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Characterization of an atrazine-degrading Pseudaminobacter sp. isolated from Canadian and French agricultural soils.

Atrazine, a herbicide widely used in corn production, is a frequently detected groundwater contaminant. Fourteen bacterial strains able to use this herbicide as a sole source of nitrogen were isolated from soils obtained from two farms in Canada and two farms in France. These strains were indistinguishable from each other based on repetitive extragenic palindromic PCR genomic fingerprinting performed with primers ERIC1R, ERIC2, and BOXA1R. Based on 16S rRNA sequence analysis of one representative isolate, strain C147, the isolates belong to the genus Pseudaminobacter in the family Rhizobiaceae. Strain C147 did not form nodules on the legumes alfalfa (Medicago sativa L.), birdsfoot trefoil (Lotus corniculatus L.), red clover (Trifolium pratense L.), chickpea (Cicer arietinum L.), and soybean (Glycine max L.). A number of chloro-substituted s-triazine herbicides were degraded, but methylthio-substituted s-triazine herbicides were not degraded. Based on metabolite identification data, the fact that oxygen was not required, and hybridization of genomic DNA to the atzABC genes, atrazine degradation occurred via a series of hydrolytic reactions initiated by dechlorination and followed by dealkylation. Most strains could mineralize [ring-U-(14)C]atrazine, and those that could not mineralize atrazine lacked atzB or atzBC. The atzABC genes, which were plasmid borne in every atrazine-degrading isolate examined, were unstable and were not always clustered together on the same plasmid. Loss of atzB was accompanied by loss of a copy of IS1071. Our results indicate that an atrazine-degrading Pseudaminobacter sp. with remarkably little diversity is widely distributed in agricultural soils and that genes of the atrazine degradation pathway carried by independent isolates of this organism are not clustered, can be independently lost, and may be associated with a catabolic transposon. We propose that the widespread distribution of the atrazine-degrading Pseudaminobacter sp. in agricultural soils exposed to atrazine is due to the characteristic ability of this organism to utilize alkylamines, and therefore atrazine, as sole sources of carbon when the atzABC genes are acquired.

Agriculture↗

Formation of natural indigo derived from woad (Isatis tinctoria L.) in relation to product purity.

There is an increasing commercial demand for naturally sourced indigo that meets the purity standards set by the synthetic product. This study concerns the indigo made from leaves of woad (Isatis tinctoria L.), and in particular its interaction with particulate impurities arising from soil and plant materials. Also, a more reliable method using N-methyl-2-pyrrolidone has been developed for the spectrophotometric determination of indigo. In a novel application of fluorescence spectroscopy, indoxyl intermediates in indigo formation are shown to be stable for minutes. The main indigo precursor from woad can be adsorbed onto Amberlite XAD16 in conformity with a Langmuir isotherm, but indigo precursors break down on this and other resin beads to yield indigo and red compounds. Indigo made from indoxyl acetate aggregates into particles, the size distribution of which can be modified by the inclusion of a fine dispersion of calcium hydroxide. Bright field microscopy of indigo products made under defined conditions and scanning electron microscopy combined with energy-dispersive X-ray analysis reveal the relationship of indigo with particulate materials. A model illustrating the interaction of indigo with particulate contaminants is developed on the basis of the results obtained, and recommendations are made for improving the purity of natural indigo.

Indigo Carmine↗

Cytotoxic and genotoxic activity of 1,3-dichloropropene in cultured mammalian cells.

1,3-Dichloropropene (DCP), a widely used soil fumigant previously found to be carcinogenic in both mice and rats, was evaluated for its cytotoxic and genotoxic effects in cultured rodent and human cells. A reduction of cell viability that was dependent on the dose and the length of treatment was observed with the trypan blue and the neutral red assay in both V79 cells and rat hepatocytes exposed to DCP concentrations ranging from 0.18 to 5.6 mM. In the absence of a metabolic activation system, a dose-dependent frequency of DNA single-strand breaks, that were only partially repaired within 24 hr, was revealed by the alkaline elution technique in V79 cells exposed to subtoxic DCP concentrations. The genotoxicity of DCP was confirmed by the results obtained in metabolically competent primary cultures of both rat and human hepatocytes which displayed similar dose-related amounts of DNA fragmentation and DNA repair synthesis, and showed, in comparison to metabolically deficient V79 cells, a somewhat greater sensitivity to the cytotoxic and DNA-damaging effects of DCP. The increase in the frequency of DNA breaks observed in rat hepatocytes after GSH depletion confirms the role of this tripeptide in DCP detoxification; its reduction in hepatocytes simultaneously exposed to metyrapone is consistent with a cytochrome P450-dependent biotransformation of DCP to more toxic metabolites.

