Isolation, characterization, identification and potentiality of fungicide thiram (TMTD) degraders under laboratory conditions.
Pedro and Walter (2006) extensively discussed the fundamental process of bioremediation and attenuation. The authors presumed a definition of bioremediation as the enhanced decontamination of polluted environments via a biological activity distinguishing it from biodegradation which they defined as an alteration in the chemical composition of a molecule mediated by a biological process. They further suggested that microorganisms are able to utilize many organic pollutants as sources of energy and carbon and use some of the highly chlorinated pollutants as electron acceptors under anoxic conditions. The rate and extent of biodegradation can be highly variable, depending on the microbial community structure and the prevailing environmental conditions. Sometimes these natural degradation processes proceed in contaminated environments without the need for human intervention, whereas in others some degree of intervention is necessary to stimulate biodegradation (Kruglov, 1991).
Thiram belongs to the ethylene bisdithiocarbamate (EBDC) chemical class. The EBDCs are fungicides used to prevent crop damage in the field and to protect harvested crops from deterioration in storage or transport (National Institute of Safety and Health (NIOSH), 1986). Thiram is used as seeds, fruit, vegetable, ornamental and turf crops protectant from a variety of fungal diseases. It is also used as an animal repellent to protect fruit trees and ornamentals from damage by rabbits, rodents and deer. Shirkot (1983) reported that Pseudomonas aeruginosa utilizes TMTD as a major source of carbon and energy. Other studies also reported the same phenomenon in field and laboratory experiments (Shirkot and Gupta, 1985 and Shirkot et al., 1990) but the isolation and characterization of other TMTD-degrading microbial species has not been pursued. Sahin and Tamer (2000) isolated a number of species of bacteria and fungi from enrichment cultures containing the fungicide Thiram. Fungal isolates were identified as Aspergillus niger, A. flavus and Penicellium steckii while the bacterial isolates were assigned to the genera: Bacillus, Arthrobacter,Moraxella and Streptomyces.
Materials and Methods
Thiram Utilizers: Thiram--degrading microorganisms were isolated by the enrichment and adaptation techniques described by Klages and Lingens (1980). Soil samples were obtained from agricultural soils (El Gadarif) that had been exposed to thiram for a duration of more than five years (85.68 g commercial formulation [ha.sup.-1]), and were stored. When required, soil was held at ambient temperature for a week and then sieved via < 2 mm mesh, oven-dried and it's pH and organic matter were determined. Ten grams of the soil were added to flasks containing 150 ml of the mineral salt medium (MSM) (Edwards et al., 1992). MSM medium contained the following ingredients in one liter of distilled water: [K.sub.2]HP[O.sub.4], 0.8 g; K[H.sub.2]P[O.sub.4], 0.2 g; MgS[O.sub.4]. 7[H.sub.2]O, 0.2 g; CaS[O.sub.4], 0.1 g [(N[H.sub.4]).sub.6]M[O.sub.24].4[H.sub.2]O, 0.001 g; (N[H.sub.4])S[O.sub.4], 5 g; yeast extract, 0.1 g and 1 ml of a stock solution of trace elements. The trace elements solution was prepared in 100 ml distilled water by dissolving ZnS[O.sub.4]. 7[H.sub.2]O, 10 mg; Mn[Cl.sub.2]. 4[H.sub.2]O, 30 mg; [H.sub.3]B[O.sub.3], 30 mg; CaCb. 6[H.sub.2]O, 20 mg; CuCb. 2[H.sub.2]O, 10 mg; Ni[Cl.sub.2]. 6[H.sub.2]O, 20 mg; NaMo[O.sub.4]. 2[H.sub.2]O, 30 mg. MSM was supplemented with 200 mg thiram [I.sup.-1] as the only source of carbon. Flasks were then incubated on a rotary shaker (50 rpm) at 27[degrees]C and pH: [+ or -]7.2. After 7 days, 1 ml soil suspension was transferred from each flask to flasks in the same medium. After another 7 days, 1 ml of soil suspension was spread into MSM Agar plate in three replicates. Plates were incubated at 27[degrees]C and examined daily for the presence of growth. Colonies present were subcultured for purification, preserved on nutrient agar slants and considered as potential thiram degrading/utilizing isolates.
