Reduction in Salt Stress Due to the Action of Halophilic Bacteria That Promote Plant Growth in Solanum lycopersicum.

Soil salinity is one of the most important factors reducing agricultural productivity worldwide. Halophilic plant growth-promoting bacteria (H-PGPB) represent an alternative method of alleviating saline stress in crops of agricultural interest. In this study, the following halophilic bacteria were evaluated: Bacillus sp. SVHM1.1, Halomonas sp. SVCN6, Halomonas sp. SVHM8, and a consortium. They were grown under greenhouse conditions in Solanum lycopersicum at different salinity concentrations in irrigation water (0, 20, 60, and 100 mM NaCl) to determine the effects on germination, fruit quality, yield, and concentration of osmoprotectors in plant tissue. Our results demonstrate the influence of halophilic bacteria with the capacity to promote plant growth on the germination and development of Solanum lycopersicum at higher salinity levels. The germination percentage was improved at the highest concentration by the inoculated treatments (from 37 to 47%), as were the length of the radicle (30% at 20 mM) and plumule of the germinated seed, this bacterium also increased the weight of the plumule (97% at 100 mM). They also improved the yield. The dry weight of the plant, in addition to having an influence on the quality of the fruit and the concentration of osmoprotectors (Bacillus sp. SVHM 1.1) had the greatest effect on fruit yield (1.5 kg/plant at 20 mM), by the otherhand, Halomonas sp. SVHM8 provided the best fruit quality characteristics at 100 mM. According to the above results, the efficiency of halophilic PGPB in the attenuation of salt stress in Solanum lycopersicum has been proven.

Aromatic compounds depurative and plant growth promotion rhizobacteria abilities of Allenrolfea vaginata (Amaranthaceae) rhizosphere microbial communities from a solar saltern hypersaline soil.

Introduction: This work investigates whether rhizosphere microorganisms that colonize halophyte plants thriving in saline habitats can tolerate salinity and provide beneficial effects to their hosts, protecting them from environmental stresses, such as aromatic compound (AC) pollution. Methods: To address this question, we conducted a series of experiments. First, we evaluated the effects of phenol, tyrosine, 4-hydroxybenzoic acid, and 2,4-dichlorophenoxyacetic (2,4-D) acids on the soil rhizosphere microbial community associated with the halophyte Allenrolfea vaginata. We then determined the ability of bacterial isolates from these microbial communities to utilize these ACs as carbon sources. Finally, we assessed their ability to promote plant growth under saline conditions. Results: Our study revealed that each AC had a different impact on the structure and alpha and beta diversity of the halophyte bacterial (but not archaeal) communities. Notably, 2,4-D and phenol, to a lesser degree, had the most substantial decreasing effects. The removal of ACs by the rhizosphere community varied from 15% (2,4-D) to 100% (the other three ACs), depending on the concentration. Halomonas isolates were the most abundant and diverse strains capable of degrading the ACs, with strains of Marinobacter, Alkalihalobacillus, Thalassobacillus, Oceanobacillus, and the archaea Haladaptatus also exhibiting catabolic properties. Moreover, our study found that halophile strains Halomonas sp. LV-8T and Marinobacter sp. LV-48T enhanced the growth and protection of Arabidopsis thaliana plants by 30% to 55% under salt-stress conditions. Discussion: These results suggest that moderate halophile microbial communities may protect halophytes from salinity and potential adverse effects of aromatic compounds through depurative processes.

Inulin from halophilic archaeon Haloarcula: Production, chemical characterization, biological, and technological properties.

Halophilic archaea are capable of producing fructans, which are fructose-based polysaccharides. However, their biochemical characterization and biological and technological properties have been scarcely studied. The aim of this study was to evaluate the production, chemical characterization, biological and technological properties of a fructan inulin-type biosynthesized by a halophilic archaeon. Fructan extraction was performed through ethanol precipitation and purification by diafiltration. The chemical structure was elucidated using Fourier Transform-Infrared Spectroscopy and Nuclear Magnetic Resonance (NMR). Haloarcula sp. M1 biosynthesizes inulin with an average molecular weight of 8.37 × 106 Da. The maximal production reached 3.9 g of inulin per liter of culture within seven days. The glass transition temperature of inulin was measured at 138.85 °C, and it exhibited an emulsifying index of 36.47 %, which is higher than that of inulin derived from chicory. Inulin from Haloarcula sp. M1 (InuH) demonstrates prebiotic capacity. This study represents the first report on the biological and technological properties of inulin derived from halophilic archaea.

