Diversity and industrial potential of hydrolaseproducing halophilic/halotolerant eubacteria

Halophilic and haloterant eubacteria have been isolated from different marine and hypersaline environments. Halophilic eubacteria also occur in environments typified by more than one soda lakes which are both hypersaline and extremely alkaline. These organisms have been shown to produce a wide array of hydrolytic enzymes including proteases, amylases, xylanases, cellulases as well as lipases and DNases. These enzymes are commonly applied in the production of fermented food and food supplements, in animal feed, laundry detergents and textile industries. Several studies have shown that enzymes derived from halophilic and halotolerant eubacteria are not only halostable but may also be thermostable and alkalistable. This extremophilicity make the enzymes suitable candidates in various fields of biotechnology and may even open up new application opportunities

Isolation of hydrolase producing bacteria from Sua pan solar salterns and the production of endo-1, 4-bxylanase from a newly isolated haloalkaliphilic Nesterenkonia sp.

Eighty seven bacterial isolates were obtained from evaporator ponds using culture enrichment technique and screened for xylanase, mannanase and cellulase activity. Based on biochemical and phenotypic characteristics, the isolates were divided into 18 groups. Thirteen groups were Bacillusspecies, four were Halomonas species, while one group belonged to the genus Nesterenkonia. Four Bacillus isolates, Sua-BAC005, Sua-BAC012, Sua-BAC017 and Sua-BAC019, as well as Nesterenkonia sp. Sua-BAC020 were studied further. Isolate Sua-BAC005 affiliated with Bacillus amyloliquefaciens secreted 12.6 U/ml and 9.0 U/ml b-mannanase and b-xylanase, respectively, while isolates Sua-BAC012, Sua-BAC017 and Sua-BAC019 affiliated with Bacillus licheniformis, produced less than 2 U/ml of xylanase, cellulase and mannanase. Nesterenkonia sp. Sua-BAC020 grew at 0 – 20% NaCl with anoptimum at 2.5% NaCl, and at pH 7 – 9.5 with an optimum at pH 9. This isolate produced 3.5 U/ml xylanase when cultivated at pH 8 in 10% NaCl. Five xylanase activity bands were detected on Native-PAGE coupled with zymogram

Yeast biodiversity in vineyard environments is increased by human intervention

One hundred and five grape samples were collected during two consecutive years from 33 locations on seven oceanic islands of the Azores Archipelago. Grape samples were obtained from vineyards that were either abandoned or under regular cultivation involving common viticultural interventions, to evaluate the impact of regular human intervention on grape yeast biota diversity in vineyards. A total of 3150 yeast isolates were obtained and 23 yeast species were identified. The predominant species were Hanseniaspora uvarum, Pichia terricola, Starmerella bacillaris and Issatchenkia hanoiensis. The species Barnettozyma californica, Candida azymoides and Pichia cecembensis were reported in grapes or wine-associated environments for the first time. A higher biodiversity was found in active vineyards where regular human intervention takes place (Shannon index: 1.89 and 1.53 in the first and second years, respectively) when compared to the abandoned ones (Shannon index: 0.76 and 0.31). This finding goes against the assumptions that human intervention can destroy biodiversity and lead to homogeneity in the environment. Biodiversity indices were considerably lower in the year with the heaviest rainfall. This study is the first to report on the grape yeast communities from several abandoned vineyards that have undergone no human intervention.Joao Drumonde Neves is the recipient of a fellowship of the Azorean Government (M321/006/F/2008) and PROEMPREGO. This work was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI), and by national funds through FCT by the projects FCOMP-01-0124-008775, PTDC/AGR-ALI/103392/2008 and PTDC/AGR-ALI/121062/2010.info:eu-repo/semantics/publishedVersio

Complexity and dynamics of the winemaking bacterial communities in berries, musts, and wines from apulian grape cultivars through time and space

Currently, there is very little information available regarding the microbiome associated with the wine production chain. Here, we used an amplicon sequencing approach based on high-throughput sequencing (HTS) to obtain a comprehensive assessment of the bacterial community associated with the production of three Apulian red wines, from grape to final product. The relationships among grape variety, the microbial community, and fermentation was investigated. Moreover, the winery microbiota was evaluated compared to the autochthonous species in vineyards that persist until the end of the winemaking process. The analysis highlighted the remarkable dynamics within the microbial communities during fermentation. A common microbial core shared among the examined wine varieties was observed, and the unique taxonomic signature of each wine appellation was revealed. New species belonging to the genus Halomonas were also reported. This study demonstrates the potential of this metagenomic approach, supported by optimized protocols, for identifying the biodiversity of the wine supply chain. The developed experimental pipeline offers new prospects for other research fields in which a comprehensive view of microbial community complexity and dynamics is desirable.Peer ReviewedPostprint (published version