Allyl Compounds↗

Fluorescence spectroscopic studies of natural organic matter fractions.

Because of the well-known molecular complexity and heterogeneity of natural organic matter (NOM), an aquatic bulk NOM was fractionated into well-defined polyphenolic-rich and carbohydrate-rich subfractions. These fractions were systematically characterized by fluorescence emission, three dimensional excitation-emission matrices, and synchronous-scan excitation spectroscopy in comparison with those of the reference International Humic Substances Society soil humic acid and Suwannee River fulvic acid. Results indicate that fluorescence spectroscopy can be useful to qualitatively differentiate not only NOM compounds from varying origins but also NOM subcomponents with varying compositions and functional properties. The polyphenolic-rich NOM-PP fraction exhibited a much more intense fluorescence and a red shift of peak position in comparison with the carbohydrate-rich NOM-CH fraction. Results also indicate that synchronous excitation spectra were able to provide improved peak resolution and structural signatures such as peak positioning, shift, and intensity among various NOM components as compared with those of the emission and excitation spectra. In particular, the synchronous spectral peak intensity and its red shift in the region of about 450-480 nm may be used to indicate the presence or absence of high molecular weight and polycondensed humic organic components, or the multicomponent nature of NOM or NOM subcomponents.

Chemical Fractionation↗

Preferential suppression of delayed-type hypersensitivity by L-156,602, a C5a receptor antagonist.

In the course of our screening for in vivo immunomodulating substances in which sheep red blood cells (SRBC) and heat-killed Brucella abortus cells (thymus dependent and independent antigens, respectively) for antibody production assays, and trinitrobenzene sulfonic acid (TNBS) for delayed-type hypersensitivity (DTH) assay were adopted as antigens, we detected a DTH-specific suppressive activity. The producing organism was isolated from a soil sample collected in Ushiku City, Ibaraki, Japan and identified with Streptomyces sp. A1502 (FERM P-12448). The active component was identified with L-156,602, a C5a receptor antagonist. L-156,602 suppressed both TNBS-induced and TNP-SRBC-induced DTH while it enhanced antibody production against SRBC, Brucella abortus, and TNP-SRBC. L-156,602 significantly suppressed DTH induced by direct injection of type 1 helper T cells and its relevant antigen into hind-footpads, indicating that the efferent phase of DTH was affected by L-156,602. The results demonstrated that L-156,602 preferentially suppressed the DTH response.

Adjuvants, Immunologic↗

Red mud and fly ash for remediation of mine sites contaminated with As, Cd, Cu, Pb and Zn.

The paper presents the results of a laboratory investigation conducted for assessing the feasibility of immobilising the heavy metals (As, Cd, Cu, Pb and Zn) contained in a disused mine tailings dam. Samples of tailings were mixed with relatively small proportions of bauxite red mud and power station fly ash. The sorption capacity of these two materials, already determined for Cd, Cu, Pb and Zn, has been determined here for arsenic by means of batch experiments on purposely prepared solutions. The results of this preliminary investigation show that sorption capacity is strongly influenced by pH. While the red mud performs better at low pH, the ability of fly ash to adsorb As increases with increasing pH. Tests carried out in leach columns containing tailings alone and tailings mixed with 15% by weight neutralized red mud or fly ash demonstrate the heavy metal sorption potential of the two materials, the red mud proving to be the more effective.

Arsenic↗

Development of simple and sensitive spectrophotometric method for the determination of bendiocarb in its formulations and environmental samples.

