SERS characterization of biochemical changes associated with biodesulfurization of dibenzothiophene using Gordonia sp. HS126-4N.

In this study, Gordonia sp. HS126-4N was employed for dibenzothiophene (DBT) biodesulfurization, tracked over 9 days using SERS. During the initial lag phase, no significant spectral changes were observed, but after 48 h, elevated metabolic activity was evident. At 72 h, maximal bacterial population correlated with peak spectrum variance, followed by stable spectral patterns. Despite 2-hydroxybiphenyl (2-HBP) induced enzyme suppression, DBT biodesulfurization persisted. PCA and PLS-DA analysis of the SERS spectra revealed distinctive features linked to both bacteria and DBT, showcasing successful desulfurization and bacterial growth stimulation. PLS-DA achieved a specificity of 95.5 %, sensitivity of 94.3 %, and AUC of 74 %, indicating excellent classification of bacteria exposed to DBT. SERS effectively tracked DBT biodesulfurization and bacterial metabolic changes, offering insights into biodesulfurization mechanisms and bacterial development phases. This study highlights SERS' utility in biodesulfurization research, including its use in promising advancements in the field.

Differentiation of different dibenzothiophene (DBT) desulfurizing bacteria via surface-enhanced Raman spectroscopy (SERS).

Fossil fuels are considered vital natural energy resources on the Earth, and sulfur is a natural component present in them. The combustion of fossil fuels releases a large amount of sulfur in the form of SO x in the atmosphere. SO x is the major cause of environmental problems, mainly air pollution. The demand for fuels with ultra-low sulfur is growing rapidly. In this aspect, microorganisms are proven extremely effective in removing sulfur through a process known as biodesulfurization. A major part of sulfur in fossil fuels (coal and oil) is present in thiophenic structures such as dibenzothiophene (DBT) and substituted DBTs. In this study, the identification and characterization of DBT desulfurizing bacteria (Chryseobacterium sp. IS, Gordonia sp. 4N, Mycolicibacterium sp. J2, and Rhodococcus sp. J16) based on their specific biochemical constituents were conducted using surface-enhanced Raman spectroscopy (SERS). By differentiating DBT desulfurizing bacteria, researchers can gain insights into their unique characteristics, thus leading to improved biodesulfurization strategies. SERS was used to differentiate all these species based on their biochemical differences and different SERS vibrational bands, thus emerging as a potential technique. Moreover, multivariate data analysis techniques such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were employed to differentiate these DBT desulfurizing bacteria on the basis of their characteristic SERS spectral signals. For all these isolates, the accuracy, sensitivity, and specificity are above 90%, and an AUC (area under the curve) value of close to 1 was achieved for all PLS-DA models.

Surface enhanced Raman spectroscopy for the characterization of the metabolites of the biodesulfurization of dibenzothiophene carried out by Tsukamurella paurometabola.

Large amount of sulphur is released by the combustion of fossil fuels in the form of SoX which affects human health and leads to acid rain. To overcome this issue, it is essential to eliminate sulphur moieties from heterocyclic organo-sulphur compounds like Dibenzothiophene (DBT) present in the petrol. In this study Surface enhanced Raman scattering (SERS) spectroscopy is used to analyze the desulfurizing activity of Tsukamurella paurometabola bacterial strain. The most prominent SERS peaks observed at 791, 837, 944 and 1032 cm-1, associated to C-S stretching, are solely observed in dibenzothiophene and its metabolite-I (DBTS) but absent in 2-Hydroxybiphenyl (metabolite-II) and extraction sample of supernatant as a result of biodesulfurization. Moreover, the SERS peaks observed at 974 (characteristic peak of benzene ring) and 1015 cm-1 is associated to C-C ring breathing while 1642 and 1655 cm-1 assigned to CC bonds of aromatic ring. These peaks are only observed in 2-Hydroxybiphenyl (metabolite-II) and extraction sample of supernatant as a result of biodesulfurization. Notably, these peaks are absent in the Dibenzothiophene and its metabolite-I which indicate that aromatic ring is carrying sulfur in this fraction. Moreover, multivariate data analytical tools like principal component analysis (PCA) and PCA-loadings are applied to further differentiate between dibenzothiophene and its metabolites that are Dibenzothiophene sulphone (metabolite-I) and 2-Hydroxybiphenyl (metabolite-II).

