Degradation of 2,4-D herbicide by microorganisms isolated from Brazilian contaminated soil

Degradação do herbicida 2,4-D por microrganismos isolados de solo contaminado do Brasil

ENVIRONMENTAL MICROBIOLOGY

SHORT COMMUNICATION

Degradation of 2,4-D herbicide by microorganisms isolated from Brazilian contaminated soil

Degradação do herbicida 2,4-D por microrganismos isolados de solo contaminado do Brasil

Tatiane M. Silva^I; Maria I. Stets^I; André M. Mazzetto^I; Fabiana D.Andrade^I; Sônia A. V. Pileggi^I; Paulo R. Fávero^II;Marcelo D. Cantú^III; Emanuel Carrilho^III; Paulo I.B. Carneiro^IV; Marcos Pileggi^I,*

^IDepartamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brasil

^IIDepartamento de Análises Clínicas e Toxicológicas, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brasil

^IIIInstituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, Brasil

^IVDepartamento de Química, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brasil

ABSTRACT

The aim of this work was to isolate microorganisms from Brazilian soil contaminated with 2,4-D herbicide, and analyze the efficiency for 2,4D degradation, using high-performance liquid chromatography (HPLC). Serratia marcescens and Penicillium sp had never been reported as able to degrade 2,4-D. The isolated strains represent a great potential for bioremediation.

Key-words: Bioremediation, Biodegradation, 2,4-dichlorophenoxyacetic acid.

RESUMO

O objetivo deste trabalho foi isolar microrganismos de solo brasileiro contaminado com o herbicida 2,4-D, e analisar a eficiência da degradação por cromatografia líquida de alta eficiência (HPLC). Serratia marcescens e Penicillium sp jamais haviam sido relatadas como degradadoras de 2,4-D. As linhagens isoladas representam um grande potencial em biorremediação.

Palavras-chave: Ácido 2,4-diclorofenoxilacético, Biorremediação, Biodegradação.

2,4-dichlorophenoxyacetic acid (2,4-D) is an organic acid with pKa of 2.6 and high water solubility (45 g L^-1). It presents a systemic mode of action and has been widely employed in wheat, rice, corn, sorghum and sugar cane cultures to control harmful wide-leaf weeds (21). Particularly in Brazil, this herbicide is extensively used in many crops (16). Because it is highly selective and systemic, this herbicide is transported through the plant, being accumulated in the growing roots, inhibiting the growth of weeds.

2,4-D is classified by both ANVISA (Brazilian National Agency for Sanitarian Vigilance) and WHO (World Health Organization) as a hormonal herbicide of level II toxicity. It is considered a carcinogen agent, affecting liver, heart and central nervous system, leading to convulsions (6,13). This herbicide is usually commercialized as salt, amine and ester formulations, and has post-emergence action. After its application in field, the excess of the herbicide is easily transferred to the groundwater, due to its high solubility in water (600 mg.L^-1 at 25ºC) (18). Even after a long period of disuse, considerable amounts of either 2,4-D or its main product of degradation, 2,4-dichlorophenol (2,4-DCF) (1), might be found in surface waters, and groundwater as well. Therefore, the development of an efficient degradation process for this herbicide is extremely relevant and necessary (10). The efficiency of the degradation methodology can be monitored and easily determined by using high-performance liquid chromatography (HPLC), since this technique allows the determination of the amount of herbicide and its metabolites in the samples (2).

2,4-D has frequently been used as a chemical model to investigate the evolution and diversity of catabolic genes involved in the degradation of anthropogenic contaminants in the environment. Although the chemical structure of 2,4-D is relatively complex, it is readily degraded and used as a carbon source by various environmental microorganisms (12).

Many investigations have suggested bioremediation as an alternative process for the removal of xenobiotic compounds from the soil, due to the smaller environmental impact, and greater efficiency (7,9,15,19,22).

