Isolation Of Soil Bacteria Research Articles

Degradation of MCPA, 2,4-D, and Other Phenoxyalkanoic Acid Herbicides Using an Isolated Soil Bacterium

A soil bacterium was isolated capable of metabolizing the five phenoxyalkanoic acid herbicides 2,4-D, 2,4-DB, MCPA, MCPB, and 2,4,5-T in mineral solution with the individual herbicides as sole carbon source. The optimum temperature for breakdown was in the range 20-30 o C. In a 500-L aerated tank, the organism metabolized kilogram quantities of both 2,4-D and MCPA dimethylamine formulations to inorganic chloride. This bacterium retained activity for at least 18 months after storage in a frozen state at -10 o C. When applied to field plots, the organism is able to survive in the soil for at least 615 days and degrade both 2,4-D and MCPA significantly more rapidly than in soil from an untreated control plot

Degradation of selected insecticides by bacteria isolated from soil.

V. Rangaswamy and K. Venkateswarlu Department of Microbiology, Sri Krishnadevaraya University, Anantapur 515 003, India In semi-arid tropics, monocropping of groundnut, involving high- yielding varieties, has led to the outbreak of serious insect pests causing major losses in crop yield (Reddy and Ghewande 1986). As a consequence, there has been the use, both extensively and intensively, of organophosphorus insecticides, particularly monocrotophos and quinalphos, for an effective control of the insects (Patel and Vora 1981). Also, there is a steady increase in the application of large quantities of synthetic pyrethroids, mainly cypermethrin and fenvalerate, in groundnut pest management (Das 1988). The entry of such widely used insecticides into soil might have far-reaching consequences because it would disturb the delicate equilibrium between a microorganism and its environment, both involved in an important biological process. It has now been well established that the major or fequently the only means of degradation for several pesticides in the environment is microbial. Also, the data available in literature indicate that soil bacteria may be more important in the degradation of certain pesticides (Tu and Miles 1976). No information is, however, available on degradation of monocrotophos, quinalphos, cypermethrin and fenvalerate by soil bacteria. The present study has, therefore, been aimed at assessing the capability of pure cultures of bacteria, isolated from insecticide-treated soil by enrichment culture technique, in degradation of insecticides commonly used in groundnut cultivation. MATERIALS AND METHODS To isolate soil bacteria, capable of degrading the selected insecticides, enrichment culture technique was followed. One millilitre aliquots of 500 ppm aqueous solutions prepared from commercial formulations of the four insecticides (Table I) were added separately to 10 g portions of a black soil, collected from a fallowlgroundnut field to provide a final concentration of 5 kg ha- . The insecticide-treated soil samples were maintained at 60% water-holding capacity and incubated at room temperature (28 • 4~ Ten days after four such additions, at Send reprint requests to Professor K. Venkateswarlu, at the above address.

Characterization of two DNA clones specific for identification of Corynebacterium sepedonicum

Escherichia coli TB1 was transformed with pUC9 containing fragmented DNA (4–10 kilobases (kb)) from Corynebacterium sepedonicum. The resulting genomic bank was screened by a dot blot assay to identify clones specifically hydridizing to C. sepedonicum DNA and not to the DNA of several other Gram-positive and Gram-negative bacteria. Two clones (III24 and III31) were selected because of their ability to strongly hybridize to C. sepedonicum DNA and weakly hybridize to the DNA of C. michiganense, Erwinia carotovora, Agrobacterium tumefaciens, Bacillus subtilis, Pseudomonas solanacearum, Micrococcus luteus, and Arthrobacter globiformis. These two clones were also specific for C. sepedonicum DNA when tested against the DNA from 30 isolates of soil bacteria. Restriction enzyme analysis has shown that the two clones have an insert of 8 kb (III24) and 4 kb (III31). On the basis of restriction enzyme patterns, one clone (III24) does not correspond to plasmid pCL 50, a cryptic plasmid found in several C. sepedonicum isolates. Because purified III24 and III31 DNA can be used to detect approximately 1 ng of C. sepedonicum genomic DNA, the two clones can complement serological or biological detection methods. This could be useful, especially when a high degree of specificity is required for detection or identification of this plant pathogen.

