Biomineralization Potential of Bacillus subtilis,Rummeliibacillus Stabekisii and Staphylococcus Epidermidis Strains In Vitro Isolated from Speleothems,Khasi Hill Caves,Meghalaya, India

Microorganisms were isolated and identified from speleothems at Khasi hill caves, Meghalaya. The aim was to understand their biomineralization potential. Analyses of the speleothems from Krem Soitan, Krem Mawpun, and Krem Lawbah using scanning electron microscope (SEM) showed evidences for microbe–mineral interactions. SEM showed microbial reticulate and beaded filaments, cells, fiber calcites, and clusters of coccoid-like structures. A total of 113 bacterial strains were isolated and identified by a combination of conventional and molecular based tools. 105 strains that were sequenced belonged to the genus: Bacillus, Rummeliibacillus, Staphylococcus, and Brevibacterium. The BLASTn sequence search of 16S rRNA sequences with the National Centre for Biotechnology Information database to establish the identity of the strains yielded similarity scores of ≥99% with the respective organisms. The strains were identified as Bacillus simplex, Bacillus gaemokensis, Bacillus subtilis, Bacillus thuringiensis, Bacillus albus, Bacillus cereus, Bacillus anthracis, Bacillus weihenstephanensis, Rummeliibacillus stabekisii, Bacillus wiedmannii, Staphylococcus epidermidis, Rummeliibacillus pycnus, Kurthia zopfii, and Brevibacterium frigoritolerans. These strains were tested for biomineralization on B-4 medium. Five strains (B. subtilis, R. stabekisii, Staphylococcus epiderdimis, B. cereus, and B. wiedmannii) had the capability to precipitate biominerals in vitro. B. subtilis, R. stabekisii, and S. epidermidis precipitated 0.24, 0.36, and 0.35 g/L of biominerals at 22°C at the end of the four week experiment period. These strains increased the pH of the medium from 7 to 8.95. The precipitated biominerals were imaged using an ultra-high resolution field emission SEM. X-ray diffraction of the biomineral precipitated by R. stabekisii showed that it was composed of vaterite and jungite. Whereas S. epidermidis showed that it was composed of calcite, vaterite, and jungite. B. subtilis produced small, circular calcite crystals. This is the first comprehensive report on the possible evidences about the role of R. stabekisii and S. epidermidis in calcite precipitation isolated from speleothems in the Indian caves. These results allow us to postulate that the identified strains have biomineralization potential. Further evidences of the coexistence of exopolysaccharides, whisker fiber calcites, microbial filaments, and coccoid-like forms point to biogenic inputs in the cave mineral formations.     

Oily sludge degradation by bacteria from Ankleshwar, India

Three bacterial strains, Bacillus sp. SV9, Acinetobacter sp. SV4 and Pseudomonas sp., SV17 from contaminated soil in Ankleshwar, India were tested for their ability to degrade the complex mixture of petroleum hydrocarbons (such as alkanes, aromatics, resins and asphaltenes), sediments, heavy metals and water known as oily sludge. Gravimetric analysis showed that Bacillus sp. SV9 degraded approx. 59% of the oily sludge in 5 days at 30 °C whereas Acinetobacter sp. SV4 and Pseudomonas sp. SV17 degraded 37% and 35%. Capillary gas chromatographic analysis revealed that after 5 days the Bacillus strain was able to degrade oily sludge components of chain length C₁₂–C₃₀ and aromatics more effectively than the other two strains. Maximum drop in surface tension (from 70 to 28.4 mN/m) was accompanied by maximum biosurfactant production (6.7 g l⁻¹) in Bacillus sp. SV9 after 72 h, these results collectively indicating that this bacterial strain has considerable potential for bioremediation of oily sludge.     

Mineralogical Footprints of Bacterial Biofilms Associated with Labit Cave,a Part of the Longest Cave System in India

The present study was an attempt to demonstrate the capabilities of the microbial strains from the unexplored Labit cave in India to precipitate calcite providing evidence for biotic processes involved in formation of speleothem deposits. Six calcifying bacterial strains majority belonging to genus Bacillus were isolated from the cave. SEM studies revealed an array of various in vitro crystal polymorphs generated by the isolated bacteria which are similar to microscopic observations on natural formations in speleothems. The EDX spectrum of the precipitated crystals predominately composed of calcium carbonate indicating the relevance of bacterial biofilm in cave geomicrobiology and biogenic evolution of cave formations in the studied cave, which is further supported by XRF analysis and Raman spectroscopy.     

