In vitro degradation of linamarin by microorganisms isolated from cassava wastewater treatment lagoons

This study aimed at isolating and characterizing of microorganisms able to use linamarin as sole carbon source. Thirty one microbial strains were isolated from manipueira, a liquid effluent of cassava processing factories. Among these strains, Bacillus licheniformis (isolate 2_2) and Rhodotorulla glutinis (isolate L1) were able to degrade 71 percent and 95 percent of added linamarin, respectively, within 7 days, showing high biodegradation activity and great potential for detoxification of cassava processing wastewaters.

In vitro degradation of linamarin by microorganisms isolated from cassava wastewater treatment lagoons.

This study aimed at isolating and characterizing of microorganisms able to use linamarin as sole carbon source. Thirty one microbial strains were isolated from manipueira, a liquid effluent of cassava processing factories. Among these strains, Bacillus licheniformis (isolate 2_2) and Rhodotorulla glutinis (isolate L1) were able to degrade 71% and 95% of added linamarin, respectively, within 7 days, showing high biodegradation activity and great potential for detoxification of cassava processing wastewaters.

Nitrogen-fixing chemo-organotrophic bacteria isolated from cyanobacteria-deprived lichens and their ability to solubilize phosphate and to release amino acids and phytohormones.

AIMS: Cyanobacteria-deprived lichens of the species Canoparmelia caroliniana, Canoparmelia crozalsiana, Canoparmelia texana, Parmotrema sancti-angeli and Parmotrema tinctorum were screened for the presence of chemo-organotrophic nitrogen-fixing bacteria. METHODS AND RESULTS: Fifty-three lichen samples subjected to enrichment selection using a nitrogen-free minimal medium were positive for acetylene reduction. Seventeen isolates, able to fix nitrogen, belonged to Gamma-proteobacteria group and were identified as: Acinetobacter sp., Pantoea sp., Pseudomonas sp., Pseudomonas stutzeri, Serratia marcescens and Stenotrophomonas maltophilia, according to 16S rRNA gene sequences and biochemical tests. The excretion of amino acid and phytohormone and the ability of mineral phosphate solubilization were determined in 14 isolates. All isolates were able to release amino acids and 3-indoleacetic acid. About 64% of the isolates solubilized phosphates and 30% released ethylene. CONCLUSIONS: These data confirm sparse evidence from the literature on the occurrence of chemo-organotrophic nitrogen-fixing bacteria in cyanobacteria-deprived lichens; the isolates presented physiologic features which might benefit the host if they are expressed when the bacteria are harboured by lichens. SIGNIFICANCE AND IMPACT OF THE STUDY: Chemo-organotrophic nitrogen-fixing bacteria were isolated from a high percentage (72.6%) of cyanobacteria-deprived lichens. All isolates presented important physiological characteristics, some of which are being described here for the first time.

Streptomyces drozdowiczii sp. nov., a novel cellulolytic streptomycete from soil in Brazil.

An actinomycete strain, isolated from a Mata Atlântica soil sample, showing cellulolytic activity was subjected to polyphasic taxonomic characterization to determine its identity. Strain M7aT presented morphological and chemotaxonomic characteristics consistent with its assignment to the genus Streptomyces. Phylogenetic analysis of its 16S rDNA sequence revealed that the strain differed from described streptomycetes available in the public databases; the most closely related species was Streptomyces laceyi, with 98.4% nucleotide similarity. It also differed from other cellulolytic strains in its phenotypic characteristics. It is therefore proposed that strain M7aT, a cellulolytic strain with biotechnological potential, represents a novel species, named Streptomyces drozdowiczii sp. nov. The type strain is M7aT (=CBMAI 0498T=CIP 107837T=NRRL B-24297T).

Drug resistance in Chromobacterium violaceum

Chromobacterium violaceum is a free-living bacterium commonly found in aquatic habitats of tropical and subtropical regions of the world. This bacterium is able to produce a large variety of products of biotechnological and pharmacological use. Although C. violaceum is considered to be non-pathogenic, some cases of severe infections in humans and other animals have been reported. Genomic data on the type strain ATCC 12472(T) has provided a comprehensive basis for detailed studies of pathogenicity, virulence and drug resistance genes. A large number of open reading frames associated with various mechanisms of drug resistance were found, comprising a remarkable feature of this organism. Amongst these, beta-lactam (penicillin and cephalosporin) and multidrug resistance genes (drug efflux pumps) were the most numerous. In addition, genes associated with bacitracin, bicyclomycin, chloramphenicol, kasugamycin, and methylenomycin were also found. It is postulated that these genes contribute to the ability of C. violaceum to compete with other bacteria in the environment, and also may help to explain the common drug resistance phenotypes observed in infections caused by this bacterium

Biodegradation of alachlor by soil streptomycetes.