Association between Grape Yeast Communities and the Vineyard Ecosystems

The grape yeast biota from several wine-producing areas, with distinct soil types and grapevine training systems, was assessed on five islands of Azores Archipelago, and differences in yeast communities composition associated with the geographic origin of the grapes were explored. Fifty-seven grape samples belonging to the Vitis vinifera grapevine cultivars Verdelho dos Acores (Verdelho), Arinto da Terceira (Arinto) and Terrantez do Pico (Terrantez) were collected in two consecutive years and 40 spontaneous fermentations were achieved. A total of 1710 yeast isolates were obtained from freshly crushed grapes and 1200 from final stage of fermentations. Twenty-eight species were identified, Hanseniaspura uvarum, Pichia terricola and Metschnikowia pulcherrima being the three most representative species isolated. Candida carpophila was encountered for the first time as an inhabitant of grape or wine-associated environments. In both sampling years, a higher proportion of H. uvarum in fresh grapes from Verdelho cultivar was observed, in comparison with Arinto cultivar. Qualitatively significant differences were found among yeast communities from several locations on five islands of the Archipelago, particularly in locations with distinctive agro-ecological compositions. Our results are in agreement with the statement that grape-associated microbial biogeography is non-randomly associated with interactions of climate, soil, cultivar, and vine training systems in vineyard ecosystems. Our observations strongly support a possible linkage between grape yeast and wine typicality, reinforcing the statement that different viti-cultural terroirs harbor distinctive yeast biota, in particular in vineyards with very distinctive environmental conditions.Joao Drumonde Neves is the recipient of a fellowship of the Azorean Government (M321/006/F/2008) and PROEMPREGO. This work was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI), and by national funds through FCT by the projects FCOMP-01-0124-008775, PTDC/AGR-ALI/103392/2008 and PTDC/AGR-ALI/121062/2010.info:eu-repo/semantics/publishedVersio

A new method for the detection of early contamination of red wine by Brettanomyces bruxellensis using Pseudomonas putida 4-ethylphenol methylene hydroxylase (4-EPMH)

Brettanomyces/Dekkera bruxellensis is a cause of major concern for the winemaking industry worldwide. If a slight presence of this spoilage yeast in red wine adds a Brett character, a strong contamination has irreversible and detrimental effects on the organoleptic qualities due to the production of volatile phenols such as 4-ethylphenol. Time is a key factor in the treatment of B. bruxellensis contaminations. Nowadays, the diagnostic and quantification resources available are time consuming and too expensive, making them either inadequate or inaccessible to most of the winemakers. This study was focused on a new, easy to use, inexpensive method that could allow winemakers to directly detect B. bruxellensis contamination in red wine at an early stage, hence, reducing wine spoilage. In this work, the ability of Pseudomonas putida 4-ethylphenol methylene hydroxylase was tested in order to catabolize the 4-ethylphenol and to elaborate an enzymatic assay with the purpose of detecting early contaminations by B. bruxellensis in red wine. We have developed a colorimetric enzymatic assay, based on the redox state of the 4-ethylphenol methylene hydroxylase co-factor, cytochrome C, that can detect and quantify low concentrations of 4-ethylphenol. The range of concentrations detected is well below the level detectable by the human nose. Combined to an enrichment step, this method allows the detection of B. bruxellensis at an initial concentration of less than 10 cells per ml

Partial purification and characterization of endo-&#946-1,4- mannanases from Scopulariopsis candida strains isolated from solar salterns

Scopulariopsis candida strains LMK004 and LMK008 previously isolated from a solar saltern were cultivated in Vogel’s medium supplemented with NaCl and locust bean gum galactomannan as carbonsource and inducer for b-mannannase production. S. candida LMK004 produced up to 180 nkat/ml whereas LMK008 produced 116 nkat/ml. These levels dropped significantly when a-cellulose was usedas carbon source. Both enzymes were partially purified by ammonium sulphate precipitation and anionexchange chromatography. The molecular mass of LMK004 and LMK008 b-mannanases were estimatedto be 41 and 28 kDa, respectively. The b-mannanase from LMK004 was most active at pH 5 and 50°C, and retained ³ 80% of its activity at pH 5 – 6.5 after 24 h of incubation at 4°C. In contrast, the LMK008 bmannanase retained ³ 60% activity between pH 6 – 7. Both enzymes remained stable for 3 h between 30 and 40°C, and showed loss of activity at higher temperatures. The LMK008 b-mannanase tolerated high NaCl concentrations with 70% activity remaining after incubation for 2 h at 20% NaCl, whereas the LMK004 b-mannanase was only active between 0 - 10% NaCl. The current study shows that fungi thatinhabit hypersaline environments produce plant cell wall degrading enzymes that display similar properties to other fungi from low-salt environments

Assessment of alkaliphilic haloarchaeal diversity in Sua pan evaporator ponds in Botswana

Cultivation-dependent and molecular-based culture-independent methods were used to assess alkaliphilic haloarchaeal diversity at Sua pan evaporator ponds in Botswana. Isolates belonging to thegenera Natrialba, Natronococcus and Natronorubrum were recovered from brine samples by enrichment and identified through a series of biochemical tests as well as sequencing of 16S rRNA fragments. In addition, an environmental 16S rRNA library was constructed from brine samples of two evaporator ponds. The library comprised members of the genera Halorubrum (65%), Natrialba (14%), Natronorubrum (7%) and new phylotypes (14%). The new phylotypes consisted of two clones that exhibited low 16S rRNA similarity (95 – 97%) with known species and could potentially represent new species in the genus Halorubrum, one clone with 91% similarity to Natronolimnobius which couldrepresent a new genus, as well as an unidentifiable phylotype which exhibited 79% similarity to Methanotorris formicicus. Two major differences were observed between cultivation- and molecularbasedmethods; firstly, Halorubrum species were largely represented in the environmental clone library but no isolates were obtained, and secondly, Natronococcus species were isolated but not detected inthe clone library. An overlap between the archaeal isolates and the ribosomal library clones was apparent although the novel phylotypes detected in this study were not recovered through cultivatio

<I>Nesterenkonia suensis </I>sp. Nov., a haloalkaliphilic actinobacterium isolated from a salt pan

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