A Transiently Hypersaline Microbial Mat Harbors a Diverse and Stable Archaeal Community in the Cuatro Cienegas Basin, Mexico.

Microbial mats are biologically diverse communities that are analogs to some of the earliest ecosystems on Earth. In this study, we describe a unique transiently hypersaline microbial mat uncovered in a shallow pond within the Cuatro Cienegas Basin (CCB) in northern México. The CCB is an endemism-rich site that harbors living stromatolites that have been studied to understand the conditions of the Precambrian Earth. These microbial mats form elastic domes filled with biogenic gas, and the mats have a relatively large and stable subpopulation of archaea. For this reason, this site has been termed archaean domes (AD). The AD microbial community was analyzed by metagenomics over three seasons. The mat exhibited a highly diverse prokaryotic community dominated by bacteria. Bacterial sequences are represented in 37 phyla, mainly Proteobacteria, Firmicutes, and Actinobacteria, that together comprised >50% of the sequences from the mat. Archaea represented up to 5% of the retrieved sequences, with up to 230 different archaeal species that belong to 5 phyla (Euryarchaeota, Crenarchaeota, Thaumarchaeota, Korarchaeota, and Nanoarchaeota). The archaeal taxa showed low variation despite fluctuations in water and nutrient availability. In addition, predicted functions highlight stress responses to extreme conditions present in the AD, including salinity, pH, and water/drought fluctuation. The observed complexity of the AD mat thriving in high pH and fluctuating water and salt conditions within the CCB provides an extant model of great value for evolutionary studies, as well as a suitable analog to the early Earth and Mars.

Characterizations of highly efficient moderately halophilic toluene degrading exiguobacterium mexicanum M7 strain isolated from Egyptian saline sediments.

Toluene and other monoaromatic compounds are released into the environment particularly saline habitats due to the inappropriate disposal methods of petroleum products. Studying the bio-removal strategy is required to clean up these hazardous hydrocarbons that threaten all ecosystem life using halophilic bacteria with higher biodegradation efficiency of monoaromatic compounds as a sole carbon and energy source. Therefore, sixteen pure halophilic bacterial isolates were obtained from saline soil of Wadi An Natrun, Egypt, which have the ability to degrade toluene and consume it as the only source of carbon and energy. Amongst these isolates, isolate M7 exhibited the best growth with considerable properties. This isolate was selected as the most potent strain and identified based on phenotypic and genotypic characterizations. The strain M7 was belonging to Exiguobacterium genus and founded to be closely matched to the Exiguobacterium mexicanum with a similarity of 99%. Using toluene as sole carbon source, strain M7 showed good growth at a wide range temperature degree (20-40ºC), pH (5-9), and salt concentrations (2.5-10%, w/v) with optimal growth conditions at 35ºC, pH 8, and 5%, respectively. The biodegradation ratio of toluene was estimated at above optimal conditions and analyzed using Purge-Trap GC-MS. The results showed that strain M7 has the potentiality to degraded 88.32% of toluene within greatly short time (48 h). The current study findings support the potential ability to use strain M7 as a biotechnological tool in many applications such as effluent treatment and toluene waste management.

Genome sequence of an uncharted halophilic bacterium Robertkochia marina with deciphering its phosphate-solubilizing ability.

The wide use of whole-genome sequencing approach in the modern genomic era has opened a great opportunity to reveal the prospective applications of halophilic bacteria. Robertkochia marina CC-AMO-30DT is one of the halophilic bacteria that was previously taxonomically identified without any inspection on its biotechnological potential from a genomic aspect. In this study, we present the whole-genome sequence of R. marina and demonstrated the ability of this bacterium in solubilizing phosphate by producing phosphatase. The genome of R. marina has 3.57 Mbp and contains 3107 predicted genes, from which 3044 are protein coding, 52 are non-coding RNAs, and 11 are pseudogenes. Several phosphatases such as alkaline phosphatases and pyrophosphatases were mined from the genome. Further genomic study (phylogenetics, sequence analysis, and functional mechanism) and experimental data suggested that the alkaline phosphatase produced by R. marina could potentially be utilized in promoting plant growth, particularly for plants on saline-based agricultural land.

