Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico

Abstract Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.

Resumo Isla Arena está localizada na coordenada 20°70N - 90°45W, de Campeche, México. Nesses estuários, o oceano se mistura com a água doce e os ecossistemas se concentram onde proliferam petenos e flamingos rosa. Crustáceos e moluscos abundam no mar. Apesar de sua enorme riqueza marinha, não há estudos realizados sobre a presença de microrganismos halofílicos nessas águas. Neste trabalho, a diversidade e estrutura da comunidade microbiana foram investigadas através de uma abordagem metagenômica e corroboradas para o sequenciamento de genes 16S rRNA. Verificou-se que o filo Fimicutes predomina com mais de 50%, quase na mesma proporção da classe Bacilli e com quase 41% de abundância relativa da ordem Bacillales. Os resultados do sequenciamento mostraram que uma das amostras apresentou alto percentual de similaridade (99,75%) pelo programa Nucleotide BLAST com um microrganismo peculiar: Bacillus subtilis. Nossos resultados demonstram que B. subtilis pode ser uma fonte eficiente de proteases, lipases e celulases, provenientes de comunidades microbianas halofílicas localizadas em áreas pouco exploradas.

Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico / Identificação por técnicas moleculares de bactérias halofílicas que produzem enzimas importantes em área intocada de Campeche, México

Abstract Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.

Resumo Isla Arena está localizada na coordenada 20°70'N - 90°45'W, de Campeche, México. Nesses estuários, o oceano se mistura com a água doce e os ecossistemas se concentram onde proliferam petenos e flamingos rosa. Crustáceos e moluscos abundam no mar. Apesar de sua enorme riqueza marinha, não há estudos realizados sobre a presença de microrganismos halofílicos nessas águas. Neste trabalho, a diversidade e estrutura da comunidade microbiana foram investigadas através de uma abordagem metagenômica e corroboradas para o sequenciamento de genes 16S rRNA. Verificou-se que o filo Fimicutes predomina com mais de 50%, quase na mesma proporção da classe Bacilli e com quase 41% de abundância relativa da ordem Bacillales. Os resultados do sequenciamento mostraram que uma das amostras apresentou alto percentual de similaridade (99,75%) pelo programa Nucleotide BLAST com um microrganismo peculiar: Bacillus subtilis. Nossos resultados demonstram que B. subtilis pode ser uma fonte eficiente de proteases, lipases e celulases, provenientes de comunidades microbianas halofílicas localizadas em áreas pouco exploradas.

Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico / Identificação por técnicas moleculares de bactérias halofílicas que produzem enzimas importantes em área intocada de Campeche, México

Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.

Isla Arena está localizada na coordenada 20°70’N - 90°45’W, de Campeche, México. Nesses estuários, o oceano se mistura com a água doce e os ecossistemas se concentram onde proliferam petenos e flamingos rosa. Crustáceos e moluscos abundam no mar. Apesar de sua enorme riqueza marinha, não há estudos realizados sobre a presença de microrganismos halofílicos nessas águas. Neste trabalho, a diversidade e estrutura da comunidade microbiana foram investigadas através de uma abordagem metagenômica e corroboradas para o sequenciamento de genes 16S rRNA. Verificou-se que o filo Fimicutes predomina com mais de 50%, quase na mesma proporção da classe Bacilli e com quase 41% de abundância relativa da ordem Bacillales. Os resultados do sequenciamento mostraram que uma das amostras apresentou alto percentual de similaridade (99,75%) pelo programa Nucleotide BLAST com um microrganismo peculiar: Bacillus subtilis. Nossos resultados demonstram que B. subtilis pode ser uma fonte eficiente de proteases, lipases e celulases, provenientes de comunidades microbianas halofílicas localizadas em áreas pouco exploradas.

