The impact of shrimp farming effluent on bacterial communities in mangrove waters, Ceará, Brazil.

The effects of shrimp farm effluents on bacterial communities in mangroves have been infrequently reported. Classic and molecular biology methods were used to survey bacterial communities from four mangroves systems. Water temperature, salinity, pH, total heterotrophic bacteria and maximum probable numbers of Vibrio spp. were investigated. Genetic profiles of bacterial communities were also characterized by polymerase chain reaction (PCR) amplification of eubacterial and Vibrio 16S rDNA using denaturing gradient gel electrophoresis (DGGE). Highest heterotrophic counts were registered in the mangrove not directly polluted by shrimp farming. The Enterobacteriaceae and Chryseomonas luteola dominated the heterotrophic isolates. Vibrio spp. pathogenic to humans and shrimps were identified. Eubacterial genetic profiles suggest a shared community structure independent of mangrove system. Vibrio genetic profiles were mangrove specific. Neither microbial counts nor genetic profiling revealed a significant decrease in species richness associated with shrimp farm effluent. The complex nature of mangrove ecosystems and their microbial communities is discussed.

New group-specific 16S rDNA primers for monitoring foaming mycolata during saline waste-water treatment.

Newly designed group-specific PCR primers for denaturing gradient gel electrophoresis (DGGE) were used to investigate foaming mycolata from a bioreactor treating an industrial saline waste-water. Genetic profiles on DGGE gels were different with NaCl at 1.65 and 8.24 g l(-1), demonstrating that mycolata community was affected by salinity. A semi-nested PCR strategy resulted in more bands in community genetic profiles than direct amplification. DNA sequencing of bands confirmed the efficacy of the novel primers with sequences recovered being most similar to foam producing mycolata. The new group-specific primers/DGGE approach is a new step toward a more complete understanding of functionally important groups of bacteria involved in biological treatment of waste-water.

Genetic diversity of Saccharomyces cerevisiae strains during the 24 h fermentative cycle for the production of the artisanal Brazilian cachaça.

AIMS: Characterization of yeast populations and genetic polymorphism of Saccharomyces cerevisiae strains collected during the short fermentative cycles from the spontaneous fermentations during the artisanal cachaça production. METHODS AND RESULTS: The prevalent S. cerevisiae strains were analysed by PFG and RAPD-PCR using primers EI1 and M13. The molecular analysis have showed a high degree of genetic polymorphism among the strains within a 24 h fermentative cycle. CONCLUSION: The genetic diversity observed in the S. cerevisiae strains may be occurring due to the existence of a large number of individual genotypes within the species. The unique characteristics of the cachaça fermentation process probably allows for a faster detection of molecular polymorphisms of yeast strains than other types of fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: Spontaneous fermentations to produce cachaça, due to their characteristics, are an excellent model for the study of molecular diversity of S. cerevisiae strains during the production of fermented beverages.

Yeast communities and genetic polymorphism of Saccharomyces cerevisiae strains associated with artisanal fermentation in Brazil.

Yeast communities and genetic polymorphism of prevalent Saccharomyces cerevisiae strains isolated from the spontaneous fermentation of the sugarcane juice during the production of aguardente in three distilleries in the state of Minas Gerais, Brazil, were studied. S. cerevisiae was the prevalent species during the process of aguardente production, but Schizosaccharomyces pombe was predominant in old fermentations in one distillery. Transient yeast species were found in a variable number, probably due to the daily addition of sugarcane juice, and they were different for each of the three distilleries studied. PFGE and PCR analysis of the predominant strains of S. cerevisiae isolated from the fermented must showed a high degree of genetic polymorphism among the three distilleries. A high molecular variability of S. cerevisae strains was also observed among strains isolated from the same vat at different fermentation ages. Our results showed that there was a succession of geneticly different strains of S. cerevisae during the process of aguardente production.

Yeast communities associated with sugarcane in Campos, Rio de Janeiro, Brazil.

