular, several reports on

Figure 7. Pesticide removal and growth of Micrococcus luteus in medium amended with methylparation. Concentration of pesticide

in media without bacterial strain: ; Concentration of pesticide in media inoculated with bacterial strain: ; Growth of bacte-

ria in media without pesticide: (–m–). Growth of bacteria in media amended with pesticide: (–n–). Values are a median of three

replicates (p £ 0.05).

Figure 8. Pesticide removal and growth of Exiguobacterium aurantiacum in medium amended with simazine. Concentration of pes-

ticide in media without bacterial strain: ; Concentration of pesticide in media inoculated with bacterial strain: ; Growth of

bacteria in media without pesticide: (–m–). Growth of bacteria in media amended with pesticide: (–n–). Values are a median of

three replicates (p £ 0.05).

310 Lopez et al.

Pseudomonas sp. strains able to degrade or com-pletely mineralize atrazine to CO2 and NH3 exist(Clausen et al., 2002).

Although no radiolabeled studies with pesti-cides have been performed here in order to dem-onstrate evolution of 14CO2 as evidence ofmineralization, our results suggest that the strainsmentioned above possess optimal growth capaci-ties for removing selected pesticides from thegrowth medium under the described experimentalconditions.

Bioremediation is a choice treatment for con-taminated waters, which may be accomplishedin situ or via bioreactor (Martirani et al., 1996). Inthe design of a bioremediation system, the choicemust be made between the use of indigenousmicroorganisms or incubation with strains adaptedto degrade specific compounds (Pollard et al.,1994). Our results showed that selected strains ableto grow and with different pesticide removalcapacities can be isolated from an oligotrophic lakeby selective enrichment. Short selection periodswere required and several types of aerobic bacteriahave been isolated. In this context, the use ofnoncomplex experimental conditions (enrichmenttechniques and selective agar media) allowed us todevelop a rapid method for selection and isolationof indigenous bacteria capable of pesticide utiliza-tion. The short selection period suggested thatpesticide-degrading bacteria were present in theaquatic environment at the time of sampling.

Agriculture activities and pesticides manufac-turing plants are two of the main pesticides sourcesthat produce the pollution of different environ-ments including surface waters. However, previousdata showed that in the oligotrophic lake used inour studies, no pesticide were detected (Benaventeet al., 1998), suggesting that the isolation ofindigenous microbiota that can tolerate high con-centrations of several pesticides was not correlatedwith chronic pesticide contamination derived fromhuman activities.

Acknowledgements

This work was supported in part by a fellowshipto B. Rodelas from the Spanish Ministerio deCiencia y Tecnologıa, Programa Ramon y Cajal

References

Abou-Arab, A.A.K. (2002). Degradation of organochloride

pesticides by meat starter in liquid media and fermented

sausage. Food Chem. Toxicol. 40, 33–41.

Aislabe, J. and Lloyd-Jones, G. (1995). A review of bacterial

degradation of pesticides. Aust. J. Soil Res. 33, 925–42.

Banat, I.M. (1993). The isolation of a thermopholic biosurf-

actant producing Bacillus sp. Biotechnol. Lett. 15, 591–4.

Bekhi, R.M., Topp, E., Warren, D. and Germon, P. (1993).

Metabolism of the herbicide atrazine by Rhodococcus strain.

Appl. Environ. Microb. 59, 1955–9.

Benavente, J., Rodriguez, M. and Cruz-San Julian, J. (1998)

Caracterization hidroquımica y cartografica de las zonas

humedas de Cadiz, Malaga y Almerıa. Informe Servicio de

Evaluacion de recursos Naturales. Conserjerıa de Medio

Ambiente. Junta de Andalucıa. Sevilla. Spain.

Sneath P.H.A., Mair N.S., Sharpe M.E. and Holt J.G. (eds,).

(1986). Bergey’s Manual of Determinative Bacteriology.

Williams and Wilkins, Baltimore, USA.

Clausen, G.B., Larsen, L., Johnsen, K., de Lipthay, J.R. and

Aamand, J. (2002). Quantification of the atrazine-degrading

Pseudomonas sp. strain ADP in aquifer sediment by quan-

titative competitive polymerase chain reaction. FEMS

Microbiol. Ecol. 41, 221–9.

Cook, A.M. (1987). Biodegradation of s-atrazine xenobiotics.

FEMS Microbiol. Rev. 46, 93–116.

Doddamani, H.P. and Ninekar, H.Z. (2001). Biodegradation of

carbaryl by aMicrococcus species.Curr.Microbiol. 43, 69–73.

Dua, M., Singh, A., Sethunathan, N. and Johri, A.K. (2002).

Biotechnology and biorremediation: successes and limita-

tions. Appl. Microbiol. Biot. 59, 143–152.

Edwards, M.L., Lilley, A.K., Timms-Wilson, T.H., Thompson,

I.P. and Cooper, I. (2001). Characterization of the cultur-

able heterotrophic bacterial community in a small eutrophic

lake (Priest Pot). FEMS Microbiol. Ecol. 35, 295–304.