The Potential Use of Ectoine Produced by a Moderately Halophilic Bacteria Chromohalobacter salexigens KT989776 for Enhancing Germination and Primary Seedling of Flax “Linum usitatissimum L.” under Salinity Conditions

The similarity between plant and microbial cells encourage the use of microbial metabolites of halophilic bacteria for the alleviation of salt stress in plants. In the current research work, a compatible solute ectoine extracted from a moderately halophilic bacteria Chromohalobacter salexigens KT989776 was used to enhance flax germination and primary seedling under different levels of salinity. Two successive experiments including germination in Petri plates under six levels of salinity (0, 3, 5, 7, 9 and 11 dS.m-1) and a pot experiment under three irrigating water salinity levels (2, 3 and 4) with two types of ectoine application (spray and soil addition) were conducted. Germination parameters were recorded for the first experiment while a fresh and dry weight of plants and peroxidase activity in addition to sodium-potassium ratio were estimated in the pot experiment. Also, ectoine accumulation in plants was detected using HPLC. Results of LC-MS proved the production of ectoine by C. salexigens KT989776 and ectoine enhanced significantly all germination parameters of flax seeds, decreased sodium accumulation in the plant, increased potassium content, and lowered peroxidase and phenoloxidase activity. Also, HPLC analysis proved that ectoine was detected in all treated samples while not detected in non-treated control.

Bacterias halófilas con potencial para la recuperación de suelos salinizados en Sáchica-Boyacá, Colombia

Es posible usar bacterias halófilas aisladas de ambientes salinos como una posible alternativa de rehabilitación de suelos salinizados para la producción agrícola y agropecuaria. En este estudio se evaluaron seis bacterias halófilas para determinar su potencial en la disminución de la conductividad eléctrica (Ce) de suelo moderadamente salino del municipio de Sáchica- Boyacá (Colombia). Las bacterias se aislaron en agar nutritivo con diferentes concentraciones de NaCl, luego fueron inoculadas en el suelo (3 kg puesto en materas) para realizar un bioensayo en invernadero durante 2 meses. Se realizaron dos fases, la primera hasta conseguir un valor de 2 ds/m (Ce) con el fin de sembrar plantas de tomate en la segunda fase. Se realizaron mediciones semanales de la (Ce) durante todo el bioensayo y solo en la fase 2 se determinaron semanalmente variables cualitativas de crecimiento de tomate (altura, número de hojas, área foliar, marchitez y clorosis). Los resultados mostraron un efecto directo de las bacterias halófilas en la disminución de la (Ce) del suelo, pasando de 5.2 a 0.8 ds/m (T1), 0.7 ds/m (T2), 0.8 ds/m (T3), 0.6 ds/m (T4), 0.7 ds/m (T5), 0.7 ds/m (T6), 3.0 ds/m (testigo), valores que corresponden a salinidad muy ligera (< 2 ds/m). En relación con las variables agronómicas, se evidenciaron mejoras en las características evaluadas en comparación con el testigo absoluto, sobre todo en altura y número de hojas de las plantas. Para el testigo absoluto se perdieron 80 % de las unidades experimentales, mientras que en el suelo con los diferentes tratamientos solo en algunos casos se perdió el 20 %. De acuerdo con esto, se evidencia que las bacterias halófilas utilizadas en esta investigación tienen potencial para la rehabilitación de suelos salinizados.