Biodesulfurization of organosulfur compounds by a trehalose biosurfactant producing Gordonia sp. isolated from crude oil contaminated soil.

Certain factors hinder the commercialization of biodesulfurization process, including low substrate-specificity of the currently reported desulfurizing bacteria and restricted mass transfer of organic-sulfur compounds in biphasic systems. These obstacles must be addressed to clean organic-sulfur rich petro-fuels that pose serious environmental and health challenges. In current study, a dibenzothiophene desulfurizing strain, Gordonia rubripertincta W3S5 (source: oil contaminated soil) was systematically evaluated for its potential to remove sulfur from individual compounds and mixture of organic-sulfur compounds. Metabolic and genetic analyses confirmed that strain W3S5 desulfurized dibenzothiophene to 2-hydroxybiphenyl, suggesting that it follows the sulfur specific 4 S pathway. Furthermore, this strain demonstrated the ability to produce trehalose biosurfactants (with an EI24 of 53%) in the presence of dibenzothiophene, as confirmed by TLC and FTIR analyses. Various genome annotation tools, such as ClassicRAST, BlastKOALA, BV-BRC, and NCBI-PGAP, predicted the presence of otsA, otsB, treY, treZ, treP, and Trehalose-monomycolate lipid synthesis genes in the genomic pool of strain W3S5, confirming the existence of the OtsAB, TreYZ, and TreP pathways. Overall, these results underscore the potential of strain W3S5 as a valuable candidate for enhancing desulfurization efficiency and addressing the mass transfer challenges essential for achieving a scaled-up scenario.

Genomic analysis and biodesulfurization potential of a new carbon-sulfur bond cleaving Tsukamurella sp. 3OW.

Direct combustion of sulfur-enriched liquid fuel oil causes sulfur oxide emission, which is one of the main contributors to air pollution. Biodesulfurization is a promising and eco-friendly method to desulfurize a wide range of thiophenic compounds present in fuel oil. Previously, numerous bacterial strains from genera such as Rhodococcus, Corynebacterium, Gordonia, Nocardia, Mycobacterium, Mycolicibacterium, Paenibacillus, Shewanella, Sphingomonas, Halothiobacillus, and Bacillus have been reported to be capable of desulfurizing model thiophenic compounds or fossil fuels. In the present study, we report a new desulfurizing bacterium, Tsukamurella sp. 3OW, capable of desulfurization of dibenzothiophene through the carbon-sulfur bond cleavage 4S pathway. The bacterium showed a high affinity for the hydrocarbon phase and broad substrate specificity towards various thiophenic compounds. The overall genome-related index analysis revealed that the bacterium is closely related to Tsukamurella paurometabola species. The genomic pool of strain 3OW contains 57 genes related to sulfur metabolism, including the key dszABC genes responsible for dibenzothiophene desulfurization. The DBT-adapted cells of the strain 3OW displayed significant resilience and viability in elevated concentrations of crude oil. The bacterium showed a 19 and 37% reduction in the total sulfur present in crude and diesel oil, respectively. Furthermore, FTIR analysis indicates that the oil's overall chemistry remained unaltered following biodesulfurization. This study implies that Tsukamurella paurometabola species, previously undocumented in the context of biodesulfurization, has good potential for application in the biodesulfurization of petroleum oils.

Dietary Supplementation of Microbial Dextran and Inulin Exerts Hypocholesterolemic Effects and Modulates Gut Microbiota in BALB/c Mice Models.