The major aim of this work was to explore the metabolic versatility of microorganisms, and identify pure microbial cultures highly specialized in the degradation of the largely employed herbicide 2,4-D.

Soil samples were collected from wheat (Triticum sp) cultivation area (Capão da Onça Farm School Universidade Estadual de Ponta Grossa, Paraná, Brazil - 25º 16'S; 50º 16'W), where the herbicide had been regularly used for a period of three years. The most recent application has occurred four months before collecting. About 50 g of soil were collected at ten different places, approximately 10 cm depth. The soil samples were thoroughly mixed, and portions of 1 g were further isolated and homogenized using a glass rod. In order to select the herbicide tolerant microorganisms, a serial dilution assay was carried out in 0.9% NaCl solution, which revealed that 10^-3 was the best dilution for obtaining isolated colonies. They were plated in a rich medium, nutrient agar, and in a selective medium, nutrient agar plus 806 g/L of 2,4-D. The applied concentration of 2,4-D herbicide was equivalent to what is recommended for field experimentation. Inocula with soil suspension of 100 µL were used for each 1 g of sample.

In order to obtain mesophilic strains of different growing velocities, the cultures were incubated at 18 and 28ºC, in duplicates. After incubation, growing 2,4-D tolerant colonies were selected and stored in rich and selective medium, as well as in criogenic tubes, according to Castellani (3).

The quantity of 2,4-D herbicide in the supernatant of bacterial cultures was measured by HPLC, using a Shimadzu LC10 Ai chromatograph, equipped with an UV detector. The separation was carried out in a Merck C-18 column (150 mm × 2.1 mm × 5 µm), and the elution was performed with methanol / 20 mM ammonium acetate (pH 4.5) - (50:50 / v:v), under a flow rate of 0.7 mL min^-1. The selected wavelength for 2,4-D detection was 230 nm.

The extension of the 2,4-D degradation process for each microorganism was accessed by monitoring the decrease of the 2,4-D peak area after incubation. For each evaluated condition, a control sample was prepared exactly in the same conditions, but with no microorganism. 2,4-D peak area achieved for the control samples was considered as 100%, and the degradation values obtained from the treatment samples were calculated based on the control value.

Five strains and the control were incubated for two weeks at 28ºC, in 100 mL of culture medium containing 2,4-D (2.35 mg mL^-1). The tubes were centrifuged at 12,100xg for 5 min, and an aliquot of 500 µL of the supernatants was submitted to purifying procedures, using C18 cartridges (100 mg Amersham/Amprep). The cartridges were washed gently with 1 mL of methanol and 1 mL of 0.5% TFA (trifluoroacetic acid) solution. The cartridges were then washed with 2 mL of 0.5% TFA. Finally, the sample was eluted with 10 mL of methanol, and 10 µL were submitted to HPLC. After the complete procedure, the final concentration of 2,4-D was 100 mg mL^-1 for the control (Nutrient Broth plus 2,4-D).

The bacterial degrading strains were identified and characterized by Gram staining and biochemical assays. Lactose fermenter bacterial strains were selected in MacConkey agar and tested for decarboxylation of L-tryptophan, glucose fermentation, gas production from glucose, sulfidric gas production, urea hydrolysis, lysine and ornithine decarboxylation, motility, indol production, rhamnose fermentation, and citrate utilization, using Enterobacteria Kit (Laborclin, Pinhais, Brazil). The non-lactose fermenter isolates were tested for glucose fermentation/oxidation, growth in BHI broth and cetrimide, gelatinase production, motility, use of nitrate, xylose, maltose and lactose, using NF-PROV Kit (Newprov, Pinhais, Brazil). Capsule production was investigated by Gins Method (17). Fungal cultures were stained with lactophenol cotton blue and submitted to morphologic identification (11).