It is possible to use halophilic bacteria isolated from saline environments as a possible alternative for the rehabilitation of salinated soils for agricultural and agricultural production. Six halophilic bacteria were evaluated to determine their potential in the reduction of the electrical conductivity (Ce) of moderately saline soil in the municipality of Sáchica-Boyacá (Colombia). The bacteria were isolated on nutritive agar with different concentrations of NaCl, then inoculated in the soil (3 kg placed in pots) to perform a bioassay in the greenhouse for 2 months. Two phases were carried out, the first until obtaining a value of 2 ds / m (Ce) in order to plant tomato plants in the second phase. Weekly measurements of the ( Ce ) were made throughout the bioassay and only in phase 2 were determined weekly qualitative variables of tomato growth (height, number of leaves, foliar area, wilting and chlorosis). The results showed a direct effect of the halophilic bacteria in the decrease of the (Ce) of the soil, going from 5.2 to 0.8 ds / m (T1), 0.7 ds / m (T2), 0.8 ds / m (T3), 0.6 ds / m (T4), 0.7 ds / m (T5), 0.7 ds / m (T6), 3.0 ds / m (Control), values that correspond to very light salinity (< 2 ds / m). Regarding the agronomic variables, improvements were observed in the characteristics evaluated in comparison with the absolute control, especially in height and number of leaves of the plants. For the absolute control 80 % of the experimental units were lost, while in the soil with the different treatments only in some cases 20 % was lost. Accordingly, it is evident that the halophilic bacteria used in this research have potential for the rehabilitation of salinated soils.

Bioprospection of proteases from Halobacillus andaensis for bioactive peptide production from fish muscle protein

BACKGROUND: Biologically active peptides produced from fish wastes are gaining attention because their health benefits. Proteases produced by halophilic microorganisms are considered as a source of active enzymes in high salt systems like fish residues. Hence, the aim of this study was the bioprospection of halophilic microorganisms for the production of proteases to prove their application for peptide production. RESULTS: Halophilic microorganisms were isolated from saline soils of Mexico and Bolivia. An enzymatic screening was carried out for the detection of lipases, esterases, pHB depolymerases, chitinases, and proteases. Most of the strains were able to produce lipases, esterases, and proteases, and larger hydrolysis halos were detected for protease activity. Halobacillus andaensis was selected to be studied for proteolytic activity production; the microorganism was able to grow on gelatin, yeast extract, skim milk, casein, peptone, fish muscle (Cyprinus carpio), and soy flour as protein sources, and among these sources, fish muscle protein was the best inducer of proteolytic activity, achieving a protease production of 571 U/mL. The extracellular protease was active at 50°C, pH 8, and 1.4 M NaCl and was inhibited by phenylmethylsulfonyl fluoride. The proteolytic activity of H. andaensis was used to hydrolyze fish muscle protein for peptide production. The peptides obtained showed a MW of 5.3 kDa and a radical scavenging ability of 10 to 30% on 2,2-diphenyl-1-picrylhydrazyl and 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and a ferric reducing ability of plasma. Conclusion: The use of noncommercial extracellular protease produced by H. andaensis for biologically active peptide production using fish muscle as the protein source presents a great opportunity for high-value peptide production.

Diversity of culturable moderately halophilic bacteria producing extracellular hydrolytic enzymes from marine sediments / Diversidade de bactérias cultiváveis moderadamente halofílicas que produzem enzimas hidrolíticas extracelulares de sedimentos marinhos

A total of 114 moderately halophilic bacteria were isolated from marine sediment environments. The isolates are belonged to 23 species based on the 16S rRNA sequence analysis. 63, 52, 47, 57, 74, 15 and 4 isolates are able to produce protease, amylase, lipase, pectinase, pulluanase, xylanase, cellulase, respectively. Combined hydrolytic enzyme activity analysis show that 15 strains present 1 hydrolytic activity, 32 strains present 2 hydrolytic activities, 21 strains present 3 hydrolytic activities, 26 strains present 4 hydrolytic activities, 11 strains present 5 hydrolytic activities and 2 strains present 6 hydrolytic activities. Hydrolase activities are widely distributed in a variety of species. The highest rates for production of protease, amylase, lipase, pectinase, pullanase, xylanase and cellulase were observed in species of B. baekryungensis, Hallobacillus sp., B. pumilus, B. megaterium or P. chungwhensis, B. amyloliquefaciens, B. pumilus, B. baekryungensis, respectively. However, the higher activities of protease, pectinase and pulluanase are frequently produced by the species of Halomonas sp. B. amyloliquefaciens or P. chungwhensis, and Vibrio sp. respectively. This investigation show that the diversity of halophilic bacteria from marine sediments could serve as a potential source of hydrolytic enzymes for industrial applications. (AU)