Microbial exopolysaccharides (EPSs), having great structural diversity, have gained tremendous interest for their prebiotic effects. In the present study, mice models were used to investigate if microbial dextran and inulin-type EPSs could also play role in the modulation of microbiomics and metabolomics by improving certain biochemical parameters, such as blood cholesterol and glucose levels and weight gain. Feeding the mice for 21 days on EPS-supplemented feed resulted in only 7.6 ± 0.8% weight gain in the inulin-fed mice group, while the dextran-fed group also showed a low weight gain trend as compared to the control group. Blood glucose levels of the dextran- and inulin-fed groups did not change significantly in comparison with the control where it increased by 22 ± 5%. Moreover, the dextran and inulin exerted pronounced hypocholesterolemic effects by reducing the serum cholesterol levels by 23% and 13%, respectively. The control group was found to be mainly populated with Enterococcus faecalis, Staphylococcus gallinarum, Mammaliicoccus lentus and Klebsiella aerogenes. The colonization of E. faecalis was inhibited by 59-65% while the intestinal release of Escherichia fergusonii was increased by 85-95% in the EPS-supplemented groups, respectively, along with the complete inhibition of growth of other enteropathogens. Additionally, higher populations of lactic acid bacteria were detected in the intestine of EPS-fed mice as compared to controls.

Genome analysis of novel Apilactobacillus sp. isolate from butterfly (Pieris canidia) gut reveals occurrence of unique glucanogenic traits and probiotic potential.

This study was conducted with a perception that fructose-rich niches may inhabit novel species of lactic acid bacteria that are gaining importance as probiotics and for the production of exopolysaccharides that have applications in food and pharmaceuticals. Recently, some Lactobacillus species have been reclassified as fructophilic lactic acid bacteria due to their preference for fructose over glucose as a carbon source. These bacteria are likely to be found in fructose rich niches such as flower nectar and insects that feed on it. We explored the butterfly gut and acquired a new isolate, designated as F1, of fructophilic lactic acid bacteria, which produces a glucan-type exopolysaccharide. Whole genome sequencing and in silico analysis revealed that F1 has significantly lower average nucleotide identity and DNA-DNA hybridization values as compared to its closest Apilactobacillus neighbors in phylogenetic analysis. Therefore, we declare the isolate F1 as a novel Apilactobacillus species with the proposed name of Apilactobacillus iqraium F1. Genome mining further revealed that F1 harbors genes for exopolysaccharide synthesis and health-promoting attributes. To this end, F1 is the only Apilactobacillus species harboring three diverse α-glucan-synthesis genes that cluster with different types of dextransucrases in the dendrogram. Moreover, many nutritional marker genes, as well as genes for epithelial cell adhesion and antimicrobial synthesis, were also detected suggesting the probiotic attributes of F1. Overall analysis suggests A. iqraium sp. F1 be a potential candidate for various health beneficial and pharmaceutical applications.

Whole Genome Sequence Analysis of a Novel Apilactobacillus Species from Giant Honeybee (Apis dorsata) Gut Reveals Occurrence of Genetic Elements Coding Prebiotic and Probiotic Traits.

Apilactobacillus spp. are classified as obligate fructophilic lactic acid bacteria (FLAB) that inhabit fructose-rich niches such as honeybee gut. Lactic acid bacteria are an important component of the gut microbiome and play a crucial role in maintaining gut health. In this study, a new FLAB strain HBW1, capable of producing glucan-type exopolysaccharide, was isolated from giant honeybee (Apis dorsata) gut and subjected to whole genome sequencing (WHS) to determine its health-beneficial traits. The genome size of the isolate was 1.49 Mb with a GC content of 37.2%. For species level identity, 16S rDNA sequence similarity, genome to genome distance calculator (dDDH), and average nucleotide identity (ANI) values were calculated. Phylogenetic analysis showed that the isolate HBW1 belongs to the Apilactobacillus genus. The dDDH and ANI values in comparison with closely clustered Apilactobacillus kunkeei species were 52% and 93.10%, respectively. Based on these values, we concluded that HBW1 is a novel species of Apilactobacillus, and we propose the name Apilactobacillus waqarii HBW1 for it. Further, WHS data mining of HBW1 revealed that it harbors two glucosyltransferase genes for prebiotic glucan-type exopolysaccharide synthesis. Moreover, chaperon (clp) and methionine sulfoxide reductase (msrA, msrB, and msrC) genes as well as nutritional marker genes for folic acid (folD) and riboflavin biosynthesis (rib operon), important for conferring probiotic properties, were also detected. Occurrence of these genetic traits make HBW1 an excellent candidate for application to improve gut function.