Among 25 isolated strains, five presented major degrading potential, and were identified as Acinetobacter sp, Serratia marcescens, Stenothrophomonas maltophilia,Flavobacterium sp and Penicillium sp. All these bacterial strains presented capsule, and were Gram negative. Capsule production could optimize the formation of a microbial consortium, where each strain would be responsible for the production of different enzymes which could increase herbicide degradation potential. The growth curve in the presence of 2,4-D demonstrated that the adaptation of the strains to the culture medium was very fast, with no lag phase. On the other hand, the curves also indicated fast growing, and therefore a good adaptability of these strains to media with herbicide, considering that log phase varied between 10 and 20 h. In order to determine the extension of the degradation by each isolated strain, samples and control solutions were analyzed by HPLC. The bacterial degradation of 2,4-D generated just one retention time peak in this approach. For this reason, the degradation efficiency was determined based on 2,4-D peak variation. Using 2,4-D peak area values obtained for samples and respective controls, the degradation values for each strain were: Acinetobacter sp 0.25%; Serratia marcescens 0.25%; Stenothrophomonas maltophilia 30.20%; Flavobacterium sp 10.10%; and Penicillium sp 29.80% (Fig. 1).

The strains isolated in this work showed to be potentially 2,4-D degrading agents. Acinetobacter sp, Stenothrophomonas maltophilia and Flavobacterium sp had been previously described as 2,4-D herbicide degrading microorganisms in other studies (4,5,8,14,20), revealing a great importance in further studies aiming the bioremediation processes. The results are also noteworthy, considering that Serratia marcescens and Penicillium sp were isolated from tropical soil, and had never been reported as able to degrade 2,4-D before, a herbicide extensively used in Brazil.

ACKNOWLEDGEMENTS

The authors wish to thank the National Research Council (CNPq), for a grant to Fabiana Diuk de Andrade; Jefferson Zagonel and Eloir Moresco, from the School-Farm "Capão da Onça", State University of Ponta Grossa, for technical information on herbicides; Daniel Kurt Lottis, for English revision of the manuscript; and Dr. João Lúcio de Azevedo, for valuable comments and suggestions.

Submitted: November 07, 2006; Returned to authors for corrections: January 04, 2007; Approved: July 27, 2007