Induction of systemic resistance in chickpea against Fusarium wilt by Bacillus strains

Plant growth promoting rhizobacteria (PGPR) strains Rb29 (B. amyloliquefaciens MF352007), Bs1 (B. subtilis MF352017) and Bt1 (B. tequilensis MF352019) were tested for growth promotion and for their ability to induce systemic resistance against Fusarium wilt, a vascular disease of chickpea, using two methods that include whole plant and a split-root system. Bacillus strains and Fusarium oxysporum f. sp. ciceris (FOC) were inoculated on separate halves of roots of chickpea seedlings at the same time and then planted in separate pots either in superposition or one side of the other. All Bacillus strains systemically induced resistance against FOC, and significantly (p < 0.05) reduced the wilt disease by 98–100%. Application of Bacillus strains effectively enhanced plant growth, leading to increased plant height, root length, a fresh and dry weight of shoots and roots. These results help to explain the role of strains of Bacillus in growth promotion and biological control of Fusarium wilt in chickpea. This is the first report of systemic-induced resistance against Fusarium wilt in chickpea obtained by application of Bacillus strains to a root system spatially separated from the FOC-inoculated root.     

Novel Hyper Antimony-Oxidizing Bacteria Isolated from Contaminated Mine Soils in China

Antimony (Sb)-oxidizing bacteria play an important role in environmental Sb bioremediation because of their ability to convert the more toxic Sb(III) to the less toxic Sb(V). So far, the information about the Sb(III)-oxidizing bacteria species is still limited. In this study, three highly Sb(III)-resistant bacterial strains were isolated from contaminated mine soils after aerobic enrichment culturing with Sb(III) (1 mM). The morphological, biochemical, and 16S rRNA gene sequencing analysis suggested that the three novel bacterial isolates fell within Cupriavidus, Moraxella, and Bacillus, respectively. Among the strains, Moraxella sp. S2 isolated from soils with the highest Sb content exhibited the highest minimum inhibitory concentration for Sb(III) but the lowest Sb(III) oxidation efficiency, which could not completely oxidize 50 μM Sb(III) in 15 days. Cupriavidus sp. S1 was able to oxidize 50 μM Sb(III) completely in 12 days, but could not oxidize 100 μM Sb(III) even with extended time of incubation, while Bacillus sp. S3 with the lowest resistance to Sb(III) could aerobically oxidize 100 µM Sb(III) within 2 days, showing high Sb(III) oxidation efficiency. Our research demonstrated that indigenous microorganisms associated with Sb mine soils were capable of Sb oxidation, and the novel bacteria isolated could represent good candidates for Sb remediation in heavily polluted sites.     

Improvement in symbiotic efficiency of chickpea (Cicer arietinum) by coinoculation of Bacillus strains with Mesorhizobium sp. Cicer

Rhizobacteria belonging to Bacillus sp. were isolated from the rhizosphere of chickpea (Cicer arietinum). Ten Bacillus strains were studied for their antifungal activity, effect on seedling emergence and plant growth promotion. Two Bacillus strains CBS127 and CBS155 inhibited the growth of all the four pathogenic fungi tested on nutrient agar medium plates in vitro. Seed inoculation with different Bacillus strains showed stimulatory effect on root and shoot growth at 10 d of observation in comparison to control whereas four Bacillus strains CBS24, CBS127, CBS129 and CBS155 caused retardation of shoot growth at 10 d. Maximum nodule-promoting effect was observed with Bacillus strains CBS106, CBS127 and CBS155. The symbiotic effectiveness of Mesorhizobium sp. Cicer strain Ca181 was further improved on coinoculation with six Bacillus strains i.e. CBS9, CBS17, CBS20, CBS106, CBS127 and CBS155 at 80 d of plant growth under sterile conditions and shoot dry weight ratios increased 1.62 to 1.74 times those of Mesorhizobium-inoculated treatments, suggesting the usefulness of introduced rhizobacteria in improving crop productivity.     