Um total de 114 bactérias moderadamente halofílicas foram isoladas de ambientes de sedimentos marinhos. Os isolados pertencem a 23 espécies com base na análise da sequência 16S rRNA. 63, 52, 47, 57, 74, 15 e 4 isolados são capazes de produzir protease, amilase, lipase, pectinase, pululanase, xilanase, celulase, respectivamente. A análise da atividade enzimática hidrolítica combinada mostra que 15 cepas apresentam 1 atividade hidrolítica, 32 cepas apresentam 2 atividades hidrolíticas, 21 cepas apresentam 3 atividades hidrolíticas, 26 cepas apresentam 4 atividades hidrolíticas, 11 cepas apresentam 5 atividades hidrolíticas e 2 cepas apresentam 6 atividades hidrolíticas. Atividades de hidrolase são amplamente distribuídas em uma variedade de espécies. As maiores taxas de produção de protease, amilase, lipase, pectinase, pululanase, xilanase e celulase foram observadas em espécies de B. baekryungensis, Hallobacillus sp., B. pumilus, B. megaterium ou P. chungwhensis, B. amyloliquefaciens, B. pumilus, B. baekryungensis, respectivamente. No entanto, as atividades mais elevadas de protease, pectinase e pululanase são freqüentemente produzidas pelas espécies de Halomonas sp. B. amyloliquefaciens ou P. chungwhensis e Vibrio sp. respectivamente. Esta investigação mostra que a diversidade de bactérias halofílicas de sedimentos marinhos pode servir como uma fonte potencial de enzimas hidrolíticas para aplicações industriais. (AU)

Proteolytic activity of recombinant DegP from Chromohalobacter salexigens BKL5

Background: DegP is a serine protease that specifically cleaves and refolds unfolding proteins in the periplasmic space of the cells. To date, there is no information regarding DegP from halophilic bacteria. Chromohalobacter salexigens BKL5 is a moderately halophilic bacterium that has the ability to grow in a media containing more than 15% salt. Therefore, the objectives of this work were to clone and overexpress DegP-encoding gene from C. salexigens BKL5 and characterize its biochemical properties. Results: DegP-encoding gene was overexpressed in Escherichia coli BL21(DE3) CodonPlus in an active form. SDS-PAGE analysis showed that the molecular weight of the recombinant DegP was 45 kDa. Size-exclusion chromatography analysis suggested that recombinant DegP was present in two multimeric states, hexameric and dodecameric, with molecular weights of 297.9 and 579.12 kDa, respectively. Both conformations were enzymatically active when casein was used as substrate for enzymatic assay. Circular dichroism analysis showed that recombinant DegP was composed of 0.21­0.29 helical content, which was comparable to the helical content in the crystal structure of E. coli DegP. The basic/acidic residue ratio of recombinant DegP was 0.56, which was slightly higher than that of DegP from extreme halophiles (average, 0.45) but significantly lower than that of DegP from nonhalophiles (average, 0.94). Conclusions: Recombinant DegP from C. salexigens BKL5 showed proteolytic activity when ß-casein was used as a substrate. In silico analysis indicated that recombinant DegP had characteristics similar to those of halophilic proteins depending on its amino acid composition.