Streptomycetes resistant to the herbicide alachlor [2-chloro-2',6'-diethyl- N-(methoxymethyl) acetanilide] were used in degradation assays to characterize the products of alachlor biodegradation. Of six strains tested, Streptomyces sp. LS166, LS177, and LS182 were able to grow at an alachlor concentration of 144 mg l(-1) and degraded approximately 60-75% of the alachlor in 14 days, as evaluated by high performance liquid chromatography. The alachlor biodegradation products were identified by gas chromatography-mass spectrometry based on mass spectral data and fragmentation patterns. All compounds detected in these assays were similar for all streptomycetes strains tested, and involved dechlorination with subsequent N-dealkylation and cyclization of the remaining N-substituent with one of the ethyl groups to produce indole and quinoline derivatives. The enzymatic pathway used by Streptomyces sp. LS182 did not generate DEA (2',6'-diethylaniline), a carcinogenic derivative of alachlor reported in other studies. Given the high degradation rates observed here, the Streptomyces strains tested may be useful in the degradation/detoxification processes of alachlor.

Characterization of Biofilm Formation by Xylella fastidiosa In Vitro.

Xylella fastidiosa colonizes the xylem of various host plants, causing economically important diseases such as Pierce's disease in grapevine and citrus variegated chlorosis (CVC) in sweet oranges. The aggregative nature of this bacterium has been extensively documented in the plant xylem and the insect's foregut. Structured communities of microbial aggregates enclosed in a self-produced polymeric matrix and attached to a surface are defined as biofilms. In this study, we characterized biofilm formation by X. fastidiosa through the use of a novel in vitro assay for studying biofilm growth in a potential mimic system of what might occur in planta. We used wood, a xylem rich material, as a surface for bacterial attachment and biofilm formation, under shear force. We demonstrated that X. fastidiosa strains isolated from various hosts formed biofilm on wood in this in vitro assay. Different biofilm morphology was detected, which seems to vary according to the strain tested and microenvironmental conditions analyzed. We observed that strains from different hosts could be grouped according to three parameters: biofilm morphology, the ability to form clumps in liquid culture, and the ability to attach to glass surfaces. We hypothesize that biofilm formation is likely a major virulence factor in diseases related to X. fastidiosa, bringing a new perspective for disease treatment.

Isolation and characterization of actinomycetes from Brazilian tropical soils.

Actinomycetes have been isolated from three Brazilian tropical soils. The dispersion and differential centrifugation procedure revealed count values 1.5 to 5.0 times greater than those obtained by the conventional dilution plate technique for all soils and media tested. Eighteen strains, promising for biotechnological applications, were submitted to chemotaxonomic procedures and numerical taxonomy for identification. Two were identified as Amycolatopsis orientalis, one as Streptomyces misakiensis, and two tentatively included or associated to S. chromofuscus and S. griseoruber. The others, all belonging to the Streptomyces genus, could not be fitted into any known species, and were arranged by the UPGMA analysis for classification, as an isolated group. This suggests that the actinomycetes in tropical soils may represent a vast unexplored resource for biotechnology.

Molecular characterization of Acidithiobacillus ferrooxidans and A. thiooxidans strains isolated from mine wastes in Brazil.

Nineteen strains of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, including 12 strains isolated from coal, copper, gold and uranium mines in Brazil, strains isolated from similar sources in other countries and the type strains of the two species were characterized together with the type strain of A. caldus by using a combination of molecular systematic methods, namely ribotyping, BOX- and ERIC-PCR and DNA-DNA hybridization assays. Data derived from the molecular fingerprinting analyses showed that the tested strains encompassed a high degree of genetic variability. Two of the Brazilian A. ferrooxidans organisms (strains SSP and PCE) isolated from acid coal mine waste and uranium mine effluent, respectively, and A. thiooxidans strain DAMS, isolated from uranium mine effluent, were the most genetically divergent organisms. The DNA-DNA hybridization data did not support the allocation of Acidithiobacillus strain SSP to the A. ferrooxidans genomic species, as it shared only just over 40% DNA relatedness with the type strain of the species. Acidithiobacillus strain SSP was not clearly related to A. ferrooxidans in the 16S rDNA tree.

Biosystematic studies on novel streptomycetes from soil.