Comparative molecular studies of halophilic bacteria from saline water and soil in the Saudi environment / Estudos moleculares comparativos de bactérias halofílicas de água salina e solo no meio ambiente saudita

Halophilic bacteria are microorganisms that grow optimally in the presence of the very high concentration of sodium chloride. Halophiles are vital sources of various enzymes including hydrolases, which are very stable and catalytically highly efficient at high salt concentration and other extreme conditions such as high temperature, pH and presence of organic solvents. Several hydrolases such as amylases, proteases, and lipases have been obtained from halophilic bacteria and are commonly used for various industrial applications. We initiated a screening to isolate and characterize the halophilic bacteria from the Red Sea, which is one of the saltiest bodies of water in the world. Water and soil samples, collected from the Red Sea coast, Jeddah, Saudi Arabia, were screened for isolation of halophilic bacteria. Ten bacterial isolates were obtained, which were characterized by biochemical tests and 16S rRNA gene sequencing. Hydrolase producing bacteria among the isolates were screened by plate assay on starch and gelatin agar plates for amylase and protease, respectively. Two bacterial isolates i.e. Bacillus glycinifermentans S3 and Enterobacter cloacae W1were found to possess significant amylase and protease activity.

Bactérias halofílicas são microrganismos que crescem de maneira ideal na presença de uma concentração muito alta de cloreto de sódio. Halófilos são fontes vitais de várias enzimas, incluindo hidrolases, que são muito estáveis e cataliticamente altamente eficientes em alta concentração de sal e outras condições extremas, como alta temperatura, pH e presença de solventes orgânicos. Várias hidrolases como amilases, proteases e lipases foram obtidas a partir de bactérias halofílicas e são comumente usadas para várias aplicações industriais. Iniciamos uma triagem para isolar e caracterizar as bactérias halofílicas do Mar Vermelho, que é um dos corpos de água mais salgados do mundo. Amostras de água e solo, coletadas na costa do Mar Vermelho, Jeddah, na Arábia Saudita, foram examinadas quanto ao isolamento de bactérias halofílicas. Foram obtidos dez isolados bacterianos, caracterizados por testes bioquímicos e seqüenciamento do gene 16S rRNA. As bactérias produtoras de hidrolase entre os isolados foram triadas por ensaio em placa em placas de amido e ágar de gelatina para amilase e protease, respectivamente. Verificou-se que dois isolados bacterianos, isto é, Bacillus glycinifermentans S3 e Enterobacter cloacae W1, possuíam significativa atividade de amilase e protease.

Insights Into the Microbiology of the Chaotropic Brines of Salar de Atacama, Chile.

Microbial life inhabiting hypersaline environments belong to a limited group of extremophile or extremotolerant taxa. Natural or artificial hypersaline environments are not limited to high concentrations of NaCl, and under such conditions, specific adaptation mechanisms are necessary to permit microbial survival and growth. Argentina, Bolivia, and Chile include three large salars (salt flats) which globally, represent the largest lithium reserves, and are commonly referred to as the Lithium Triangle Zone. To date, a large amount of information has been generated regarding chemical, geological, meteorological and economical perspectives of these salars. However, there is a remarkable lack of information regarding the biology of these unique environments. Here, we report the presence of two bacterial strains (isolates LIBR002 and LIBR003) from one of the most hypersaline lithium-dominated man-made environments (total salinity 556 g/L; 11.7 M LiCl) reported to date. Both isolates were classified to the Bacillus genera, but displayed differences in 16S rRNA gene and fatty acid profiles. Our results also revealed that the isolates are lithium-tolerant and that they are phylogenetically differentiated from those Bacillus associated with high NaCl concentration environments, and form a new clade from the Lithium Triangle Zone. To determine osmoadaptation strategies in these microorganisms, both isolates were characterized using morphological, metabolic and physiological attributes. We suggest that our characterization of bacterial isolates from a highly lithium-enriched environment has revealed that even at such extreme salinities with high concentrations of chaotropic solutes, scope for microbial life exists. These conditions have previously been considered to limit the development of life, and our work extends the window of life beyond high concentrations of MgCl2, as previously reported, to LiCl. Our results can be used to further the understanding of salt tolerance, most especially for LiCl-dominated brines, and likely have value as models for the understanding of putative extra-terrestrial (e.g., Martian) life.