Degradation of Pyrethroid Insecticides in the Environment

The environmental fates of fenvalerate and fenpropathrin have been studied in comparison with other agricultural pyrethroids such as permethrin, cypermethrin and deltamethrin. Al-though photostability of these pyrethroids has much improved, they are still susceptible to photolysis in the environment. The major photochemical reactions are isomerization, ester bond cleavage and decarboxylation. The mechanism of decarboxylation reaction, which is unique to fenvalerate photolysis, has been clarified by elucidation of the chemical structures of free radical intermediates, using a spin trap method combined with HPLC, ESR and GC-MS analyses. The metabolic profiles in plants are similar to those in mammals. The trans isomers are more rapidly hydrolyzed than the cis isomers, and oxidation occurs to a greater extent with the more stable cis isomers. The resultant products are converted to conjugates by coupling with glucose, gentiobiose, sophorose, glucosylxylose, malonylglucose, and two types of tri-glucose. Hydrogen cyanide, which would be released via ester hydrolysis, is rapidly detoxified in plants by metabolic conversion to β-cyanoalanine, asparagine, aspartic acid, and γ-glutamyl-β-cyanoalanine. The ester hydrolysis is also a main degradation route in soils. A slight modification in chemical structures significantly alters the degradation rate. Either replacement of chlorine with bromine or introduction of nitrile group to benzyl group retards the degradation. Further, the traps and αS isomers are degraded more rapidly than the cis and αR antipodes, respectively. Most of the isolated soil bacteria possess different types of pyrethroid-hydrolyzing enzymes with high substrate specificity. The stereoselective degradation could be rationalized by involvement of these enzymes. These environmental fate studies are useful not only in evaluating the safety but to facilitate creation of the more bioacceptable, biorational pesticide.

Degradation of the insecticide azinphos-methyl in soil and by isolated soil bacteria

Degradation of the insecticide azinphos-methyl in soil and by isolated soil bacteria

Microbial Transformation of the Labdanes,cis-Abienol and Sclareol

The microbial transformation of cis-abienol and sclareol by a recently isolated soil bacterium, JTS–131, was studied. In the presence of metabolic inhibitors, three new compounds were isolated from the culture broth as degradation products of cis-abienol. They were determined to be (12Z)-labda-12,14-dien-18-ol, (12Z)-labda-12,14-dien-18-oic acid and its methylester. Two new compounds were also formed from sclareol in the presence of metabolic inhibitors. They were determined to be 13β-hydroxylabd-14-en-18-oic acid and 13β-hydroxylabd-14-en-18-oic acid methylester. As a result of sequence determination experiments, the biotransformation pathways of cis-abienol and sclareol by the bacterium were proposed.

Effect of glucose on the amount of bacteria mineralizing 2,4-dichlorophenoxyacetic acid in soil.

Plate numbers of bacteria and relative incidence of strains capable of mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) in chernozem samples incubated for 14d with the herbicide (50 ppm) in the presence or absence of glucose (1000 ppm) were compared. Whereas the total number of bacteria increased 1.2-fold in the variant with 2,4-D and 2.4-fold in the variant with glucose and the herbicide, the number of 2,4-D-mineralizing bacteria increased 12.1-fold and 34.2-fold, respectively. In a collection of 96 isolates of soil bacteria substantially more strains capable of degradation of 2,4-D in the presence of glucose were detected as compared with the variant without it, indicating that processes of cometabolic type are involved during the degradation of this herbicide in the soil.

Microbial transformation of the labdanes, cis-abienol and sclareol.

The microbial transformation of cis-abienol and sclareol by a recently isolated soil bacterium, JTS–131, was studied. In the presence of metabolic inhibitors, three new compounds were isolated from the culture broth as degradation products of cis-abienol. They were determined to be (12Z)-labda-12,14-dien-18-ol, (12Z)-labda-12,14-dien-18-oic acid and its methylester. Two new compounds were also formed from sclareol in the presence of metabolic inhibitors. They were determined to be 13β-hydroxylabd-14-en-18-oic acid and 13β-hydroxylabd-14-en-18-oic acid methylester. As a result of sequence determination experiments, the biotransformation pathways of cis-abienol and sclareol by the bacterium were proposed.

Dehalogenases in Soil Bacteria

Sixteen bacterial strains isolated from soil were able to grow on either 2-monochloropropionic acid or monochloroacetic acid as the sole carbon and energy source. The isolates were divided into five groups on the basis of differences in their dehalogenase activities towards four substrates - monochloroacetic acid, dichloroacetic acid, 2-monochloropropionic acid and 2,2-dichloropropionic acid. Disc gel electrophoresis of crude extracts identified four distinct dehalogenases with different electrophoretic mobilities: three isolates contained one, three or four dehalogenases, respectively, and the remaining 13 isolates contained different combinations of two dehalogenases. In some cases, dehalogenases with the same mobilities from different isolates appeared to be identical enzymes. In others, enzymes from different isolates with the same electrophoretic mobility had different substrate activity profiles. Pseudomonas putida PP3 was shown to contain one enzyme which was comparable with one of the dehalogenases detected in several of the newly isolated soil bacteria. The second enzyme was not found in soil bacteria and represented a fifth dehalogenase. The significance of these results in terms of the evolution of dehalogenase activity is discussed.

Multiple forms of chondroitinase AC from Arthrobacter aurescens.