Alleviation of salt stress by halotolerant and halophilic plant growth-promoting bacteria in wheat (Triticum aestivum)

ABSTRACT In the current study, 18 halotolerant and halophilic bacteria have been investigated for their plant growth promoting abilities in vitro and in a hydroponic culture. The bacterial strains have been investigated for ammonia, indole-3-acetic acid and 1-aminocyclopropane-1-carboxylate-deaminase production, phosphate solubilisation and nitrogen fixation activities. Of the tested bacteria, eight were inoculated with Triticum aestivum in a hydroponic culture. The investigated bacterial strains were found to have different plant-growth promoting activities in vitro. Under salt stress (200 mM NaCl), the investigated bacterial strains significantly increased the root and shoot length and total fresh weight of the plants. The growth rates of the plants inoculated with bacterial strains ranged from 62.2% to 78.1%.Identifying of novel halophilic and halotolerant bacteria that promote plant growth can be used as alternatives for salt sensitive plants. Extensive research has been conducted on several halophilic and halotolerant bacterial strains to investigate their plant growth promoting activities. However, to the best of my knowledge, this is the first study to inoculate these bacterial strains with wheat.

A rare glimpse into the morbid world of necrotising fasciitis: Flesh‑eating bacteria Vibrio vulnificus.

Necrotising fasciitis is one of the fatal skin and soft tissue infections. Vibrio vulnificus is a rare cause of necrotising fasciitis; however, the disease is one of the major manifestations of the bacteria. Here, we report one such case in a middle‑aged male patient. He presented with the signs of bilateral lower limb cellulitis and altered sensorium. V. vulnificus was isolated from blood culture and also from debrided tissue. Though the organism is well characterised, it is a rare causative agent of necrotising fasciitis. This case is a re‑emphasis on active look out for this bacterium in patients presenting with necrotizsing fasciitis.

Biology of Boeckella poopoensis Marsh, 1906 (Copepoda, Calanoida) in natural conditions in temporary saline lakes of the central Argentina / Biología de Boeckella poopoensis Marsh, 1906 (Copepoda, Calanoida) en condiciones naturales en lagos salinos temporarios del centro de Argentina

Boeckella poopoensis Marsh, 1906 is the dominant copepod in saline lakes from northern Patagonia to southern Peru. It is a tolerant species, which has been registered at salinities between 20 and 90 g.L-1, and is important because it integrates the diet of flamingos and fishes of commercial and sport interest. The aims of this study were to analyze the characteristics of populations of B. poopoensis in the central region of Argentina and to establish their relationships with environmental parameters. Monthly samples during 2007 were taken in four temporary lakes of La Pampa province. Environmental parameters and density, size, biomass, number and size of eggs were determined. The salinity ranged between 13.38 and 32.90 g.L-1. In three lakes that had previously continuously contained water, B. poopoensis was registered throughout the whole study. In the fourth lake, which was filled in January, it was present only when salinity exceeded 15 g.L-1. The population of the lake that was filled differed from that of the other lakes in terms of the density and biomass of adults and copepodites. The number of ovigerous females represented a higher percentage of the population during the colonization of the lake that had been dried and these produced the highest number of eggs. In the three lakes in which B. poopoensis was always recorded, its characteristics were more influenced by the availability of food than by temperature or salinity. It was found that the strategies of the species vary throughout the hydroperiod; at the beginning, thrives when the salinity rises and impedes the presence of less tolerant species. At this point, the production of relatively small eggs is high, allowing rapid colonization. When the lakes become relatively stable, B. poopoensis allocates more energy to reach larger sizes and although egg production is not so high, they are larger, allowing it to maintain stable populations.