Genomic analysis provides insights into the Gordonia sp. W3S5 taxonomy and sulfur metabolism-related genes.

The Gordonia sp. W3S5, isolated from oil-polluted soil samples can remove sulfur from a variety of symmetric and asymmetric thiophenic compounds and diesel oil. Its draft genome sequence was comprised of 49 contigs, total genome size 4.86 Mb, and a G + C content of 67.50%. According to the current bacterial taxonomy procedures (16S rRNA gene sequence and overall genome-related index), the W3S5 was affiliated to Gordonia rubripertincta. Rapid Annotation using Subsystem Technology (ClassicRAST) server revealed that the W3S5 contains 4435 coding sequences, 404 subsystems and 60 sulfur metabolism genes. The RAST comparative genomic analysis showed that the genes connected with organic sulfur metabolism are majorly related to ssu and dszABC operons. Moreover, the comparison of orthologous gene clusters using OrthoVenn2 web server revealed a total of 4869 clusters, 2685 core orthologs, 632 shared orthologs and 112 unique ortholog clusters among the W3S5 and other type strains of Gordonia. This is the first report describing genome-based characterization of a Gordonia rubripertincta strain desulfurizing thiophenic compounds and diesel oil. The desulfurization potential of Gordonia rubripertincta W3S5 and genomic analyses revealed it as a valuable biocatalyst for process development to desulfurize a broad range of thiophenic sulfur-containing compounds, which are a major component of organic sulfur in petroleum oil. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02850-4.

Conventional genetic manipulation of desulfurizing bacteria and prospects of using CRISPR-Cas systems for enhanced desulfurization activity.

Highly active and stable biocatalysts are the prerequisite for industrial scale application of the biodesulfurization process. Scientists are making efforts for increasing the desulfurizing activity of native strains by employing various genetic engineering approaches. Nevertheless, the achieved desulfurization rate is lower than the industrial requirements. Thus, there is a dire need to use efficient genetic tools for precise genome editing of desulfurizing bacteria for enhanced efficiency. In comparison to the previously used genetic engineering tools the newly developed CRISPR-Cas is a more efficient and simple genetic tool that has been successfully applied for targeted genome modification of eukaryotes as well as prokaryotes. In this paper, we have reviewed the approaches, previously used to enhance the biodesulfurization rates of the sulfur metabolizing microorganisms and have discussed the potential of CRISPR-Cas systems in engineering desulfurizing biocatalysts. We have also proposed a model to construct competent desulfurizing recombinants involving use of CRISPR-Cas technology. The model can be used to over-express the dsz genes under a constitutive promoter in a suitable heterologous host, to get a steady expression of desulfurization pathway. This may serve as an inducement to develop better performing desulfurizing recombinant strains using CRISPR-Cas systems, which can be helpful in increasing the rate of biodesulfurization in future.

Comparative Analysis of Draft Genome Sequence of Rhodococcus sp. Eu-32 with Other Rhodococcus Species for Its Taxonomic Status and Sulfur Metabolism Potential.