ADP-Dependent Kinases From the Archaeal Order Methanosarcinales Adapt to Salt by a Non-canonical Evolutionarily Conserved Strategy.

Halophilic organisms inhabit hypersaline environments where the extreme ionic conditions and osmotic pressure have driven the evolution of molecular adaptation mechanisms. Understanding such mechanisms is limited by the common difficulties encountered in cultivating such organisms. Within the Euryarchaeota, for example, only the Halobacteria and the order Methanosarcinales include readily cultivable halophilic species. Furthermore, only the former have been extensively studied in terms of their component proteins. Here, in order to redress this imbalance, we investigate the halophilic adaptation of glycolytic enzymes from the ADP-dependent phosphofructokinase/glucokinase family (ADP-PFK/GK) derived from organisms of the order Methanosarcinales. Structural analysis of proteins from non-halophilic and halophilic Methanosarcinales shows an almost identical composition and distribution of amino acids on both the surface and within the core. However, these differ from those observed in Halobacteria or Eukarya. Proteins from Methanosarcinales display a remarkable increase in surface lysine content and have no reduction to the hydrophobic core, contrary to the features ubiquitously observed in Halobacteria and which are thought to be the main features responsible for their halophilic properties. Biochemical characterization of recombinant ADP-PFK/GK from M. evestigatum (halophilic) and M. mazei (non-halophilic) shows the activity of both these extant enzymes to be only moderately inhibited by salt. Nonetheless, its activity over time is notoriously stabilized by salt. Furthermore, glycine betaine has a protective effect against KCl inhibition and enhances the thermal stability of both enzymes. The resurrection of the last common ancestor of ADP-PFK/GK from Methanosarcinales shows that the ancestral enzyme displays an extremely high salt tolerance and thermal stability. Structure determination of the ancestral protein reveals unique traits such as an increase in the Lys and Glu content at the protein surface and yet no reduction to the volume of the hydrophobic core. Our results suggest that the halophilic character is an ancient trait in the evolution of this protein family and that proteins from Methanosarcinales have adapted to highly saline environments by a non-canonical strategy, different from that currently proposed for Halobacteria. These results open up new avenues for the search and development of novel salt tolerant biocatalysts.

Microbial community composition and trophic role along a marked salinity gradient in Laguna Puilar, Salar de Atacama, Chile.

The geological, hydrological and microbiological features of the Salar de Atacama, the most extensive evaporitic sedimentary basin in the Atacama Desert of northern Chile, have been extensively studied. In contrast, relatively little attention has been paid to the composition and roles of microbial communities in hypersaline lakes which are a unique feature in the Salar. In the present study biochemical, chemical and molecular biological tools were used to determine the composition and roles of microbial communities in water, microbial mats and sediments along a marked salinity gradient in Laguna Puilar which is located in the "Los Flamencos" National Reserve. The bacterial communities at the sampling sites were dominated by members of the phyla Bacteroidetes, Chloroflexi, Cyanobacteria and Proteobacteria. Stable isotope and fatty acid analyses revealed marked variability in the composition of microbial mats at different sampling sites both horizontally (at different sites) and vertically (in the different layers). The Laguna Puilar was shown to be a microbially dominated ecosystem in which more than 60% of the fatty acids at particular sites are of bacterial origin. Our pioneering studies also suggest that the energy budgets of avian consumers (three flamingo species) and dominant invertebrates (amphipods and gastropods) use minerals as a source of energy and nutrients. Overall, the results of this study support the view that the Salar de Atacama is a heterogeneous and fragile ecosystem where small changes in environmental conditions may alter the balance of microbial communities with possible consequences at different trophic levels.