Members of three putatively novel Streptomyces species, designated Streptomyces groups A, B and C, were repeatedly isolated from environmental samples taken from four hay meadow plots at Cockle Park Experimental Farm, Northumberland (UK). Representative isolates were found to have properties consistent with their classification in the genus Streptomyces and were recovered in three taxa using different phenotypic criteria, namely morphological and pigmentation properties, rapid enzyme tests, and whole-organism fatty acid, protein electrophoretic and pyrolysis mass-spectrometric data. The isolates were rapidly characterised as three taxonomic groups using pyrolysis mass spectrometry. The three taxa were also distinguished from one another and from validly described species of Streptomyces using rapid enzyme tests based on the fluorophores 7-amino-methylcoumarin and 4-methylumbelliferone, and computer-assisted identification procedures. The results indicate that selective isolation and rapid characterisation of streptomycetes using pyrolysis mass spectrometry provide a practical way of determining the phenotypic species diversity of streptomycetes in natural habitats. The experimental data also indicate that representative sampling of cultivable streptomycetes from soil can best be achieved using a multi-step extraction procedure coupled with the use of selective isolation procedures.

Streptomyces malaysiensis sp. nov., a new streptomycete species with rugose, ornamented spores.

The taxonomic position of a streptomycete strain isolated from Malaysian soil was established using a polyphasic approach. The organism, designated strain ATB-11T, was found to have chemical and morphological properties consistent with its classification in the genus Streptomyces. An almost complete 16S rRNA gene (rDNA) sequence determined for the test strain was compared with those of previously studied streptomycetes by using two treeing algorithms. The 16S rDNA sequence data not only supported classification of the strain in the genus Streptomyces but also showed that it formed a distinct phyletic line. At maturity, the aerial hyphae of strain ATB-11T differentiated into tight, spiral chains of rugose, cylindrical spores. The organism was readily distinguished from representatives of validly described Streptomyces species with rugose spores by using a combination of phenotypic features. It is proposed, therefore, that strain ATB-11T be classified in the genus Streptomyces as Streptomyces malaysiensis sp. nov.

Evaluating the microbial diversity of soil samples: methodological innovations.

This manuscript is a review of the innovative methodologies that enable more precise evaluations of soil microbial diversity. Highlighting the molecular approach, which does not require the isolation of microorganisms and allows the inclusion of non-culturable genotypes in the analyses, the described methodologies revolutionised the environmental microbiology and opened gateways for an accurate understanding of the ecology and diversity of microorganisms. The application of techniques based on soil total DNA extraction, PCR amplification of genes or gene fragments, and sequence analysis revealed that the microbial universe is far more complex than ever imagined. Examples of applications of the molecular approach to study the diversity of soil diazotrophic bacteria are given.

First evaluation of the Brazilian microorganisms biocatalytic potential.

The biocatalytic potential of two novel Brazilian strains of Aspergillus niger and Rhodotorula glutinis, revealed enantioselective epoxide hydrolase activity in the asymmetrization of meso-epoxide and monosubstituted epoxides respectively. These two types of oxirane derivatives are not usually good substrates for biocatalytic enantioselective conversion.

Discrimination of Rhizobium tropici and R. leguminosarum strains by PCR-specific amplification of 16S-23S rDNA spacer region fragments and denaturing gradient gel electrophoresis (DGGE).

With the aim of detecting Rhizobium species directly in the environment, specific PCR primers for Rh. tropici and Rh. leguminosarum were designed on the basis of sequence analysis of 16S-23S rDNA spacer regions of several Rh. tropici, Rh. leguminosarum and Agrobacterium rhizogenes strains. Primer specificity was checked by comparison with available rDNA spacer sequences in databases, and by PCR using DNA from target and reference strains. Sequence polymorphisms of rDNA spacer fragments among strains of the same species were detected by denaturing gradient gel electrophoresis (DGGE). The specific PCR primers designed in this study could be applied to evaluate the diversity of Rh. tropici and Rh. leguminosarum by analysing the polymorphisms of 16S-23S spacer rDNA amplified from either whole-cell or soil-extracted DNA.

Molecular evidence for shifts in polysaccharide composition associated with adaptation of soybean Bradyrhizobium strains to the Brazilian Cerrado soils.

Pyrolysis mass spectrometry (PyMS) and DNA fingerprinting (RAPD and RSalpha hybridization) were used to characterize soybean inoculant strains and root nodule isolates of bradyrhizobia from the Brazilian Cerrado soils. Most isolates were shown to be derived from the inoculant strains on the basis of genotype comparisons by DNA fingerprinting. Phenotypic analysis (using PyMS) of the strains and separately of the polysaccharides derived from them showed that the nodule isolates differed from the parental strains, suggesting adaptation to the Cerrado soil environment. The extent of the differences between the derivatives and inoculant strains was similar for comparisons made on the basis of whole-cell preparations or from the isolated polysaccharides, indicating that the adaptation was caused by changes in the composition of the polysaccharides produced.