Yeast communities associated with sugarcane leaves, stems and rhizosphere during different phases of plant development were studied near Campos, in Rio de Janeiro, Brazil. Atmospheric temperature, soil granulometry and pH, and sugar cane juice degree Brix and pH were determined. Yeast communities associated with sugarcane were obtained after cellular extraction by shaking, blending and shaking plus sonication, and cultured on Yeast Nitrogen Base Agar plus glucose (0.5%) and Yeast Extract-Malt Extract Agar. No significant differences in yeast counts were found among the cellular extraction treatments and culture media. 230 yeast cultures were identified according to standard methods, and distinct yeast communities were found for each substrate studied. The prevalent species isolated from sugarcane were Cryptococcus laurentii, Cryptococcus albidus, Rhodotorula mucilaginosa and Debaryomyces hansenii.

The incidence of killer activity and extracellular proteases in tropical yeast communities.

The presence of killer and proteolytic yeasts was studied among 944 isolates representing 105 species from tropical yeast communities. We found 13 killer toxin producing species, with Pichia kluyveri being the most frequent. Other killer yeast isolates were Candida apis, Candida bombicola, Candida fructus, Candida krusei, Candida sorbosa, Hanseniaspora uvarum, Issatchenkia occidentalis, Kloeckera apis, Kluyveromyces marxianus, Pichia membranaefaciens, Pichia ohmeri-like, and Sporobolomyces roseus. The communities from which killer yeasts were isolated had strains sensitive to them, and there were interspecific and intraspecific differences in the spectra of their killer activities. Pichia kluyveri had the broadest spectra of activity against sensitive isolates, and it apparently produced different toxins. The coexistence of sensitive and killer yeasts using the same substrate suggests that there is spatial separation in microhabitats or temporal separation in different stages of successions. Basidiomycetous yeasts were more frequently proteolytic than ascomycetous yeasts. Extracellular proteases could be important for the yeasts to have access to more nitrogen nutrients and obtain a better balance with available carbon sources.

PCR amplification of the rDNA internal transcribed spacer region for differentiation of Saccharomyces cultures.

The size of the internal transcribed spacer (ITS) region as measured by gel electrophoresis of PCR products, amplified by primers ITS1 and ITS4, was over 800 bp for all Saccharomyces sensu stricto species, but yeasts belonging to other Saccharomyces species had a shorter ITS region, making this characteristic potentially useful in the identification of Saccharomyces isolates. The ITS product length was homogeneous within the species Saccharomyces cerevisiae.

Selection of rhizosphere-competent Pseudomonas strains as biocontrol agents in tropical soils.

Pseudomonas strains were isolated from the rhizosphere of maize grown in yellow-red latosol from Rio de Janeiro, Brazil, to serve as a delivery system for heterologous genes and for risk assessment studies in tropical soils. Selected strains were modified by insertion of the cryIVB gene from Bacillus thuringiensis and tested for pathogenicity gene expression against larvae of a susceptible model species, Anopheles aquasalis. Modified strains Br8 and Br12 showed similar survival performance to their parental strains, and presented a viable density of 10(7) c.f.u./g dry soil 30 days after release. A strain of P. fluorescens (Br12) that presented positive results for gene expression and the best survival performance, was selected for risk assessment studies in soil microcosms.

Yeast succession in the Amazon fruit Parahancornia amapa as resource partitioning among Drosophila spp.

The succession of yeasts colonizing the fallen ripe amapa fruit, from Parahancornia amapa, was examined. The occupation of the substrate depended on both the competitive interactions of yeast species, such as the production of killer toxins, and the selective dispersion by the drosophilid guild of the amapa fruit. The yeast community associated with this Amazon fruit differed from those isolated from other fruits in the same forest. The physiological profile of these yeasts was mostly restricted to the assimilation of a few simple carbon sources, mainly L-sorbose, D-glycerol, DL-lactate, cellobiose, and salicin. Common fruit-associated yeasts of the genera Kloeckera and Hanseniaspora, Candida guilliermondii, and Candida krusei colonized fruits during the first three days after the fruit fell. These yeasts were dispersed and served as food for the invader Drosophila malerkotliana. The resident flies of the Drosophila willistoni group fed selectively on patches of yeasts colonizing fruits 3 to 10 days after the fruit fell. The killer toxin-producing yeasts Pichia kluyveri var. kluyveri and Candida fructus were probably involved in the exclusion of some species during the intermediate stages of fruit deterioration. An increase in pH, inhibiting toxin activity and the depletion of simple sugars, may have promoted an increase in yeast diversity in the later stages of decomposition. The yeast succession provided a patchy environment for the drosophilids sharing this ephemeral substrate.