Potentiality of Individual Isolates in Degrading Thiram: The ability of each of the five pure isolates to degrade thiram was tested in MSM broth medium supplemented with 200 mg thiram per litre as the only source of carbon and nitrogen. One hundred and fifty millilitres of MSM broth medium was transferred into 500 ml Erlenmeyer flasks and sterilized in the autoclave at 121[degrees]C under a pressure of 15 Ib/[in.sup.2] for 15 minutes. Each flask was then inoculated with one of the isolates in three replicates and incubated in the dark at 27[degrees]C onto a rotary shaker operating at 50 rpm for 4 days. An un inoculated control set was also prepared in a similar manner. Residues of thiram were determined after 20, 60 and 90 hours and recorded.
Determination of Thiram Residues in MSM Broth: One ml of broth culture of each isolate and of the control were transferred each to a 250 ml conical flask in three replicates. Fifty ml of Acetonitrile (HPLC grade) were added to each flask, the contents were vigorously shaken for 15 minutes and 20 [micro]l were transferred and injected into the chromatograph (Waters, USA) with UV detector, column: Lichromosorb [RPC.sub.18] (25 cm x 3.9-mm-ID stainless steel), column temperature 25[degrees]C, mobile phase: 60% acetonitrile: 40% water, injection volume: 20 [micro]l, Mobile phase flow rate 1 ml/min, detection: UV at 254 nm (1-cm cell), retention time: 3.8 min, Calibration: standard solutions of Thiram in acetonitrile. Residues of thiram were determined after 20, 60 and 90 hours of incubation.
Identification of Thiram Utilizers
Preliminary taxonomic classification of the thiram degraders/utilizers was performed using classical, cultural, biochemical and microscopical characteristics.
Results and Discussion
Potentiality of Different Pure Cultures for Degrading Thiram: Different species of microorganisms were isolated from soils using method of enrichment and adaptation techniques. The potentiality of these isolates to utilize TMTD as a sole source of energy and carbon was examined in mineral salt liquid media (MSM). The results are shown in Fig 1, where it is clear that, after 20 hours of incubation isolate 1 and isolate 2 have displayed 90 and 94 degradation percentage respectively. It is also clear that other tested isolates compared to the control, did not show any ability for degrading the fungicide. An explanation for the loss of TMTD-utilizing ability of pure cultures of isolates 3, 4 and 5 may be that the genes for degrading TMTD may be located on plasmids which were lost during prolonged storage and sub-culturing. This phenomenon was also noticed by workers handling EPTC ((S--ethyl N, N dipropylthiocarbamate) --degrading microorganisms (Lee, 1984). In addition MSM is a homogenous liquid while soil is a heterogeneous system that comprises a gaseous portion (soil air), liquid solution (soil water or solution) and a solid portion Kruglov, 1991). Persistence of pesticide degrading mutants requires the presence of the pesticide at higher concentrations. Disappearance of pesticide degrading abilitiles by mutants indicates that such characters are controlled by plasmid rather than by chromosomal genes. A more plausible explanation is that a group of microorganisms may work in concert to degrade a certain metabolite in such away that an intermediate or an end product in the pathway of one microbe may be used as a substrate for the other.
Sahin and Tamer (2000) found that Streptomyces sp. strain W 1 and Bacillus sp. strain SA 2 degraded thiram by 43.7% and 25.0% respectively after 80 hours storage. Elsaid (2010) isolated mixed cultures of organic and inorganic nitrogen users, actinomycetes and microrganisms capable of growing on poor medium, he found that all microorganisms used caused relatively similar effects on a-endosulfan isomers. Moreover, he found that bacteria which live in poor media are more efficient in degrading a-endosulfan than others, this group degraded endosulfan by 97.2-99.7% within 45 days of incubation.