Facile, selective and sensitive spectrophotometric method has been developed for the determination of bendiocarb in its insecticidal formulations, fortified water, food grains, agriculture wastewater and agriculture soil samples with prepared reagents. The method was based on alkaline hydrolysis of the bendiocarb pesticide, and the resultant hydrolysis product of bendiocarb was coupled with 2,6-dibromo-4-methylaniline to give a yellow color product with lambda max of 457 nm or coupling with 2, 6-dibromo-4-nitroaniline to produce a red colored product with lambda max of 474 nm or coupling with 2, 4, 6-tribromoaniline to form orange red colored product has a lambda max of 465 nm. Under optimal conditions, Beer's law range for 2, 6-dibromo-4-methylaniline (DBMA) was found to be 0.6-14.0 microg mL (-1), 0.8-10.0 microg mL (-1) for 2, 6-dibromo-4-nitroaniline (DBNA) and 0.4-10.0 microg mL (-1) for 2, 4, 6-tribromoaniline (TBA). The molar absorptivity of the color systems were found to be 4.126 x 10(4) l mol (-1) cm (-1) for DBMA, 3.254 x 10(4) l mol (-1) cm (-1) for DBNA and 2.812 x 10(4) l mol (-1) cm (-1) for TBA. Sandell's of the color reactions are 0.018 microg cm (-2)(DBMA), 0.052 microg cm (-2)(DBNA) and 0.065 microg cm (-2) (TBA) respectively. The effect of the non-target species on the determination of bendiocarb was studied. The formation of colored derivatives with the coupling agents is instantaneous and stable for 18 h, 30 h, and 12 h. Performance of the proposed methods were compared statistically in terms Student's F and t-tests with the reported methods.

Environmental Monitoring↗

Economy of fertilizer nitrogen through organic sources in rain-fed rice-legume cropping systems in West Bengal, India.

Field experiments were conducted at a farmers" plot adjacent to the Regional Research Station, red and laterite zone, Sub-center Sekhampur (Birbhum district) of West Bengal, India, situated 23 degrees 24' N latitude, 87 degrees 24' E longitude, to study the effect of different bio- and organic sources of nutrients instead of total fertilizer N in terms of crop productivity in the sequence and building up of soil fertility. During the wet seasons of 1997 and 1998, 12 combinations of bio- and organic sources (crop residues, well decomposed cow dung, dhanicha as green manure) were substituted for 25-50% of N fertilizer applied on transplanted rice (Cv. IR 36). Subsequently, during the winters of 1997-1998 and 1998-1999, leguminous pulse crops like lentil (Lens culinaris [L.] Medic.), gram ( Cicer arietinum L.) and lathyrus (Lathyrus sativus L.) were grown with and without inoculation of Rhizobium. Results revealed that the application of inorganic N in combination with organic sources exhibited a significant increase in rice yield (3.60-3.84 t ha(-1) ) compared to the yield from sole application of N (3.19-3.26 t ha(-1) ). The study showed that about 25% of total applied N was saved without significant yield reduction with simultaneous improvement of soil physical properties (pH, organic matter, available N, P, K, and CEC). Seed yield of pulses (lentil, gram, and lathyrus) were more pronounced in the treatment inoculated with Rhizobium, with a saving of 42.6-48.4 kg N ha(-1). Therefore, the results suggest that the combined application of inorganic and organic N sources in a 75:25 ratio is a superior N-management practice with regards to crop yields as well as improvement of soil fertility.

Agriculture↗

A gene encoding multidrug resistance (MDR)-like protein is induced by aluminum and inhibitors of calcium flux in wheat.

A cDNA clone exclusively induced by aluminum (Al) was isolated from root apices of wheat (Triticum aestivum L.) by the differential display method. The predicted amino acid sequence exhibited homology to the multidrug resistance (MDR) proteins that is known as a member of the ATP-binding cassette (ABC) protein superfamily. Thus this gene was named TaMDR1 (Triticum aestivum MDR). TaMDR1 was induced as a function of Al concentration in the range from 5 to 50 microM, which is in the range of Al content in natural acid soil environment. The concentration required for the induction was lower in the Al-sensitive cultivar than in the Al-tolerant cultivar, indicating that the accumulation of TaMDR1 mRNA was associated with the events caused by Al toxicity rather than Al tolerance. TaMDR1 was significantly induced by the exposure to lanthanum, gadolinium and ruthenium red, which are known as inhibitors of calcium channels. Furthermore, decreasing of calcium ion in growth medium caused stimulation of the gene expression. These results suggested that the induction of TaMDR1 is caused by the breaking of calcium homeostasis which occurred at early stage of Al toxicity.