Rhodococcus sp. Eu-32 has shown an extended novel dibenzothiophene desulfurization sulfur-specific 4S pathway and could remove significant amounts of organic sulfur from coal. Here, we present the draft genome sequence of Eu-32 with a genome size of approximately 5.61 Mb, containing 5065 protein coding sequences with a G+C content of 65.1%. The Rhodococcus sp. Eu-32 showed ~ 99% identity at the 16S rRNA gene sequence level while < 34% digital DNA-DNA hybridization and < 81% average nucleotide identity values with the genome sequence of most closely related known Rhodococcus species, suggesting that it is taxonomically different from the already reported Rhodococcus species. Among the annotated genes, 90 are involved in the metabolism of sulfur. Comparative genome analysis suggests many commonalities in sulfur metabolism gene sets that may have evolved due to many factors including ecological pressures. Our study and the genome sequence data will be available for further research and will provide insights into potential biotechnological and industrial applications of this bacterium.

Biodesulfurization of Thiophenic Compounds by a 2-Hydroxybiphenyl-Resistant Gordonia sp. HS126-4N Carrying dszABC Genes.

Microorganisms can metabolize or transform a range of known chemical compounds present in fossil fuels by naturally having highly specific metabolic activities. In this context, the microbial desulfurization of fuels is an attractive and alternative process to the conventional hydrodesulfurization (HDS) process, since the thiophenic sulfur containing compounds such as dibenzothiophene (DBT) and benzothiophene (BT) cannot be removed by HDS. A DBT desulfurizing mesophilic bacterium, identified on the basis of 16S rRNA gene sequence as Gordonia sp. HS126-4N (source: periphery soil of a coal heap) has been evaluated for its biodesulfurization traits and potential to desulfurize the thiophenic compounds. The HPLC and LC/MS analyses of the metabolites produced from DBT desulfurization and PCR-based nucleotide sequence confirmation of the key desulfurizing genes (dszA/dszB/dszC) proved that HS126-4N could convert DBT to 2-hydroxybiphenyl (2-HBP) via the 4S pathway. The isolate could convert 0.2 mM of DBT to 2-HBP within 48 h and was reasonably tolerant against the inhibitory effect of 2-HBP (retained 70% of growth at 0.5 mM 2-HBP). The isolated biocatalyst desulfurized/degraded 100% of 0.2 mM of 4-methyl DBT, 2,8-dimethyl DBT, BT and 3-methyl BT within 108 h. The capabilities to survive and desulfurize a broad range of thiophenic sulfur containing substrates as well as less inhibition by the 2-HBP suggest that HS126-4N could be a potential candidate for improved biodesulfurization/organic sulfur removal from fossil fuels.

Dibenzothiophene desulfurization capability and evolutionary divergence of newly isolated bacteria.

Metabolically microorganisms are diverse, and they are capable of transforming almost every known group of chemical compounds present in coal and oil in various forms. In this milieu, one of the important microbial metabolic processes is the biodesulfurization [cleavage of carbon-sulfur (C-S) bond] of thiophenic compounds, such as dibenzothiophene (DBT), which is the most abundant form of organic sulfur present in fossil fuels. In the current study, ten newly isolated bacterial isolates, designated as species of genera Gordonia, Amycolatopsis, Microbacterium and Mycobacterium, were enriched from different samples in the presence of DBT as a sole source of organic sulfur. The HPLC analysis of the DBT grown cultures indicated the consumption of DBT and accumulation of 2-hydroxybiphenyl (2-HBP). Detection of 2-HBP, a marker metabolite of 4S (sulfoxide-sulfone-sulfinate-sulfate) pathway, suggested that the newly isolated strains harbored metabolic activity for DBT desulfurization through the cleavage of C-S bond. The maximum 2-HBP formation rate was 3.5 µmol/g dry cell weight (DCW)/h. The phylogenetic analysis of the new isolates showed that they had diverse distribution within the phylogenetic tree and formed distinct clusters, suggesting that they might represent strains of already reported species or they were altogether new species. Estimates of evolutionary divergence showed high level of nucleotide divergence between the isolates within the same genus. The new isolates were able to use a range of heterocyclic sulfur compounds, thus making them suitable candidates for a robust biodesulfurization system for fossil fuels.