Pullulanases of alkaline and broad pH range from a newly isolated alkalophilicBacillus sp. S-1 and aMicrococcus sp. Y-1

Summary Two highly alkalophilic bacteria, and potent producers of alkaline pullulanase, were isolated from Korean soils. The two isolates, identified asBacillus sp. S-1 andMicrococcus sp. Y-1, grow on starch under alkaline conditions and effectively secrete extracellular pullulanases. The two isolates were extremely alkalophilic since bacterial growth and enzyme production occurred at pH values ranging from pH 6.0 to 12.0 forMicrococcus sp. Y-1 and pH 6.0 to 10.0 forBacillus sp. S-1. Both strains secrete enzymes that possess amylolytic and pullulanolytic acitivities. Extracellular crude enzymes of both isolates gave maltotriose as the major product formed from soluble starch and pullulan hydrolysis. Compared to other alkalophilic microbes such asMicrococcus sp. (0.57 units ml^–1),Bacillus sp. KSM-1876 (0.56 units ml^–1) andBacillus No. 202-1 (1.89 units ml^–1) these isolates secreted extremely high concentrations (7.0 units ml^–1 forBacillus sp. S-1 and 7.6 units ml^–1 forMicrococcus sp. Y-1) of pullulanases in batch culture. The pullulanase activities from both strains were mostly found in the culture medium (85–90%). The extracellular enzymes of both bacteria were alkalophilic and moderately thermoactive; optimal activity was detected at pH 8.0–10.0 and between 50 and 60°C. Even at pH 12.0, 65% of original Y-1 pullulanase activity and 10% of S-1 pullulanase activity remained. The two newly isolated strains had broad pH ranges and moderate thermostability for their enzyme activities. These result strongly indicate that these new bacterial isolates have potential as producers of pullulanases for use in the starch industry.     

Endophytic Bacillus sp. Isolated from the Interior of Balloon Flower Root

A bacterial strain, designated CY22, was isolated from the interior of balloon flower (Platycodon grandiflorum) root in the Republic of Korea. The isolate coproduced an iturin-like antifungal compound and a surfactin-like potent biosurfactant. Analysis of the 16S-rDNA of strain CY22 showed that the isolate was a member of Bacillus. High similarities were observed between strain CY22 and Bacillus sp. TKSP 24, and between strain CY22 and B. subtilis 168. Phylogenetic analysis based on 16S-rDNA sequences showed that strain CY22 was closely related to Bacillus sp. The main whole-cell fatty acids were anteiso-C_15:0 (37%), C_17:0 (5.1%), and iso-C_15:0 (27.7%). DNA G + C content was 54 mol%. Based on phylogenetic inference, phenotypic and chemotaxonomic characteristics, this endophytic strain Bacillus sp. CY22 was assigned to the genus Bacillus.     

Amino Acid Substitutions and Intragenic Duplications of Bacillus sp. PS3 Flagellin Cause Complementation of the Bacillus subtilis Flagellin Deletion Mutant

Bacillus sp. PS3 produces a glycosylated flagellin. In this study, a number of the glycosylated residues of the flagellin protein were found to be located in the central variable region of this protein. We also report that the motility defect of the Bacillus subtilis flagellin mutant was complemented by Bacillus sp. PS3 flagellin variants without glycosylation, which contained amino acid substitutions and intragenic duplications in the variable region of flagellin.     

Sequence Analysis of a 32-kb Region Including the Major Ribosomal Protein Gene Clusters from Alkaliphilic Bacillus sp. Strain C-125

Forty-one open reading frames (ORFs) were identified in a 32-kb DNA fragment of alkaliphilic Bacillus sp. C-125. A similarity search using the BSORF database found 37 ORFs with significant sequence similarity to B. subtilis RNA polymerase subunits, elongation factor G, elongation factor Tu, and ribosomal proteins. Each ORF product showed more than 70% identity to those of B. subtilis. Gene organization in the region of str, S10, spc, and the α cluster was highly conserved among three strains, C-125, B. subtilis, and B. stearothermophilus.     

Biogenic Evidences of Moonmilk Deposition in the Mawmluh Cave,Meghalaya, India

Moonmilk, a microcrystalline secondary cave deposit, actively forms on the floor of Krem Mawmluh – a limestone cave in Meghalaya, Northeastern India. Due to the abundance of micrite and calcified microbial filaments, we hypothesize that these deposits form as a result of ongoing microbial interactions. Consistent with this idea, we report electron microscopic and microbiological evidences for the biological origin of moonmilk in Krem Mawmluh. Scanning electron microscopy indicated abundant calcified microbial filaments, needle calcite, fibre calcites (micro-fibre and nano-fibre calcite crystals), biofilm and microbial filaments in the moonmilk. The total viable culturable microbes showed high population densities for microbes in the moonmilk and moonmilk pool waters. In vitro culture experiments, confirmed the capability of many of the isolated strains to precipitate calcite and some of the identified isolates belonged to the Bacillus sp. and Actinomycetes. These results clearly support the biogenic nature of the deposits.     