Ascomycetous yeast communities of marine invertebrates in a southeast Brazilian mangrove ecosystem.

The ascomycetous yeast communities associated with 3 bivalve mollusk, and 4 crab species were studied in the mangrove at Coroa Grande on Sepetiba Bay in Rio de Janeiro, Brazil. These were made up mostly of diverse but sparse and apparently allochtonous yeast populations. The striking exception was a prevalent population of the species Kluyveromyces aestuarii, which predominated the yeast communities of 2 detritus feeding crabs, Sesarma rectum and Uca spp., and the shipworm Neoteredo reynei. However, K. aestuarii was absent from the omnivorous crabs Aratus pisonii and Goniopsis cruentata, and the clam Anomalocardia brasiliana, and was rare in the clam Tagelus plebeius from mostly submerged more sandy sediments. Pichia membranaefaciens, Candida valida-like, Candida krusei, Candida sorbosa, Candida colliculosa-like, Candida famata-like, Kloeckera spp., Candida guilliermondii, Candida albicans, Candida silvae, Geotrichum spp., Rhodotorula spp., Cryptococcus spp., and the methylotrophic yeast Candida boidinii were frequently isolated. The 322 ascomycetous yeast cultures representing 252 isolates from crabs and mollusks were classified as 40 species that fit standard descriptions, and 44 putative new species. The ascomycetous yeast communities of the mangrove ecosystem include many new biotypes that require better taxonomic definition.

A new genetically isolated population of the Saccharomyces sensu stricto complex from Brazil.

Genetic and karyotypic studies of some Saccharomyces sensu stricto yeasts from Brazil revealed a genetically isolated population which apparently represents a new sibling species of S. cerevisiae.

Yeast communities of the cactus Pilosocereus arrabidae as resources for larval and adult stages of Drosophila serido.

The feeding behavior of Drosophila serido on the yeast communities of necrotic stem tissue of Pilosocereus arrabidae were studied in a sand dune ecosystem of Rio de Janeiro, Brazil. The prevalence of cactophilic yeasts including Pichia barkeri, Candida sonorensis and Geotrichum sp. in the crops and external surfaces of D. serido reflected its association with the cactus habitat. The effective number of yeasts vectored on the surface of flies was higher than that in the crops. Also overlap between the yeasts from stems and from crops was partial suggesting selective feeding by the flies in the substrates visited. The females had a higher effective number of yeast species and a lower similarity than males with the yeast community of P. arrabidae. This was probably related to the search for oviposition sites by females. The presence of Pichia thermotolerans-like and Pichia amethionina var pachycereana in the flies, but not in P. arrabidae stems, indicated that D. serido was not limited to this cactus species. The larvae and adults lived in different patches with the adults feeding in patches with higher yeast species richness. The larvae had a narrower feeding niche and higher overlap with P. arrabidae, and preferred P. barkeri and Pichia cactophila as food. Adult flies fed on patches with the most frequent yeasts except for P. cactophila. Pichia caribaea was found in higher frequency in the adult crops than in the stems. Our data suggested that there was food selection and diet partitioning between adult and larval stages of D. serido.

Yeast communities of the cactus Pilosocereus arrabidae and associated insects in the Sandy coastal plains of southeastern Brazil.

The yeast communities from necrotic tissues, decaying flowers and fruits, and from larval feeding sites of the moth Sigelgaita sp. in the cactus Pilosocereus arrabidae were surveyed in three restinga ecosystems in Southeastern Brazil. Insects associated with these substrates were sampled to verify the vectoring of yeasts. The cactus Pilosocereus arrabidae was shown to have four different yeast communities associated with it. Necrotic stems had a diverse yeast community with the prevalent species Pichia barkeri, Candida sonorensis, Pichia cactophila, Geotrichum sp., Myxozyma mucilagina and Sporopachydermia sp. A, representing about 80% of the total isolates. Pichia sp. A and a Candida domercqii-like species represented more than 90% of the yeast isolates from decaying flowers. Fruits had a heterogeneous yeast community with typical fruit yeasts of the genus Kloeckera, basidiomicetous anamorphs of the genus Cryptococcus, the black yeast Aureobasidium pullulans, Pichia sp. A, a Candida domercqii-like species, and some cactophilic yeasts, especially Clavispora opuntiae. The feeding site of Sigelgaita sp. larvae had Clavispora opuntiae as the prevalent species. Insect vectors are suggested as one the most important factors influencing the composition of these yeast communities.