Phylogenetic characterization and novelty of organic sulphur metabolizing genes of Rhodococcus spp. (Eu-32).

Rhodococcus spp. (Eu-32) has the unique ability to metabolize organic sulphur containing compounds like dibenzothiophene through an extended sulphur specific pathway (Akhtar et al., in FEMS Microbiol Lett 301:95-102, 2009). Efforts were made to isolate and characterize the presumed desulphurizing genes (dszABC) involved in the sulphur specific pathway of isolate Eu-32 by employing standard and degenerate polymerase chain reaction primers. The partial dszA gene sequence of isolate Eu-32 showed 92% sequence identity with a putative FMNH-2 dependent monooxygenase of Rhodococcus erythropolis PR4. The dszC gene sequence showed 99% homology with the dibenzothiophene monooxygenase desulphurizing enzyme of another Rhodococcus species. The dszB gene was not unambiguously identified. A phylogenetic analysis by maximum likelihood method of the 16S rRNA gene and deduced DszA and C amino acid sequences suggest that horizontal gene transfer events might have taken place during the evolution of desulphurizing genes of Rhodococcus spp. (Eu-32).

Polyhexamethylene biguanide functionalized cationic silver nanoparticles for enhanced antimicrobial activity.

Polyhexamethylene biguanide (PHMB), a broad spectrum disinfectant against many pathogens, was used as a stabilizing ligand for the synthesis of fairly uniform silver nanoparticles. The particles formed were characterized using UV-visible spectroscopy, FTIR, dynamic light scattering, electrophoretic mobility, and TEM to measure their morphology and surface chemistry. PHMB-functionalized silver nanoparticles were then evaluated for their antimicrobial activity against a gram-negative bacterial strain, Escherichia coli. These silver nanoparticles were found to have about 100 times higher bacteriostatic and bactericidal activities, compared to the previous reports, due to the combined antibacterial effect of silver nanoparticles and PHMB. In addition to other applications, PHMB-functionalized silver nanoparticles would be extremely useful in textile industry due to the strong interaction of PHMB with cellulose fabrics.

Analysis of the dibenzothiophene metabolic pathway in a newly isolated Rhodococcus spp.

Out of 17 samples collected from diverse environments, 110 bacterial isolates of varied characteristics were screened for their dibenzothiophene-desulphurizing activity. A single isolate, Eu-32, originating from a soil sample taken from the roots of a eucalyptus tree, displayed dibenzothiophene-desulphurizing activity. This isolate metabolized dibenzothiophene to 2-hydroxybiphenyl (2-HBP), as detected by HPLC, and was also able to use other organic sulphur compounds as a sole sulphur source. Based on morphological, biochemical and molecular studies, it was found that the organism belongs to the genus Rhodococcus, with a maximum of 95% identity to species in this genus for the partial sequence of the 16S rRNA gene. Isolate Eu-32 could desulphurize 0.2 mM dibenzothiophene to 2-HBP in 72 h at a temperature of 30 degrees C and pH 7.0. The structure and molecular mass of metabolites produced from dibenzothiophene desulphurization were identified by GC-MS, and two sulphur-free products, 2-HBP and biphenyl, were detected in ethyl acetate extract. It was concluded that isolate Eu-32 is a unique desulphurizing biocatalyst that desulphurizes dibenzothiophene through an extended, sulphur-specific degradation pathway with the selective cleavage of C-S bonds.