While the isolated soil bacterium, Arthrobacter aurescens, had been found to secrete a chondroitinase AC into the culture medium, it was recognized that the chondroitinase preparation obtained from the broth of the strain cultured in a jar fermentor contained at least three electrophoretically separable components of the enzyme. Each component (named chondroitinase I, II, and III, respectively) was separated from the others by use of isoelectric focusing and then the enzymic properties of each component were examined. The value of isoelectric point of each component differed from one another (I, 5.5; II, 5.9; III, 6.4). However, no difference could be detected in pH-stability (pH 5.0 to 7.5), optimum pH (pH 6.0), thermal stability (below 45°C), optimum temperature (50°C), substrate specificity (chondroitin A and C lyase), mode of action (endo type, the degree of multiple attack was 3.0 to 3.1), and the dissociation constant of the enzyme-substrate complex (Km for chondroitin sulfate C was 3.3_??_3.6×10-4M). Thus the electrophoretically separable components with the chondroitinase activity were thought to be the multiple forms of the enzyme, chondroitinase AC. There was also no appreciable difference in the molecular weight values of those chondroitinase components, however, considerable difference was detected in the carbohydrate content of those components.

Isolation and Crystallization of Extracellular 3β-Hydroxysteroid Oxidase ofBrevibacterium sterolicumnov. sp.

Among about 500 strains tested, a newly isolated soil bacterium, Brevibacterium sterolicum nov. sp. KY 3463 (ATCC 21387) showed the highest potency in production of 3β-hydroxysteroid oxidase in the culture fluid. The 3β-hydroxysteroid oxidase was purified from the culture filtrate by a procedure involving ammonium sulfate fractionation, DEAE-cellulose and hydroxyapatite column chromatographies and Sephadex G–75 gel filtration. Crystals of the enzyme were obtained from solutions of the purified preparation by the addition of ammonium sulfate. The crystals appeared as fine rods, with a bright yellow color. The enzyme is homogeneous by disc gel electrophoresis and ultracentrifugation. Sedimentation velocity yields a value of . It exhibits a typical flavoprotein spectrum of absorption maxima at 280, 390, and 470 mμ.

Isolation and Crystallization of Extracellular 3β-Hydroxysteroid Oxidase of Brevibacterium sterolicum nov. sp.

Among about 500 strains tested, a newly isolated soil bacterium, Brevibacterium sterolicum nov. sp. KY 3463 (ATCC 21387) showed the highest potency in production of 3β-hydroxysteroid oxidase in the culture fluid. The 3β-hydroxysteroid oxidase was purified from the culture filtrate by a procedure involving ammonium sulfate fractionation, DEAE-cellulose and hydroxyapatite column chromatographies and Sephadex G–75 gel filtration. Crystals of the enzyme were obtained from solutions of the purified preparation by the addition of ammonium sulfate. The crystals appeared as fine rods, with a bright yellow color. The enzyme is homogeneous by disc gel electrophoresis and ultracentrifugation. Sedimentation velocity yields a value of . It exhibits a typical flavoprotein spectrum of absorption maxima at 280, 390, and 470 mμ.

Denitrification by Isolated Soil Bacteria Under Various Environmental Conditions

AbstractNitrate reducing microorganisms were isolated from soil and their denitrifying properties were compared under various environmental conditions. The isolated denitrifiers cultured in a liquid medium under anaerobic conditions responded differently to changing pH, temperature, nitrate or nitrite concentration. Unfavorable growth conditions led to the accumulation of nitrite. Various combinations of the selected bacteria revealed that interactions occur among the microbes during growth in relation to their denitrifying activity. Variation among bacterial species and specific environmental conditions resulted in different denitrifying characteristics; therefore, generalizations cannot be made regarding microbial denitrification in soil for possible applied uses.

Inositol Phosphate Phosphatases of Microbiological Origin. The Isolation of Soil Bacteria Having Inositol Phosphate Phosphatase Activity

The isolation of three soil bacteria exhibiting inositol phosphate phosphatase activity is described. One isolate, a member of the genus Pseudomonas, had pronounced phytase activity but the others had not. The latter were slightly active against pentaphosphates, however, and they easily dephosphorylated tetraphosphates and lower esters.

QUALITATIVE STUDIES OF SOIL MICROORGANISMS: XI. FURTHER OBSERVATIONS ON THE NUTRITIONAL CLASSIFICATION OF BACTERIA

A review of the method developed in this laboratory in 1943 for the nutritional classification of soil bacteria has suggested slight amendments in certain differential media: (1) the substitution of vitamin-free casamino acids for a combination of amino acids, and (2) the addition of vitamin B12 to the growth factor media. In a comparative study with a newly proposed scheme of classification, the more selective plating medium advocated was found to be less suitable for the isolation of soil bacteria than the nonselective soil extract agar in the original method. Furthermore, the replacement of potassium nitrate with diammonium phosphate as source of inorganic nitrogen in the basal medium failed to cause any significant change in the nutritional grouping. Results from the nutritional classification of some 600 isolates by the two methods showed that the new procedure represents only a slight modification of the original system.