Biodegradation of the herbicide Diuron in soil by indigenous actinomycetes.

Three actinomycete strains isolated from soil treated with 2,4-D were able to degrade the herbicide Diuron in vitro. Strain CCT 4916 was the most efficient, degrading up to 37% of applied Diuron (100 mg Kg-1 soil) in 7 days, as measured by HPLC and UV/VIS spectroscopy. All strains showed protease and urease activity; intracellular activity of metapyrocatechase and pyrocatechase were not found. Actinomycete strain CCT 4916 produced manganese peroxidase, which could be potentially related to degradation of Diuron.

Classification of photobacteria associated with spoilage of fish products by numerical taxonomy and pyrolysis mass spectrometry.

Forty strains of luminous and non-luminous Photobacterium phosphoreum isolates from cod (Gadus morhua) and seven reference strains of psychrotolerant and mesophilic photobacteria were examined for 156 unit characters in a numerical taxonomic study. The fish strains were isolated from the intestines, from spoiled products and by using a specific detection method. The data were analysed using the similarity coefficient and the unweighted pair-group with arithmetic averages algorithm. In addition twenty-six of the fish isolates and five reference strains were analysed by Curie-point pyrolysis mass spectrometry. The mesophilic and psychrotolerant photobacteria were clearly separated in each of the analyses. Both procedures indicated that the spoilage isolates of P. phosphoreum had originated from the live fish as strains from the fish intestines and from the spoiled products were recovered in the same sub-groups. One sub-group of psychrotolerant P. phosphoreum strains, which was selected in modified atmosphere packed fish stored at low temperature, was also highlighted using each of the methods. The importance of classifying food spoilage bacteria has been shown and a simple key generated for the identification of luminous and non-luminous isolates of Photobacterium phosphoreum.

An investigation of the intra-generic structure of Rothia by pyrolysis mass spectrometry.

Previous studies of the genus Rothia have indicated that members of the only species, Rothia dentocariosa, are heterogeneous and may form more than one species. To study the intrageneric taxonomic structure of the Rothia taxon eighteen strains identified as R. dentocariosa, including reference organisms from culture collections (3 strains), isolates from healthy subjects (5 strains) and from clinical sources (10 strains) were examined using pyrolysis mass spectrometry. The ordination plots of the pyrolysis data indicated that all the strains clustered in a closely related group, with the exception of three strains which out-grouped. Phenotypic testing and fatty acid data indicated that the latter three strains are probably misclassified in the genus Rothia. Reanalysis of the PyMS data including only the fifteen authentic Rothia strains indicated that ten of these organisms formed a group which included the type strain, R. dentocariosa NCTC 10917. Four out of the remaining five organisms formed a diffuse group; the remaining strain was recovered as a single member cluster. These data indicate that R. dentocariosa is heterogeneous though at present there are no suitable criteria for assigning members of this taxon to more than one species.

Typing of the fish pathogen Listonella (Vibrio) anguillara by pyrolysis mass spectrometry.

Twenty-eight representatives of Listonella (Vibrio) anguillara serovars O1, O2 and O3 were compared by Curie-point pyrolysis mass spectrometry (PyMS). The representatives of serovars O1 and O3 formed discrete, homogeneous groups in ordination plots of the PyMS data. Strains from serovar O2 were recovered in two groups, one of which encompassed six strains including the type strain of the species and the reference strain for serovar O2, and the other included two strains which showed cross-reactions between serovars O2 and O5. The almost complete agreement found between the PyMS and the serological data suggests that pyrolysis mass spectrometry will prove to be an effective method for interstrain comparison within the species Listonella anguillara.

Rapid identification of streptomycetes by artificial neural network analysis of pyrolysis mass spectra.

An artificial neural network was trained to distinguish between three putatively novel species of Streptomyces using normalised, scaled prolysis mass spectra from three representative strains of each of the taxa, each sampled in triplicate. Once trained, the artificial neural network was challenged with spectral data from the original organisms, the 'training set', from additional members of the putative novel taxa and from over a hundred strains representing six other actinomycete genera. All of the streptomycetes were correctly identified but many of the other actinomycetes were mis-identified. A modified network topology was developed to recognise the mass spectral patterns of the non-streptomycete strains. The resultant neural network correctly identified the streptomycetes, whereas all of the remaining actinomycetes were recognised as unknown organisms. The improved artificial neural network provides a rapid, reliable and cost-effective method of identifying members of the three target streptomycete taxa.