Boeckella poopoensis Marsh, 1906 es el copépodo dominante en lagos salinos desde el norte de Patagonia hasta el sur del Perú. Es una especie halotolerante registrada con salinidades entre 20 y 90 g.L-1 y es importante dado que integra la dieta de flamencos y de peces de interés comercial o deportivo. El objetivo del estudio fue analizar características de poblaciones de B. poopoensis en la región central de Argentina y establecer sus relaciones con los parámetros ambientales. Durante 2007 se tomaron muestras mensuales en cuatro lagos temporarios de la provincia de La Pampa. Se determinaron parámetros ambientales y la densidad, espectro de tallas, biomasa, número y tamaão de los huevos. La salinidad varió entre 13,38 and 32,90 g.L-1. En tres lagos que habían contenido agua en forma continua, B. poopoensis se registró durante todo el estudio. En el cuarto, que se llenó en enero, sólo estuvo presente cuando la salinidad superó 15 g.L-1. La densidad y biomasa de los adultos y copepoditos de la población del lago que se llenó difirieron de las de los otros. El número de hembras ovígeras representó un mayor porcentaje del total de la población durante la colonización del lago que había estado seco y fueron las que produjeron el mayor número de huevos. En los tres lagos en los que B. poopoensis se registró siempre, sus características fueron influidas más por la disponibilidad de alimento que por la temperatura o salinidad. Este estudio mostró que las estrategias de la especie varían a lo largo del hidroperíodo; al principio, prospera cuando la salinidad aumenta e impide la presencia de especies menos tolerantes. En este momento, la producción de abundantes huevos relativamente pequeãos permite una rápida colonización. Cuando los lagos alcanzan una relativa estabilidad, B. poopoensis destina más energía a alcanzar tamaãos mayores y, aunque la producción de huevos no es tan elevada son más grandes, permitiéndole mantener poblaciones estables.

Topological analysis of carbon flux during multi-stress adaptation in Halomonas sp. AAD12

Background Osmolytes with their effective stabilizing properties are accumulated as protectants not only against salinity but also against denaturing harsh environmental stresses such as freezing, drying, high temperatures, oxygen radicals and radiation. The present work seeks to understand how Halomonas sp. AAD12 cells redirect carbon flux specifically to replenish reactions for biomass and osmolyte synthesis under changing salinity and temperature. To accomplish this goal, a combined FBA-PCA approach has been utilized. Results Experimental data were collected to supply model constraints for FBA and for the verification of the model predictions, which were satisfactory. With restrictions on the various combinations of selected anaplerotic paths (reactions catalyzed by phosphoenolpyruvate carboxylase, pyruvate carboxylase or glyoxylate shunt), two major phenotypes were found. Moreover, under high salt concentrations, when the glucose uptake rate was over 1.1 mmoL DCW- 1 h- 1, an overflow metabolism that led to the synthesis of ethanol caused a slight change in both phenotypes. Conclusions The operation of the glyoxylate shunt as the major anaplerotic pathway and the degradation of 6-phosphogluconate through the Entner-Doudoroff Pathway were the major factors in causing a distinction between the observed phenotypes.

Halotolerant bacteria in the São Paulo Zoo composting process and their hydrolases and bioproducts

Halophilic microorganisms are able to grow in the presence of salt and are also excellent source of enzymes and biotechnological products, such as exopolysaccharides (EPSs) and polyhydroxyalkanoates (PHAs). Salt-tolerant bacteria were screened in the Organic Composting Production Unit (OCPU) of São Paulo Zoological Park Foundation, which processes 4 ton/day of organic residues including plant matter from the Atlantic Rain Forest, animal manure and carcasses and mud from water treatment. Among the screened microorganisms, eight halotolerant bacteria grew at NaCl concentrations up to 4 M. These cultures were classified based on phylogenetic characteristics and comparative partial 16S rRNA gene sequence analysis as belonging to the genera Staphylococcus, Bacillus and Brevibacterium. The results of this study describe the ability of these halotolerant bacteria to produce some classes of hydrolases, namely, lipases, proteases, amylases and cellulases, and biopolymers. The strain characterized as of Brevibacterium avium presented cellulase and amylase activities up to 4 M NaCl and also produced EPSs and PHAs. These results indicate the biotechnological potential of certain microorganisms recovered from the composting process, including halotolerant species, which have the ability to produce enzymes and biopolymers, offering new perspectives for environmental and industrial applications.