ATP Binding Cassette Transporter, Subfamily B↗

Selenium and iodine in soil, rice and drinking water in relation to endemic goitre in Sri Lanka.

Endemic goitre has been reported in the climatic wet zone of south-west Sri Lanka for the past 50 years, but rarely occurs in the northern dry zone. Despite government-sponsored iodised salt programmes, endemic goitre is still prevalent. In recent years, it has been suggested that Se deficiency may be an important factor in the onset of goitre and other iodine deficiency disorders (IDD). Prior to the present study, environmental concentrations of Se in Sri Lanka and the possible relationships between Se deficiency and endemic goitre had not been investigated. During the present study, chemical differences in the environment (measured in soil, rice and drinking water) and the Se-status of the human population (demonstrated by hair samples from women) were determined for 15 villages. The villages were characterised by low (< 10%), moderate (10-25%) and high (> 25%) goitre incidence (NIDD, MIDD and HIDD, respectively). Results show that concentrations of soil total Se and iodine are highest in the HIDD villages, however, the soil clay and organic matter content appear to inhibit the bioavailability of these elements. Concentrations of iodine in rice are low (< or = 58 ng/g) and rice does not provide a significant source of iodine in the Sri Lankan diet. High concentrations of iodine (up to 84 microg/l) in drinking water in the dry zone may, in part, explain why goitre is uncommon in this area. This study has shown for the first time that significant proportions of the Sri Lankan female population may be Se deficient (24, 24 and 40% in the NIDD, MIDD and HIDD villages, respectively). Although Se deficiency is not restricted to areas where goitre is prevalent, a combination of iodine and Se deficiency could be involved in the pathogenesis of goitre in Sri Lanka. The distribution of red rice cultivation in Sri Lanka is coincident with the HIDD villages. Varieties of red rice grown in other countries contain anthocyanins and procyanidins, compounds which in other foodstuffs are known goitrogens. The potential goitrogenic properties of red rice in Sri Lanka are presently unknown and require further investigation. It is likely that the incidence of goitre in Sri Lanka is multi-factorial, involving trace element deficiencies and other factors such as poor nutrition and goitrogens in foodstuffs.

Adult↗

Blood-gas properties of plateau zokor (Myospalax baileyi).

Plateau zokor (Myospalax baileyi) is one of the blind subterranean mole rats that spend their life solely underground in sealed burrows. It is one of the special species of the Qinghai-Tibet plateau. In their burrows, oxygen is low and carbon dioxide is high and their contents fluctuate with the change of seasons, soil types, rain and depth of burrows. However, plateau zokors show successful adaptation to that extreme environment. In this study, their adapting mechanisms to the hypoxic hypercapnic environment were analyzed through the comparison of their blood-gas properties with that of pikas (Ochotona curzniae) and Sprague-Dawley rats. The results indicated that plateau zokors had higher red blood corpuscle counts (8.11+/-0.59 (10(12)/L)) and hemoglobin concentrations (147+/-9.85 g/L), but hematocrit (45.9+/-3.29%) and mean corpuscular volume (56.67+/-2.57 fL) were lower than the other rodents. Their arterial blood and venous blood pH were 7.46+/-0.07 and 7.27+/-0.07. Oxygen pressure in arterial blood of plateau zokors was about 1.5 times higher than that of pikas and rats, and it was 0.36 and 0.26 times in their venous blood. Partial pressure for carbon dioxide in arterial and venous blood of plateau zokors was 1.5-fold and 2.0-fold higher, respectively, than in rats and pikas. Oxygen saturation of plateau zokors was 5.7 and 9.3 times lower in venous blood than that of pikas and rats, respectively. As result, the difference of oxygen saturation in arterial blood to venous blood was 2- and 4.5-fold higher in plateau zokors as that of pikas and rats, respectively. In conclusion, plateau zokors had a high tolerance to pH changes in tissues, together with strong capabilities to obtain oxygen from their hypoxic-hypercapnic environment.

Adaptation, Physiological↗

Measuring stem water content in four deciduous hardwoods with a time-domain reflectometer.