• 1. Amarante Junior, O.P.; Brito, N.M.; Santos, T.C.R.; Nunes, G.S.; Ribeiro, M.L. (2003-a). Determination of 2,4-dichlorophenoxyacetic acid and its major transformation product in soil samples by liquid chromatographic analysis. Talanta, 60, 115-121.
• 2. Amarante Junior, O.P.; Santos, T.C.R.; Nunes, G.S. (2003-b). Concise revision of methods for determination of the herbicide dichlorophenoxyacetic acid (2,4-D). Quím. Nova, 26, 223-229.
• 3. Castellani, A. (1939). Viability of some pathogenic fungi in distilled water. J. Trop. Med. Hyg., 42, 225-226.
• 4. Chaudhry, G.R.; Huang, G.H. (1988). Isolation and characterization of a new plasmid from a Flavobacterium sp. which carries the genes for degradation of 2,4-dichlorophenoxyacetate. J. Bacteriol, 170, 3897-3902.
• 5. Don, R.H.; Pemberton, J.M. (1981). Properties of six pesticide degradation plasmids isolated from Alcaligenes paradoxus and Alcaligenes eutrophus.J. Bacteriol, 145, 681-686.
• 6. Garcia, G.B.; Konjuh, C.; Duffard, R.O.; Evangelista de Duffard A.M. (2006). Dopamine-beta-hydroxylase immunohistochemical study in the locus coeruleus of neonate rats exposed to 2,4-dichlorophenoxyacetic acid through mother's milk. Drug Chem. Toxicol., 29(4): 435-42.
• 7. Getenga, Z.M.; Madadi, V.; Wandiga, S.O. (2004). Studies on biodegradation of 2,4-D and metribuzin in soil under controlled conditions. Bull. Environ. Contam. Toxicol 72, 504-513.
• 8. Goris, J.; Dejonghe, W.; Falsen, E.; De Clerck, E.; Geeraerts, B.; Willems, A.; Top E.M.; Vandamme, P.; DE VOS, P. (2002). Diversity of transconjugants that acquired plasmid pJP4 or pEMT1 after inoculation of a donor strain in the A- and B-horizon of an agricultural soil and description of Burkholderia hospita sp. nov. and Burkholderia terricola sp. nov. Syst. Appl. Microbiol., 25, 340-352.
• 9. Grotzschel, S.; Koster, J.; De Beer, D. (2004). Degradation of 2,4-dichlorophenoxyacetic acid (2,4-d) by a hypersaline microbial mat and related functional changes in the mat community. Microb. Ecol, 48, 254-262.
• 10. Kundua, S.; Pala, A.; Dikshitb, A.K. (2005). UV induced degradation of herbicide 2,4-D: kinetics, mechanism and effect of various conditions on the degradation. Sep. Purif. Technol., 44, 121-129.
• 11. Larone, H.D. (1993). Medically Important Fungi, A guide to identification, 2^nd ed., Am. Soc. Microbiol., Washington, D.C.
• 12. Lee, T.H.; Kurata, S.; Nakatsu, C.H.; Kamagata, Y. (2005). Molecular Analysis of Bacterial Community Based on 16S rDNA and Functional Genes in Activated Sludge Enriched with 2,4-Dichlorophenoxyacetic Acid (2,4-D) under Different Cultural Conditions. Microb. Ecol, 49, 151-162.
• 13. Maloney, E.K.; Waxman, D.J. (1999). trans-Activation of PPARalpha and PPARgamma by structurally diverse environmental chemicals. Toxicol. Appl. Pharmacol., 161(2), 209-218.
• 14. Motosugi, K.; Soda, K. (1983). Microbial degradation of synthetic organochlorine compounds. Experientia, 39, 1214-1220.
• 15. Pandey, G.; Jain, R.K. (2002). Bacterial Chemotaxis toward Environmental Pollutants: Role in Bioremediation. Appl.Environ.Microbiol, 68, 5789-5795.
• 16. Prado, A.G.S.; Airoldi, C. (2001). Toxic effect caused on microflora of soil by pesticide picloram application. J. Environ. Monit, 3, 394-397.
• 17. Ribeiro, M.C.; Soares, M.M.S.R. (1993). Microbiologia prática roteiro e manual para bactérias e fungos, 1^st ed., Atheneu, São Paulo, SP.
• 18. Rodrigues, B.N.; Almeida, F.S. (2005). Guia de herbicidas. Grafmarke. 5. ed. Londrina, PR.
• 19. Shaw, L.J.; Burns, R.G. (2005). Rhizodeposition and the enhanced mineralization of 2,4-dichlorophenoxyacetic acid in soil from the Trifolium pratense rhizosphere. Environ. Microbiol., 7, 191-202.
• 20. Smejkal, C.W.; Seymour, F.A.; Burton, S.K.; Lappin-Scott, H.M. (2003). Characterization of bacterial cultures enriched on the chlorophenoxyalkanoic acid herbicides 4-(2,4-dichlorophenoxy) butyric acid and 4-(4-chloro-2-methylphenoxy) butyric acid. J. Ind. Microbiol. Biotechnol, 30, 561-567.
• 21. Thill, D. (2003). Growth regulator herbicides. In: Herbicide action course West Lafayette: Purdue University. p.267-291.
• 22. Torang, L.; Nyholm, N.; Albrechtsen, H.J. (2003). Shifts in biodegradation kinetics of the herbicides MCPP and 2,4-D at low concentrations in aerobic aquifer materials. Environ. Sci. Technol, 37, 3095-3103.

Publication Dates

• Publication in this collection
  17 Oct 2007
• Date of issue
  Sept 2007

History

• Accepted
  27 July 2007
• Received
  07 Nov 2006
• Reviewed
  04 Jan 2007