Genomic comparison between members of the Salinibacteraceae family, and description of a new species of Salinibacter (Salinibacter altiplanensis sp. nov.) isolated from high altitude hypersaline environments of the Argentinian Altiplano.

The application of tandem MALDI-TOF MS screening with 16S rRNA gene sequencing of selected isolates has been demonstrated to be an excellent approach for retrieving novelty from large-scale culturing. The application of such methodologies in different hypersaline samples allowed the isolation of the culture-recalcitrant Salinibacter ruber second phylotype (EHB-2) for the first time, as well as a new species recently isolated from the Argentinian Altiplano hypersaline lakes. In this study, the genome sequences of the different species of the phylum Rhodothermaeota were compared and the genetic repertoire along the evolutionary gradient was analyzed together with each intraspecific variability. Altogether, the results indicated an open pan-genome for the family Salinibacteraceae, as well as the codification of relevant traits such as diverse rhodopsin genes, CRISPR-Cas systems and spacers, and one T6SS secretion system that could give ecological advantages to an EHB-2 isolate. For the new Salinibacter species, we propose the name Salinibacter altiplanensis sp. nov. (the designated type strain is AN15T=CECT 9105T=IBRC-M 11031T).

Use of a mixed culture strategy to isolate halophilic bacteria with antibacterial and cytotoxic activity from the Manaure solar saltern in Colombia.

BACKGROUND: Water evaporation in solar salterns creates salinity gradients that promote the adaptation of microbial species to different salinities. This competitive habitat challenges the metabolic capabilities of microorganisms and promotes alterations in their production of secondary metabolites. Thus, solar salterns are a potentially important source of new natural products. In Colombia, the most important and representative solar saltern is located in Manaure (La Guajira) in the north of Colombia. The aim of this study was to develop an alternative screening strategy to select halophilic bacteria as producers of bioactive compounds from mixed microbial cultures rather than individual environmental isolates. Brine and sediment samples from different ponds (across a salinity gradient) were inoculated in seven different culture media to grow bacteria and archaea, allowing for a total of 40 different mixed cultures. An organic extract from each mixed culture was obtained and tested against multidrug resistant pathogens, including Klebsiella pneumoniae, vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus and Bacillus subtilis. In addition, the extracts were tested against two human cancer cell lines, cervical adenocarcinoma (SiHa) and lung carcinoma (A-549). RESULTS: Twenty-four of the forty extracts from mixed cultures obtained from brine and sediment samples from the Manaure solar saltern showed antibacterial activity against Bacillus subtilis. Two extracts, referred to as A1SM3-29 and A1SM3-36, were also active against a methicillin-resistant Staphylococcus aureus, with the latter extract also showing slight cytotoxic activity against the assayed human lung cancer cell line. From this mixed culture, nine isolates were cultivated, and their extracts were tested against the same pathogens, resulting in the identification of a Vibrio sp. strain (A1SM3-36-8) with antimicrobial activity that was similar to that observed for the mixed culture extract. The extract of this strain was subjected to a bioautography assay, and 3 different fractions exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus. Based on the amount obtained for each fraction, F3 was selected to isolate and identify its metabolites. The major compound was identified by NMR and HRMS as 13-cis-docosenamide, an amide that has been previously reported to be an antimicrobial and cytotoxic compound. CONCLUSIONS: Our results shows the utility of our strategy in detecting bioactive molecules in initial mixed cultures by biological assays, resulting in the isolation and characterization of Vibrio sp. A1SM3-36-8, a halophilic strain with great antibacterial and cytotoxic potential.

Extremozymes: A Potential Source for Industrial Applications.

Extremophilic microorganisms have established a diversity of molecular strategies in order to survive in extreme conditions. Biocatalysts isolated by these organisms are termed extremozymes, and possess extraordinary properties of salt allowance, thermostability, and cold adaptivity. Extremozymes are very resistant to extreme conditions owing to their great solidity, and they pose new opportunities for biocatalysis and biotransformations, as well as for the development of the economy and new line of research, through their application. Thermophilic proteins, piezophilic proteins, acidophilic proteins, and halophilic proteins have been studied during the last few years. Amylases, proteases, lipases, pullulanases, cellulases, chitinases, xylanases, pectinases, isomerases, esterases, and dehydrogenases have great potential application for biotechnology, such as in agricultural, chemical, biomedical, and biotechnological processes. The study of extremozymes and their main applications have emerged during recent years.