New technologies in time-domain reflectometry offer a reliable means of measuring soil water content. Whether these same technologies can be used or adapted to estimate the water content of other porous media, such as the woody tissue of forest trees, has not been thoroughly addressed. Therefore, curves relating the apparent dielectric constant (K(a)) to volumetric water content (g cm(-3)) were constructed for large-diameter stems of red maple (Acer rubrum L.), white oak (Quercus alba L.), chestnut oak (Q. prinus L.), and black gum (Nyssa sylvatica Marsh.). This information was combined with previously published data and a proposed "universal" calibration equation for wood was derived. Stainless-steel rods (15-cm wave guides) were inserted into 160 trees (30 to 49 per species) growing in an upland oak-hickory forest and stem water contents estimated monthly during 1994 and 1995 with a time-domain reflectometer (TDR). Volumetric water contents in April ranged from 0.28 g cm(-3) for red maple to 0.43 g cm(-3) for black gum, with no evidence that water content changed as a function of stem diameter. Stem water contents estimated during 1994 (a wet year) increased from May to July, reached a maximum in midsummer (0.41 to 0.50 g cm(-3)), and then decreased in November. During 1995 (a dry year), stem water contents for red maple and black gum (two diffuse-porous species) decreased from May to August, reached a minimum in September (0.29 to 0.37 g cm(-3)), slightly increased in October and November, and then decreased in December. A different trend was observed during 1995 for white oak and chestnut oak (two ring-porous species), with water contents remaining fairly stable from May to August, but decreasing abruptly in September and again in December. Stem water contents estimated with a TDR broadly agreed with gravimetric analyses of excised stem segments and increment cores, although there was evidence that overestimation of water content was possible with TDR as a result of wounding following wave guide installation. Nonetheless our results hold promise for the application of TDR to the study of stem water content and to the study of whole-plant water storage.

Journal Article↗

[The influence of acidic conditions on polar exopolysaccharides which bind Rhizobium japonicum to soybean root hairs].

The influence of acidic conditions on rhizobial exopolysaccharides (EPS) and their symbiotic association in host plants, were investigated. Light micrographs of soybean root hairs were taken with a Fahraeus' slide assembly after the inocubation of rhizobial bacteria; curled and swollen root hairs, and the typical shepherd's crook with an infection thread were observed. The colonizations of soybean root hairs by rhizobia were different under acidic and neutral conditions. For the Rhizobium japonicum strain USDA 136, and the indigenous acidic soil strain KR 23, morphologically distinct types of cells occurred in the acidic and neutral yeast-extract-mannitol broths. The surface structure of R. japonicum, as examined with the carbon replica and negative stain techniques, was rod shaped and polar flagellated only when the cells were incubated under neutral conditions. The materials which reacted with the ruthenium red positive reagent are in all likelihood an acidic EPS, and are believed to effect the adhesive properties of the cells. Bacterial polarity, which could be more readily observed under neutral conditions than under acidic conditions, was related to the distribution of EPS near one end of the bacteria. EPS was found to effect the initial step of polar attachment in the effective infection of host roots. By using DEAE-Sephacel ion exchange chromatography, the capsular polysaccharides from both strains of R. japonicum were also found to have different chemical components when observed in acidic and neutral cultures.

Adhesiveness↗

[Disappearance of ALS from Guam: implications for exogenous causes].