Yeasts and coliform bacteria of water accumulated in bromeliads of mangrove and sand dune ecosystems of southeast Brazil.

Yeasts and coliform bacteria were isolated from water that accumulated in the central cups and adjacent leaf axilae of two bromeliads, Neoregelia cruenta of a coastal sand dune and Quesnelia quesneliana of a mangrove ecosystem near the city of Rio de Janeiro, Brazil. The mean total coliform counts were above 10,000 per 100 mL for waters of both plants, but the mean fecal coliform counts were only 74 per 100 mL for Q. quesneliana and mostly undetected in water from N. cruenta. Of 90 fecal coliform isolates, 51 were typical of Escherichia coli in colony morphology and indol, methyl red, Volges-Proskauer, and citrate (IMViC) tests. Seven representatives of the typical E. coli cultures were identified as this species, but the identifications of nine other coliform bacteria were mostly dubious. The yeast community of N. cruenta was typical of plant surfaces with basidiomycetous yeasts anamorphs, and the black yeast Aureobasidium pullulans was prevalent. Quesnelia quesneliana had a substantial proportion of ascomycetous yeasts and their anamorphs, including a probable new biotype of Saccharomyces unisporus. Our results suggested that the microbial communities in bromeliad waters are typically autochtonous and not contaminants.

Phylogeny of Metschnikowia species estimated from partial rRNA sequences.

Phylogenetic relationships of species assigned to the genus Metschnikowia were estimated from the extents of divergence among partial sequences of rRNA. The data suggest that the aquatic species (Metschnikowia australis, Metschnikowia bicuspidata, Metschnikowia krissii, and Metschnikowia zobellii) and the terrestrial species (Metschnikowia hawaiiensis, Metschnikowia lunata, Metschnikowia pulcherrima, and Metschnikowia reukaufii) form two groups within the genus. M. lunata and M. hawaiiensis are well separated from other members of the genus, and M. hawaiiensis may be sufficiently divergent that it could be placed in a new genus. Species of the genus Metschnikowia are unique compared with other ascomycetous yeasts because they have a deletion in the large-subunit rRNA sequence that includes nucleotides 434 to 483.

Yeasts associated with Drosophila in tropical forests of Rio de Janeiro, Brazil.

The distribution and diversity of yeast species vectored by and from the crop of eight species groups of Drosophila is described for two rain forest sites and an urban wooded area in Rio de Janeiro, Brazil. The typical forest Drosophila groups guarani, tripunctata, and willistoni showed a higher diversity of yeasts than the cosmopolitan melanogaster species group, suggesting different strategies of utilization of substrates. Apiculate yeasts, including Kloeckera apis, Kloeckera javanica, and Kloeckera japonica, were the prevalent species. Geotrichum spp. and Candida citea were also frequent isolates in the forest sites. Similarities between the yeasts from the external surfaces and crops of Drosophila suggested that the feeding substrates were the main source of the yeasts vectored by these flies. Most of the yeasts were strong fermenters and assimilated few compounds, usually sucrose, cellobiose, and glycerol. This indicated a preference of the flies for food sources such as fruits. Some yeasts were primarily isolated from one group of Drosophila; for example, Kloeckera javanica from the melanogaster group, Debaryomyces vanrijiae var. yarrowii from the tripunctata group, and Kluyveromyces delphensis from the willistoni group. These associations and differences in the yeast communities among the fly groups suggested a differentiation of diets and specialization of the yeast-Drosophila association in the tropical forests.

Clavispora opuntiae and other yeasts associated with the moth Sigelgaita sp. in the cactus Pilosocereus arrabidae of Rio de Janeiro, Brazil.

Clavispora opuntiae was the prevalent yeast associated with the feeding sites of Sigelgaita sp. larvae in the cactus Pilosocereus arrabidae. Also associated with this habitat were Candida sonorensis, Pichia cactophila, Pichia barkeri, Candida sp. A, Geotrichum sp., Geotrichum sericeum and the yeast like organisms Prototheca zopfii and Acremonium sp. Atypical yeast biotypes that may represent new species of Pichia, Sporopachydermia and Candida were isolated. Mating types of Clavispora opuntiae were at a ratio 70 h- to 3 h- and reduced levels of sporulation suggested low pressure for sexual reproduction in this habitat. Sigelgaita sp. probably was not an important vector for Clavispora opuntiae because it was not isolated from an adult or eggs of this moth.