Culturable diversity of halophilic bacteria in foreshore soils

Halophilic bacteria are commonly found in natural environments containing significant concentration of NaCl such as inland salt lakes and evaporated sea-shore pools, as well as environments such as curing brines, salted food products and saline soils. Dependence on salt is an important phenotypic characteristic of halophilic bacteria, which can be used in the polyphasic characterization of newly discovered microorganisms. In this study the diversity of halophilic bacteria in foreshore soils of Daecheon, Chungnam, and Saemangeum, Jeonbuk, was investigated. Two types of media, namely NA and R2A supplemented with 3%, 5%, 9%, 15%, 20% and 30% NaCl were used. More than 200 halophilic bacteria were isolated and BOX-PCR fingerprinting analysis was done for the typing of the isolates. The BLAST identification results showed that isolated strains were composed of 4 phyla, Firmicutes (60%), Proteobacteria (31%), Bacteriodetes (5%) and Actinobacteria (4%). Isolates were affiliated with 16 genera and 36 species. Bacillus was the dominant genus in the phylum Firmicutes, comprising 24% of the total isolates. Halomonas (12%) and Shewanella (12%) were also found as the main genera. These findings show that the foreshore soil of Daecheon Beach and Saemangeum Sea of Korea represents an untapped source of bacterial biodiversity.

Evaluation of Halobacterial Extracellular Hydrolytic Activities in Several Natural Saline and Hypersaline Lakes from Romania.

This work presents the evaluation of extracellular hydrolytic activities involved in the degrading of organic compounds from several natural salt lakes located in the southern part of Romania. The paper approaches the extracellular enzymatic decomposition activity in accordance with several physico-chemical parameters and microbial abundance. The chloride content was 13 g/l in Balta Albă salt lake, around 15 – 17 g/l in Amara salt lake, 80 g/l in Movila Miresei hypersaline lake and close to saturation, 252 g/l respectively in the case of Ocnele Mari hypersaline sample. The chemical composition revealed that salinity is due to compounds of sulphur, chloride and potassium in lakes Balta Alba and Amara. In Movila Miresei, potassium is replaced by sodium and in Ocnele Mari the salinity is due mainly to chloride and sodium. The enzyme α –glucosidase showed high values in the hypersaline lakes Movila Miresei and Ocnele Mari and a similar profile was recorded also for β-glucosidase. The activity of the enzyme alkaline phosphatase is directly correlated with the chloride content. The enzyme aminopeptidase showed high values if compared with previously mentioned enzymes.

Extracellular proteases of Halobacillus blutaparonensis strain M9, a new moderately halophilic bacterium

Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9), a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i) molecular masses ranging from 30 to 80 kDa, (ii) better hydrolytic activities under neutral-alkaline pH range, (iii) expression modulated according to the culture age, (iv) susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v) specific cleavage over the chymotrypsin substrate, and (vi) enzymatic stability in the presence of salt (up to 20% NaCl) and organic solvents (e.g., ether, isooctane and cyclohexane). The proteases described herein are promising for industrial practices due to its haloalkaline properties.

Production of Extracellular Enzymes by Halophilic Bacteria Isolated from Solar Salterns.

During the course of survey of halophilic microorganisms, a total of sixteen bacterial isolates were obtained from coastal solar salterns of Orissa and West Bengal, India. Morphological, physiological and biochemical characteristics of these isolates indicate that majority of them belong to the genus Halomonas, however, members belonging to Cobetia and Halococcus were not uncommon. These isolates were screened for the production of extracellular enzymes such as amylase, glutaminase, asparaginase, xylanase, cellulase, gelatinase, inulinase, caseinase, pectinase, urease and lipase. Among these hydrolytic enzymes, glutamine and asparagine hydrolytic activities were predominant, although lipid and casein degrading activities were not inferior. However, amylase and gelatinase production were rare. None of these halophiles was able to degrade cellulose, inulin, pectin and xylan and only one isolate was capable of hydrolyzing urea.