Ellis, P.A. and Camper, N.D. (1982). Aerobic degradation of

diuron by aquatic microorganisms. J. Environ. Sci. Heal. 17,

277–89.

Ellis, B.M.L. (2000). Environmental Biotechnology Infor-

matics. Curr. Opin. Biotechnol. 11, 232–35.

Gee, J.M., Lund, B.M., Metcalf, G. and Peel, J.L. (1980).

Properties of a new group of alkalophilic bacteria. J. Gen.

Micro. 117, 9–17.

Glazer, A.N. and Nikaido, H. (1995). Microbial biotechnology:

fundamentals of applied microbiology. Freeman, New York.

Howard, P.H. (1993). Progress report: determining ‘‘real

world’’ biodegradation rates. Environ. Toxicol. Chem. 12,

1135–7.

Janssen, D.B., Oppentocht, J.E. and Poelarends, G. (2001).

Microbial dehalogenation. Curr. Opin. Biotech. 12, 254–8.

Johnsen, K., Jacbsen, C.S., Torrsvik, V. and Sørensen, J.

(2001). Pesticide effects on bacterial diversity in agricultural

soils-a review. Biol. Fertil. Soils 33, 443–53.

Kazt, I., Dosoretz, C.G., Mandelbaum, R.T. and Green, M.

(2001). Atrazine degradation under denitrifying conditions

in continuous culture of Pseudomonas ADP. Water Res. 13,

3272–5.

Lopez, L., Pozo, C., Gomez, M.A., Calvo, C. and Gonzalez-

Lopez, J. (2002). Studies on the effects of the insecticide


aldrin on aquatic microbial populations. Int. Biodeter.

Biodegr. 50, 83–87.

Martirani, L., Guardina, P., Marzullo, L. and Sannia, G.

(1996). Reduction of phenol content and toxicity in olive oil

mill waste waters with the ligninolytic fungus Pleorotus

ostreatus. Water Res. 30, 1914–18.

Mougin, C., Laugero, C., Asther, M., Dubroca, J., Frasse, P.

and Asther, M. (1994). Biotransformation of the herbicide

atrazine by the white root fungus Phanerochaete chrysos-

porium. Appl. Environ. Microb. 60, 705–8.

Pearson, W.R. and Lipman, D.J. (1988). Improved tools for

biological sequence comparison. Proc. Natl. Acad. Sci. USA

85, 2444–8.

Pollard, S.J.T., Hrudey, S.E. and Fedorak, P.M. (1994). Bior-

remediation of petroleum and creosote contamined soils: a

review of constrains. Waste Manage. Res. 12, 173–94.

Readman, J.W., Albanis, T.A., Barcelo, D., Galassi, S., Tron-

czynski, J. and Grabrielidesi, G.P. (1997). Fungicide con-

tamination of Mediterranean estuarine waters: results from

a MED POL pilot survey. Mar. Pollut. Bull. 34, 259–63.

Saagua, M.C., Vieira, G., Paveia, H. and Anselmo, A. (1998).

Isolation and preliminary characterization of Bacilllus sp.

MCS, a Gram positive 4-chlorobiphenyl degrading bacte-

rium. Int. Biodeter. Biodegr. 42, 39–43.

Salmeron, V., Martınez-Toledo, M.V. and Gonzalez-Lopez, J.

(1991). Effects of alachlor and metolachlor on the biological

activity of Azospirillum brasilense grown in chemically

defined and dialyzed-soil media. Environ. Toxicol. Chem. 10,

493–9.

Spain, J.C., Van Veld, P.A., Monti, C.A., Pritchard, P.H. and

Cripe, C.R. (1984). Comparison of p-nitrophenol biodeg-

radation in field and laboratory test systems. Appl. Environ.

Microbiol. 48, 944–50.

Thurman, E.M., Goolsby, D.A., Meyer, M.T., Mills, M.S. and

Pomes, M.L. (1992). A reconnaissance study of herbicides

and their metabolites in surface water in the mid-western

United States using immunoassay and gas chromatography/

mass spectrometry. Environ. Sci. Technol. 26, 2440–7.

Vinuesa, P., Rademaker, J.L.W., de Bruijn, F.J. and Werner,

D. (1998). Genotypic characterization of Bradyrhizobium

strains nodulating endemic woody legumes of the Canary

Islands by PCR-restriction fragment length polymorphism

analysis of genes encoding 16 S rRNA (16S rDNA) and

16 S-23S rDNA intergenic spacers, repetitive extragenic

palindromic PCR genomic fingerprinting, and partial 16 S

rDNA sequencing. Appl. Environ. Microbiol. 64, 2096–

104.

Weisburg, W.G., Barns, S.M., Pelletier, D.A. and Lane, D.J.

(1991). 16 S ribosomal DNA amplification for phylogenetic

study. J. Bacteriol. 173, 697–703.

312 Lopez et al.

identiﬁcation of bacteria isolated from an oligotrophic...

Documents