A combined treatment using ethylmethane sulfonate and ultraviolet light to compare amylase production by three Bacillus sp. isolates

Abstract

In this study, three Bacillus sp.-producing amylase enzymes were isolated from soil samples and identified using 16S rDNA sequence analysis. Amylase production and total protein productions were spectrophotometrically measured. The following media were tested to increase enzyme production: LB medium and molasses. Three Bacillus sp. were identified as follows: Bacillus subtilis subtilis, Bacillus thuringiensis, and Bacillus cereus. Amylase production levels were in the range of 10?U/mL, whereas total protein production levels were at 15?mg/mL. Higher amylase activity was found in the Bacillus subtilis isolate. Ethylmethane sulfonate (EMS) and ultraviolet (UV) mutagenesis in combination were applied to compare amylase production. Amylase activity was increased to around 58% in the treatment with 0.03?mL of EMS and UV when compared to the control group. A pilot scale bioreactor with a total working volume of 10 liters was used to produce amylase by B. subtilis subtilis. In conclusion, B. subtilis subtilis can be used to produce amylase enzyme for various industrial purposes, and, for the first time, the amylase activities of B. subtilis can be enhanced with EMS and UV treatment.     

Production,purification and characterisation of a novel halostable xylanase from Bacillus sp. NTU-06

Bacillus sp. NTU-06 was used to produce xylanase, which is an important industrial enzyme used in the pulp and paper industry. The enzyme was purified by fast protein liquid chromatography (FPLC) and had a molecular mass of 24 kDa. The enzyme was active over a concentration range of 0–20% sodium chloride in culture broth, although its activity was optimal in 5% sodium chloride. A salinity stability test showed that 43% of the enzyme activity was retained after 4 h in 20% sodium chloride. Xylanase activity was maximal at pH 8.0 and 40°C. The enzyme was somewhat thermostable, retaining 20% of the original activity after incubation at 70°C for 4 h. The xylanase had K_m and V_max values of 3.45 mg mL⁻¹ and 387.3 µmol min⁻¹mg⁻¹, respectively. The deduced internal amino acid sequence of Bacillus sp. NTU-06 xylanase resembled the sequence of beta-1,4-endoxylanase, which is a member of glycoside hydrolase family 11. Some of the novel characteristics that make this enzyme potentially effective in xylan biodegradation are discussed.     

Restriction and modification in Bacillus species: genetic transformation of bacteria with DNA from different species, part I.

Summary Host specific restriction was detected in 13 Bacillus strains, when 63 strains of Bacillus subtilis and 15 other Bacillus strains were tested with phage 105C. These 13 strains were classified into 8 groups (M,H,C,N,E,F,G,P) by the type of restriction. M-type strains (B. subtilis Marburg 168, its derivatives, and two other strains) showed relatively weak restriction, restricting 105C from other groups of Bacillus by ratios of 10^-1 to 10^-3. Strains of groups H,C,N,E,F,G, and P restricted 105C from other groups by ratios of 10^-2 to 10^-8. It was confirmed with some of the strains that type-specific modification was endowed only by the last host. Furthermore, we isolated one restriction deficient mutant of B. subtilis marburg 168-YS11, which had also lost its modification phenotype.     

Alkaline protease from <Emphasis Type="Italic">Bacillus sp.</Emphasis> isolated from coffee bean grown on cheese whey

Two strains of Bacillus, one from a culture collection (B. subtilis ATCC 6633) and a wild type (Bacillus sp. UFLA 817CF) isolated during coffee fermentation in the south of Minas Gerais, Brazil, were evaluated in relation to secretion of alkaline proteases. The strains were grown on nutrient broth, nutrient broth with sodium caseinate and nutrient broth with three different concentrations of cheese whey powder for 72 h. Samples were collected at 24-h intervals to evaluate the proteolytic activity, protein content and cell population. Maximum protease activity was observed after 24-h growth for both the microorganisms, a period that coincided with the end of the exponential phase. The specific activity values were, respectively, 839.8 U/mg for B. subtilis ATCC 6633 and 975.9 U/mg for Bacillus sp. UFLA 817CF. The 60% saturation presented the best results for specific protease activity in all the growth culture media tested with B. sp. UFLA 817CF. Bacillus sp. UFLA 817CF showed highest enzymatic activity at pH 9.0 and 40°C in the three culture media tested. The protease obtained from culture of the wild Bacillus strain presented stability at pH 7.0 and considerable heat stability at 40°C and 50°C, and could be an alternative for the industry to utilize cheese whey to produce proteolytic enzymes.     