Degradation of 4-Chlorophenol by a Moderately Halophilic Bacterial Consortium under Saline Conditions.

Aims: To investigate the degradation of 4-chlorophenol by a moderately halophilic bacterial consortium and to study the effect of nitrogen sources on the degradation of 4- chlorophenol. Study Design: 4-chlorophenol degradation by moderate halophiles. Place and Duration of Study: Centre for Environmental studies, Microbiology Lab, Anna University, Chennai, Tamil Nadu, between Jan 2009 and May 2009. Methodology: 4-chlorophenol was degraded with the moderately halophilic bacterial consortium in the minerals salts medium and degradation was analysed by gas chromatography. Effect of NaCl concentrations and alternate nitrogen sources on the degradation of 4-chlorophenol was determined. Results: The bacterial consortium was able to degrade 4-Chlorophenol at a range of NaCl concentrations, where the optimum degradation was obtained at 50 g/L of NaCl. Addition of alternate nitrogen sources like tryptone and urea did not enhance the degradation of 4- Chlorophenol. Conclusion: Bacterial consortium degraded 4-chlorophenol at optimum NaCl concentration of 50 g/L of NaCl. Addition of yeast extract as nitrogen source showed higher degradation than the nitrogen sources.

Isolation and Characterization of Secondary Metabolites from Halophilic Bacillus Species from Marin drive in Mumbai.

The present study was focussed on production of novel antibiotics from Halophilic bacterial species in Marin drive Mumbai. Bacillus pumilus were isolated from soil and screened for the production of antibiotics by plate assay and then cultured in shake flask fermentation at 30ºC for further studies. The bioactive secondary metabolites producing bacterial isolates were studied for their ability to tolerate 3% NaCl. Identification of Bacillus pumilus strains was done by using biochemical test as well as 16S r-RNA sequencing method. Identification of antibiotics was done by column chromatography as well as thin layer chromatography. Antibiotics was found to be produced by three are bacterial and one are fungal pathogen strains against E-coli (ATCC#2939), Staphylococcus aureus (ATCC# 96), Pseudomonas aeruginosa (ATCC# 2488) and one are fungal pathogen strains against Candida albicans (ATCC# 227) proved to be resistant to antibiotics produced by Bacillus pumilus. The maximum production of antibiotics from Bacillus pumilus against E-coli, Staphylococcus aureus and Candida albicans. Maximum zones of inhibition were observed after 48 hours of incubation at 30ºC against E-coli, Staphylococcus aureus and Candida albicans. After that structural elucidation was done by using IR, MS and NMR spectroscopy respectively.

Expression analysis of genes responsible for amino acid biosynthesis in halophilic bacterium Salinibacter ruber.

The degeneracy of the genetic code allows for multiple codons to encode the same amino acid. However, alternative codons and amino acids are used unevenly among genes, a phenomenon termed codon-usage bias. Genes regulating amino acid biosynthesis of Salinibacter ruber, an extremely halophilic bacterium were studied in order to determine the synonymous codon usage patterns. Factors responsible for codon usage variation among the genes were investigated using codon usage indices and multi-variate statistical approach. Overall codon usage data analysis indicated that codons ending in G and/or C were predominant among the genes. Multi-variate statistical analysis showed that there was a single major trend in the codon usage variation among the genes, which had a strong positive correlation (r = 0.93, P<0.01) with (G + C) content of the genes. Further, correlation analysis indicated that genes with higher expression level and showing a greater degree of codon usage bias were GC-rich and preferred codons with C or G nucleotides at the third position. A set of thirteen codons were identified through Chi-square test as optimal codons, which were preferred in highly expressed genes. It could be concluded that mutational bias had a profound effect on codon usage pattern. In addition, translational selections also operated with a proper balance, making the genes translationally more efficient. The frequency of these codons appeared to be correlated with the level of gene expression and might be a useful indicator in the case of genes (or open-reading-frames) whose expression levels are unknown.