Moderate halophilic bacteria colonizing the phylloplane of halophytes of the subfamily Salicornioideae (Amaranthaceae).

Halophytes accumulate large amounts of salt in their tissues, and thus are susceptible to colonization by halotolerant and halophilic microorganisms that might be relevant for the growth and development of the plant. Here, the study of 814 cultured strains and 14,189 sequences obtained by 454 pyrosequencing were combined in order to evaluate the presence, abundance and diversity of halophilic, endophytic and epiphytic microorganisms in the phytosphere of leaves of members of the subfamily Salicornioideae from five locations in Spain and Chile. Cultures were screened by the tandem approach of MALDI-TOF/MS and 16S rRNA gene sequencing. In addition, differential centrifugation was used to enrich endophytes for further DNA isolation, 16S rRNA gene amplification and 454 pyrosequencing. Culturable and non-culturable data showed strong agreement with a predominance of Proteobacteria, Firmicutes and Actinobacteria. The most abundant isolates corresponded to close relatives of the species Chromohalobacter canadensis and Salinicola halophilus that comprised nearly 60% of all isolates and were present in all plants. Up to 66% of the diversity retrieved by pyrosequencing could be brought into pure cultures and the community structures were highly dependent on the compartment where the microorganisms thrived (plant surface or internal tissues).

Screening and isolation of halophilic bacteria producing industrially important enzymes

Halophiles are excellent sources of enzymes that are not only salt stable but also can withstand and carry out reactions efficiently under extreme conditions. The aim of the study was to isolate and study the diversity among halophilic bacteria producing enzymes of industrial value. Screening of halophiles from various saline habitats of India led to isolation of 108 halophilic bacteria producing industrially important hydrolases (amylases, lipases and proteases). Characterization of 21 potential isolates by morphological, biochemical and 16S rRNA gene analysis found them related to Marinobacter, Virgibacillus, Halobacillus, Geomicrobium, Chromohalobacter, Oceanobacillus, Bacillus, Halomonas and Staphylococcus genera. They belonged to moderately halophilic group of bacteria exhibiting salt requirement in the range of 3-20%. There is significant diversity among halophiles from saline habitats of India. Preliminary characterization of crude hydrolases established them to be active and stable under more than one extreme condition of high salt, pH, temperature and presence of organic solvents. It is concluded that these halophilic isolates are not only diverse in phylogeny but also in their enzyme characteristics. Their enzymes may be potentially useful for catalysis under harsh operational conditions encountered in industrial processes. The solvent stability among halophilic enzymes seems a generic novel feature making them potentially useful in non-aqueous enzymology.

Screening and isolation of halophilic bacteria producing industrially important enzymes.

Halophiles are excellent sources of enzymes that are not only salt stable but also can withstand and carry out reactions efficiently under extreme conditions. The aim of the study was to isolate and study the diversity among halophilic bacteria producing enzymes of industrial value. Screening of halophiles from various saline habitats of India led to isolation of 108 halophilic bacteria producing industrially important hydrolases (amylases, lipases and proteases). Characterization of 21 potential isolates by morphological, biochemical and 16S rRNA gene analysis found them related to Marinobacter, Virgibacillus, Halobacillus, Geomicrobium, Chromohalobacter, Oceanobacillus, Bacillus, Halomonas and Staphylococcus genera. They belonged to moderately halophilic group of bacteria exhibiting salt requirement in the range of 3-20%. There is significant diversity among halophiles from saline habitats of India. Preliminary characterization of crude hydrolases established them to be active and stable under more than one extreme condition of high salt, pH, temperature and presence of organic solvents. It is concluded that these halophilic isolates are not only diverse in phylogeny but also in their enzyme characteristics. Their enzymes may be potentially useful for catalysis under harsh operational conditions encountered in industrial processes. The solvent stability among halophilic enzymes seems a generic novel feature making them potentially useful in non-aqueous enzymology.