Yeasts associated with the white shrimp Penaeus schmitti, sediment, and water of Sepetiba Bay, Rio de Janeiro, Brasil.

Samples were collected in a polluted site with mean fecal coliform counts of the water being 9.0 x 10(2)/100 ml and less polluted areas including commercial fishing areas with fecal coliforms typically below 2.0 x 10(1)/100 ml. Yeast populations from the polluted site were 4.6 x 10(2) to 9.2 x 10(3)/g of shrimp intestine, 1.0 x 10(1) to 2.4 x 10(2)/g of sediment and 4.0 x 10(-1) to 4.6 x 10(1)/ml of water, and in the less polluted site 3.0 x 10(1) to 2.3 x 10(2)/g of intestines and 3.0 x 10(-1) to 2.4 x 10(1)/ml of water. About half of these yeast populations were fermentative. Prevalent yeasts isolated from shrimp were Candida famata, Candida intermedia, Candida tropicalis, Candida krusei, Geotrichum sp., Trichosporon sp., Debaryomyces hansenii, Rhodotorula rubra, Kloeckera apiculata, Cryptococcus sp., Hansenula anomala, and Candida spp. The species found in water and sediment were similar to those of shrimp except that C. tropicalis was more prevalent in sediment with 25% of the population and D. hansenii in water with 12%. Yeasts normally associated with warm blooded animals and sewage were predominant in the shrimp from the polluted site, but yeasts from the less polluted site were more typical of clean water. Yeasts were concentrated in the shrimp intestine suggesting that this is a favorable environment for them but no species were found to be selectively associated with this niche.

Microbial pollution indicators in Brazilian tropical and subtropical marine surface waters.

The specificity of indicators that depend on elevated incubation temperature as a selective factor for their enumeration was questioned because of the possibility of interference from autochthonous microorganisms adapted to high ambient temperatures. Lactose-fermenting cultures isolated from fecal coliform tests of tropical marine surface waters were identified as consisting of about 70% Escherichia coli, and most of the remaining cultures being Klebsiella, Enterobacter or Citrobacter species. This confirmed the taxonomic specificity of fecal coliform tests for these waters. Fecal and total coliforms, fecal streptococcus, heterotrophic bacteria and yeast counts had correlations of above 99% confidence levels with most other microbial and chemical parameters studied. Waters with fecal coliform counts above 1000 per 100 ml had increased incidence of presumptive pathogenic yeasts, Pseudomonas aeruginosa and Salmonella. Our data support the use of coliforms or fecal streptococci as indicators of recent fecal pollution in tropical marine waters and yeast or heterotrophic bacteria counts as complementary indicator methods for these waters.

DNA relatedness among aquatic yeasts of the genus Metschnikowia and proposal of the species Metschnikowia australis comb. nov.

DNA hybridization studies were conducted to determine the taxonomic status of the aquatic group of Metschnikowia species and their varieties. Among the DNAs of the four varieties of Metschnikowia bicuspidata, that of Metschnikowia bicuspidata var. australis showed 37 to 51% relative binding with the DNAs of Metschnikowia bicuspidata var. bicuspidata and of the varieties chathamia and californica. On this basis, low intervarietal fertility, and unique habitat in antarctic seawater, we have proposed to raise M. bicuspidata var. australis to the rank of species, Metschnikowia australis, comb. nov. DNA complementarity of Metschnikowia bicuspidata var. californica and Metschnikowia bicuspidata var. chathamia DNA was greater than 80% with that of M. bicuspidata var. bicuspidata. Metschnikowia australis can be differentiated from other Metschnikowia species and varieties by its inability to form chlamydospores, the formation of two needle-shaped ascospores per ascus, lack of glucose fermentation, and lack of assimilation of both methyl-alpha-D-glucoside and glucono-delta-lactone. Other DNA-DNA reassociation experiments showed that Metschnikowia zobellii is a distinct species when compared with both aquatic and terrestrial species of the genus.