Biodiversity and phylogenetic analysis of culturable bacteria indigenous to Khewra salt mine of Pakistan and their industrial importance

Culturable bacterial biodiversity and industrial importance of the isolates indigenous to Khewra salt mine, Pakistan was assessed. PCR Amplification of 16S rDNA of isolates was carried out by using universal primers FD1 and rP1and products were sequenced commercially. These gene sequences were compared with other gene sequences in the GenBank databases to find the closely related sequences. The alignment of these sequences with sequences available from GenBank database was carried out to construct a phylogenetic tree for these bacteria. These genes were deposited to GenBank and accession numbers were obtained. Most of the isolates belonged to different species of genus Bacillus, sharing 92-99 percent 16S rDNA identity with the respective type strain. Other isolates had close similarities with Escherichia coli, Staphylococcus arlettae and Staphylococcus gallinarum with 97 percent, 98 percent and 99 percent 16S rDNA similarity respectively. The abilities of isolates to produce industrial enzymes (amylase, carboxymethylcellulase, xylanase, cellulase and protease) were checked. All isolates were tested against starch, carboxymethylcellulose (CMC), xylane, cellulose, and casein degradation in plate assays. BPT-5, 11,18,19 and 25 indicated the production of copious amounts of carbohydrates and protein degrading enzymes. Based on this study it can be concluded that Khewra salt mine is populated with diverse bacterial groups, which are potential source of industrial enzymes for commercial applications.

Avaliou-se a biodiversidade e a importância industrial de bactérias indígenas da mina de sal Khewra, Paquistão. Efetuou-se a amplificação do 16S rDNA dos isolados por PCR empregando-se os iniciadores universais FD1 e rP1, e os produtos foram seqüenciados comercialmente. Essas seqüências de genes foram comparadas com outras seqüências disponíveis no GenBank a fim de encontrar seqüências relacionadas, construindo-se uma árvore filogenética para essas bactérias. Os genes foram depositados no GenBank obtendo-se os números de acesso. A maioria dos isolados pertenceu a diferentes espécies do gênero Bacillus, apresentando 92-99 por cento de identidade de 16S rDNA com a respectiva cepa de referencia. Outros isolados apresentaram alta similaridade com Escherichia coli, Staphylococcus arlettae e Staphylococcus gallinarum, com 97 por cento, 98 por cento e 99 por cento de similaridade de16S rDNA, respectivamente. A capacidade dos isolados produzirem enzimas industriais (amilase, carboximetilcelulase, xilanase, celulase e protease) foi verificada. Todos os isolados foram testados em placas quanto a degradação de amido, carboximetilcelulose, xilana, celulose e caseína. Os isolados BPT-5, 11, 18, 19 e 25 produziram grandes quantidades de enzimas degradadoras de carboidratos e proteínas. Conclui-se que a mina de Sal Khewra apresenta diferentes grupos de bactérias, que são fontes potenciais de enzimas industriais de aplicação comercial.

Biodiversity and phylogenetic analysis of culturable bacteria indigenous to Khewra salt mine of pakistan and their industrial importance.

Culturable bacterial biodiversity and industrial importance of the isolates indigenous to Khewra salt mine, Pakistan was assessed. PCR Amplification of 16S rDNA of isolates was carried out by using universal primers FD1 and rP1and products were sequenced commercially. These gene sequences were compared with other gene sequences in the GenBank databases to find the closely related sequences. The alignment of these sequences with sequences available from GenBank database was carried out to construct a phylogenetic tree for these bacteria. These genes were deposited to GenBank and accession numbers were obtained. Most of the isolates belonged to different species of genus Bacillus, sharing 92-99% 16S rDNA identity with the respective type strain. Other isolates had close similarities with Escherichia coli, Staphylococcus arlettae and Staphylococcus gallinarum with 97%, 98% and 99% 16S rDNA similarity respectively. The abilities of isolates to produce industrial enzymes (amylase, carboxymethylcellulase, xylanase, cellulase and protease) were checked. All isolates were tested against starch, carboxymethylcellulose (CMC), xylane, cellulose, and casein degradation in plate assays. BPT-5, 11,18,19 and 25 indicated the production of copious amounts of carbohydrates and protein degrading enzymes. Based on this study it can be concluded that Khewra salt mine is populated with diverse bacterial groups, which are potential source of industrial enzymes for commercial applications.