Chemotactic responses to gas oil of Halomonas spp. strains isolated from saline environments in Argentina / Respuesta quimiotáctica hacia gas oil de cepas de Halomonas spp. aisladas de ambientes salinos de Argentina

In this study, two halophilic bacterial strains isolated from saline habitats in Argentina grew in the presence of gas oil. They were identified as Halomonas spp. and Nesterenkonia sp. by 16S ribosomal RNA sequencing. Chemotaxis towards gas oil was observed in Halomonas spp. by using swimming assays.

En el presente trabajo se aislaron dos cepas bacterianas halofílicas a partir de muestras obtenidas en ambientes salinos de Argentina, que crecieron en presencia de gasoil como única fuente de carbono. Las cepas aisladas se identificaron como Halomonas spp. y Nesterenkonia sp. mediante secuenciación del gen del ARN ribosomal 16S. En ensayos de swimming, las cepas del genero Halomonas spp. mostraron una respuesta quimiotáctica hacia el gas oil.

Characterization and stability of extracellular alkaline proteases from halophilic and alkaliphilic bacteria isolated from saline habitat of coastal Gujarat, India

The present study deals with the isolation and characterization of the moderately halophilic-alkaliphilic bacteria from a saline habitat in western India. Eight different bacterial strains were isolated using enrichment techniques at 20% (w/v) NaCl and pH 10. The isolates exhibited diversity towards gram's reaction, colony and cell morphology. They were able to grow and produce alkaline protease over a broad range of NaCl, 5-20% (w/v) and pH, 8-10. None of the isolates could grow at pH 7, and one could not grow even at pH 8. Crude and partially purified proteases from strain S5 were subjected to characterization with reference to pH, salt stability and protein folding. Optimum protease activity and stability was recorded at 10% salt and pH 9-9.5. Denaturation kinetics of S5 alkaline protease along with a reference protease was studied at 8M urea followed by renaturation. The S5 alkaline protease could be partially renatured up to 32% of the original activity. Despite of the fact that all the 8 isolates were from the same site, they displayed significant diversity with respect to their salt requirement for growth and enzyme secretion. While the effect of pH was less demarcated on growth, the protease production was significantly affected. Isolate S5 produced substantial amount of halotolerant and alkaline protease. The activity and stability of the alkaline protease in a broader range of pH and salt would definitely make this enzyme an important candidate for various industrial applications.

O presente estudo relata o isolamento e caracterização de bactérias moderadamente halofilicas e alcalífilicas de um habitat salino no oeste da Índia. Oito cepas diferentes de bactérias foram isoladas empregando técnicas de enriquecimento em NaCl a 20% (p/v) e pH 10. As cepas apresentaram diversidade em relação à coloração de Gram e à morfologia das colônias e células. As cepas foram capazes de multiplicar e produzir protease alcalina em uma ampla faixa de concentração de NaCl (5 a 20%) e pH (8 a 10). Nenhuma das cepas foi capaz de se multiplicar em pH 7, e uma não se multiplicou nem em pH 8.0. Proteases naturais e parcialmente purificadas da cepa S5 foram submetidas à caracterização com relação ao pH, estabilidade salina, e estrutura protéica. Atividade e estabilidade ótimas da protease foram obtidas com 10% de sal e pH 9-9,5. A cinética de denaturação da protease de S5, juntamente com uma protease de referencia, foi avaliada com uréia 8M seguida de renaturação. A protease alcalina de S5 foi renaturada a 32% da atividade original. Apesar de provenientes do mesmo local, as oito cepas mostraram grande diversidade em relação à exigência de sal para multiplicação e secreção enzimática. Enquanto o efeito do pH na multiplicação foi menos marcante, o efeito na produção de protease foi significativamente afetada. A cepa S5 produziu uma quantidade substancial de protease alcalina e halotolerante. A atividade e estabilidade da protease alcalina em uma faixa mais ampla de pH e sal tornam essa enzima uma importante candidata para diversas aplicações industriais.