[FIGURE 1 OMITTED]
Identification of Thiram-Utilizers: Five bacterial isolates which proved to have a potential for TMTD degradation were characterized by various cultural, microscopical and biochemical traits. Tentative identification, to the genus level, based on these characteristics was performed according to Bergey's Manual of Determinative Bacteriology (Bergey and Holt, 1994). Isolates1, 2 and 5 were isolated from El Gadarif soils that had been exposed to TMTD for a duration of more than 10 years while isolates3 and 4 were isolated from El Geraif soils, using enrichment and adaptation techniques. All isolates were capable of growing at the expense of TMTD (0.2 mg/ml). All isolates were catalase, oxidase, urease, indole, Methyl red--voges proskauer and Citrate utilization positive. The isolates designated as Iso2 and Iso3 were Gram--positive, aerobic, endospore forming rods. According to their properties the organisms were assigned to the genus Bacillus. Three other isolates (isolate 1, 4 and 5) showed Gram--negative, non endospore forming rods, produced a yellowgreen pigment (pyocyanin) and fluoresce under UV light and they were assigned to the genus Pseudomonas (Table 1, 2).
Sahin and Tamer (2000) obtained a number of species of bacteria from enrichment clutures containing the fungicide thiram. Bacterial isolates were assigned to the genera: Bacillus, Arthrobacter, Moraxella and Streptomyces. Odeyemi et al. (1998) in a study of pesticide contamination of Nigerian soils reported that at more than 31 figjg soil, thiram was extremely toxic to microorganisms. Sup and Gupta (1972) found that Rhizobium isolates from urid (Phaseolus radiatus) were resistant to TMTD at pH 7 but proved sensitive to its degradation product. They also reported that Azotobacter chroococcum isolates proved to be more sensitive as compared to Rhizobium spp. Pseudomonas aeruginosa which utilizes TMTD as a major source of carbon and energy was isolated from soil (Shirkot, 1983). Shirkot and Gupta (1985) reported that in a non-autoclaved alluvial sandy loam (pH 7.3) fortified and inoculated with the bacterium Pseudomonas aeruginosa displayed 40 and 86% degradation after 4 and 24 days incubation, respectively.
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A.A. Elhussein (1), A.G. Osman (2) and A.M. Sherif (2)
(1) Botany Department, Faculty of Science, University of Khartoum, Khartoum, Sudan
(2) Biofertilization Department, Environment and Natural Resource Research Institute, National Center for Research
Table 1: Cultural Characteristics of Bacterial Isolates. Isolate Colony Colony Colony Elevation Edge shape size surface 1 Circular Small Smooth Umbonate Entire 2 Circular Small Smooth Umbonate Entire 3 Circular Medium Smooth Drop-like Entire 4 Circular Medium Smooth Drop-like Entire 5 Circular Medium Rough Umbonate Entire Isolate Chromogensis Opacity Texture 1 Light yellow Translucent Gummy 2 Cream Transparent Gummy 3 Light yellow Translucent Gummy 4 Cream Opaque Gummy 5 Cream Translucent Gummy Table 2: Biochemical Characteristics and Tentative Identification of Bacterial Isolates. Isolates/Tests Iso 1 Iso 2 Iso 3 Gram stain - ve + ve + ve Shape Short Rod Short Short Rod Rod Endospore - + + Motility + + + Oxidase + + + Catalase + + + Urease + + + Indole - - - MR--VP - - - Citrate + + + Utilization TSI Butt A K K Slant K K K [H.sub.2]S - - - Gas + - - Gelatin Liquification + + + Starch Hydrolysis - - - ONPG - - - Lysine decarboxylase + + + Ornithine decarboxylase + + + Tentative identification Pseudomonas Bacillus Bacillus aeruginosa cereus cereus (St1) (St2) (St3) Isolates/Tests Iso 4 Iso 5 Gram stain - ve - ve Shape Short Rod Short Rod Endospore - - Motility + + Oxidase + + Catalase + + Urease + + Indole - + MR--VP - - Citrate + + Utilization TSI Butt A K Slant K K [H.sub.2]S - + Gas + - Gelatin Liquification + - Starch Hydrolysis - - ONPG + - Lysine decarboxylase + + Ornithine decarboxylase + + Tentative identification Pseudomonas Pseudomonas aeruginosa mendocina (St4) (St5) ONPG = Hydrolysis of ONPG by beta galactosidase TSI = Triple Sugar Iron Agar A = Acid K = Alkaline