The author reports the disappearance of amyotrophic lateral sclerosis (ALS) from Guam over past 30 years, which coincided with rapid changes in the ecology, socioeconomy, and westernization of the life style. This slow but steady decline is believed to be the consequences of radical changes from food collection to wage-based life style and dietary improvement in recent years and elimination of exogenous factors. Those risk factor(s) are believed to be the environmental trace metals which must have triggered the accelerated oxidative stresses in the motor neurons of genetically susceptible population. Changing Epidermiology: 1. The annual incidence of 70/100,000 in 1960s down to 7/100,000 in 1990s, and remained unchanged for past 15 years. 2. Upward shift of age at onset by 10 years and at death by 8 years and even out of sex ratio. 3. Birth cohort analysis showed less risks for those born after 1920. No ALS cases born after 1945. 4. No increase in the incidence of ALS among non-Chamorros transients of Guam and Marianas during W.W.II. 5. Long-term resident non-Charmorro and half-Chamorros on Guam are also affected. 6. Charmorro migrants to U.S. Mainland are affected after long absence from Guam. 7. Incubation period for both ways is estimated to be 18 approximately 20 years. 8. Other forms of dementias like Alzheimer disease (AD) and vascular dementias are on the rise and the leading cause of death is cerebro- and cardiovascular diseases. 9. ALS is also declining in past 10 approximately 15 years in Kii peninsula, and West New Guinea. Changing Ecology of Guam: 1. One third of Island land was used for construction of huge military bases after W.W.II. 2. Urbanization of villages including concrete houses, deep well water supply, sewage, and electrification. 3. Tourism boom: high-rise hotels, development of 7 golf courses and other recreational facilities resulted in loss of flora and erosions of soil. Socioeconomic Changes: 1. Shift in population demography; Efflux of Chamorros and influx of aliens; Chamorros less than 50% by 1990. 2. Tourists passed 1 million in 1994. 3. Automobiles 1 car/1.5 person. 4. Westernization: After W.W.II, almost free access to Military Commissary for imported food and appliances. 5. Life style: from food collection to wage-based society. Genetic Studies: 1. Familial aggregations, but no clear-cut Mendelian inheritance. 2. Segregation analysis: no absolute genetic or environmental cause but additive gene component may play a role in genetic susceptibility and basis for geographical clustering. 3. Absence of Apo-E or Mu/Zn SOD genes. 4. Recent discovery of mtDNA Complex I deficiency in Parkinsonism-dementia cases suggests mitochondrial DNA abnormality. Comparative Environmental Studies: 1. Environmental studies in three hyperendemic areas in the Western Pacific--Kii, Marianas, and west New Guinea, where strikingly high incidences of ALS is known to occur, found the identical geochemical environment--low Ca, Mg, and Zn and high A1, Mn, Fe, Si, in the garden soil and drinking water. 2. Exogenous etiologic factors that are absent from primitive culture of Auyu and Jackai tribes in West New Guinea were eliminated. 3. Cycad neurotoxicity has been excluded. 4. Suspected exogenous agents that are common in these 3 hyperendemic areas are (a) locally grown vegetables, starchy roots, and reef fish; (b) surface water containing soluble organic minerals from red laterites; (c) rain water that is chemically pure and lack of essential minerals. Pathogenic Speculation: Chronic dietary deficiency since birth in Ca, Mg and Zn induced excessive absorption of divalent cations which accelerates oxidant-mediated neuronal degenerations in a genetically susceptible population. The process is probably carried through interactions between cytoskeletal abnormality of the neuron, aging process, abnormal proteins, and mitochondrial dysfunction.

Amyotrophic Lateral Sclerosis↗

Reduction of oxamyl and related pesticides by FeII: influence of organic ligands and natural organic matter.

The reduction of oxamyl and related oxime carbamate pesticides (OCPs; methomyl and aldicarb) by FeII is an important pathway for the degradation of these compounds in soil and groundwater. A series of batch kinetic experiments was carried out to assess the effects that selected carboxylate and aminocarboxylate ligands have on these reactions. In the absence of FeII, no OCP reduction by the ligands is observed. In the presence of FeII, the rate of OCP reduction varies by several orders of magnitude and can be described by the expression k(red) = [FeII]sigma(i)k(i)alpha(i) where k(red) is the observed pseudo-first-order rate constant for OCP reduction, [FeII] is the total FeII concentration, alpha(i) is the fraction of each FeII species in solution, and k(i) is the second-order rate constant for OCP reduction by each FeII species. The reactivity of individual FeII species is dependent upon the standard one-electron reduction potential of the corresponding FeIII/FeII redox couple (E(H)o) and the availability of inner-sphere FeII coordination sites for bonding with Lewis base donor groups within the OCP structure. A linear free energy relationship is proposed. Kinetic measurements demonstrate that natural organic matter from the Great Dismal Swamp facilitates OCP reduction by FeII in the same manner as the individual organic ligands. Results from this study improve our understanding of the pathways and rates of pesticide degradation in reducing subsurface environments, especially those rich in organic matter.

Carbamates↗

Biodegradation of polyethylene by the thermophilic bacterium Brevibacillus borstelensis.

AIM: To select a polyethylene-degrading micro-organism and to study the factors affecting its biodegrading activity. METHODS AND RESULTS: A thermophilic bacterium Brevibaccillus borstelensis strain 707 (isolated from soil) utilized branched low-density polyethylene as the sole carbon source and degraded it. Incubation of polyethylene with B. borstelensis (30 days, 50 degrees C) reduced its gravimetric and molecular weights by 11 and 30% respectively. Brevibaccillus borstelensis also degraded polyethylene in the presence of mannitol. Biodegradation of u.v. photo-oxidized polyethylene increased with increasing irradiation time. Fourier Transform Infra-Red (FTIR) analysis of photo-oxidized polyethylene revealed a reduction in carbonyl groups after incubation with the bacteria. CONCLUSIONS: This study demonstrates that polyethylene--considered to be inert--can be biodegraded if the right microbial strain is isolated. Enrichment culture methods were effective for isolating a thermophilic bacterium capable of utilizing polyethylene as the sole carbon and energy source. Maximal biodegradation was obtained in combination with photo-oxidation, which showed that carbonyl residues formed by photo-oxidation play a role in biodegradation. Brevibaccillus borstelensis also degraded the CH2 backbone of nonirradiated polyethylene. SIGNIFICANCE AND IMPACT OF THE STUDY: Biodegradation of polyethylene by a single bacterial strain contributes to our understanding of the process and the factors affecting polyethylene biodegradation.

Bacteriological Techniques↗

Repair detection procedure for enumeration of fecal coliforms and enterococci from seafoods and marine environments.

The repair detection procedure of Speck et al. (Appl. Microbiol. 29:549-550, 1975) was adapted for the enumeration of coliforms, fecal coliforms, and enterococci in seafood and environmental samples. Samples were pour plated with Trypticase soy agar, followed by a 1- to 2-h incubation to effect repair; the plates were then overlaid with the selective medium and incubated. Violet red bile agar and an incubation temperature of 45 degrees C were used as the selective conditions for fecal coliforms, and KF streptococcal agar was used for the enumeration of enterococci. The method was more efficient than the standard most-probable-number method for fecal coliform enumeration and also allowed enumeration of the injured cells, which might have remained undetected when selective medium in the most-probable-number method was used. The repair detection method effectively recovered the injured portion of the population of enterococci capable of growing on KF streptococcal agar. The repair enumeration method was not suitable for coliforms in marine samples because associative marine bacteria mimicked coliforms in violet red bile agar plates incubated at 35 degrees C. The marine bacteria did not grow at 45 degrees C and therefore did not interfere with fecal coliform enumeration.

Escherichia coli↗

Molecular cloning and characterization of the gene coding for azoreductase from Bacillus sp. OY1-2 isolated from soil.

Azo dyes are regarded as pollutants because they are not readily reduced under aerobic conditions. Bacillus sp. OY1-2 transforms azo dyes into colorless compounds, and this reduction is mediated by a reductase activity for the azo group in the presence of NADPH. A 1.2-kbp EcoRI fragment containing the gene that encodes azoreductase was cloned by screening the genomic library of Bacillus sp. OY1-2 with digoxigenin-labeled probe designed from the N-terminal amino acid sequence of the purified enzyme. An open reading frame encoding the azoreductase, consisting of 178 amino acids, was predicted from the nucleotide sequence. In addition, because only a Bacillus subtillis hypothetical protein was discovered in the public databases (with an amino acid identity of 52.8%), the gene encoding the azoreductase cloned in this study was predicted to be a member of a novel family of reductases. Southern blot analysis revealed that the azoreductase gene exists as a single copy gene on a chromosome. Escherichia coli-expressing recombinant azoreductase gave a ten times greater reducing activity toward azo dyes than the original Bacillus sp. OY1-2. In addition, the expressed azoreductase purified from the recombinant E. coli lysate by Red-Sepharose affinity chromatography showed a similar activity and specificity as the native enzyme. This is the first report describing the sequencing and characterization of a gene encoding the azo dye-reducing enzyme, azoreductase, from aerobic bacteria and its expression in E. coli.

Amino Acid Sequence↗