Impact of PGPR inoculation on growth and antioxidant status of wheat under saline conditions

Two plant growth‐promoting rhizobacterial (PGPR) strains, Bacillus subtilis SU47 and Arthrobacter sp. SU18, were found to tolerate 8% NaCl. Wheat co‐inoculated with these two PGPR strains, and grown under different salinity regimes (2–6 dS m^?1), showed an increase in dry biomass, total soluble sugars and proline content. Wheat sodium content was reduced under co‐inoculated conditions but not after single inoculation with either strain or in the control. The activity of antioxidant enzymes in wheat leaves decreased under salinity stress after PGPR co‐inoculation, suggesting these PGPR species could be used for amelioration of stress in wheat plants. Activity of three antioxidant enzymes in wheat grown with both PGPR strains was reduced, most notably that of catalase activity at a salinity of 6 dS m^?1, when compared with the control. The results indicate that co‐inoculation with B. subtilis and Arthrobacter sp. could alleviate the adverse effects of soil salinity on wheat growth.     

Sequencing of three lambda clones from the genome of alkaliphilic Bacillus sp. strain C-125

The nucleotide sequences of three independent fragments (designated no. 3, 4, and 9; each 15–20 kb in size) of the genome of alkaliphilic Bacillus sp. C-125 cloned in a λ phage vector have been determined. Thirteen putative open reading frames (ORFs) were identified in sequenced fragment no. 3 and 11 ORFs were identified in no. 4. Twenty ORFs were also identified in fragment no. 9. All putative ORFs were analyzed in comparison with the BSORF database and non-redundant protein databases. The functions of 5 ORFs in fragment no. 3 and 3 ORFs in fragment no. 4 were suggested by their significant similarities to known proteins in the database. Among the 20 ORFs in fragment no. 9, the functions of 11 ORFs were similarly suggested. Most of the annotated ORFs in the DNA fragments of the genome of alkaliphilic Bacillus sp. C-125 were conserved in the Bacillus subtilis genome. The organization of ORFs in the genome of strain C-125 was found to differ from the order of genes in the chromosome of B. subtilis, although some gene clusters (ydh, yqi, yer, and yts) were conserved as operon units the same as in B. subtilis. Received: April 17, 1998 / Accepted: June 23, 1998     

Isolation and evaluation of autochthonous Bacillus subtilis strains as probiotics for fat snook (Centropomus parallelus Poey, 1860)

The objective of this study was to isolate and select autochthonous strains of Bacillus subtilis from the fat snook, Centropomus parallelus, and examine the viability of the Bacillus bacteria to determine their beneficial effect on gut colonization in reared fish. Twenty strains of Bacillus were isolated and further confirmed as B. subtilis using PCR. Among the 20 strains, two strains (B02 and B03) exhibited an inhibitory performance against five tested pathogens. The Bacillus strains B02 and B03 were added to the fish ration, and after 30 days the number of viable colonies were maintained or increased under the conditions of refrigeration (5°C), freezing (?18°C), or room temperature (30°C). These strains showed a growth rate of 0.18–0.21 h and a doubling time of 3.34–3.9 h. Both strains were tolerant to variations in NaCl, and B03 was also tolerant to bile exposure. The ability to colonize the gastrointestinal tract was also examined in healthy fat snook juveniles fed diets enriched for 30 days with strains B02 and B03. The amount of B. subtilis in the gastrointestinal juvenile tract was significantly higher in fish fed the enriched ration compared to controls. Based on these results, strains B02 and B03 were considered as candidate probiotics for fat snook.     

Isolation and characterization of chromium(VI)-reducing Bacillus sp. FY1 and Arthrobacter sp. WZ2 and their bioremediation potential

Two native bacterial strains, FY1 and WZ2, that showed high chromium(VI)-reducing ability were respectively isolated from electroplating and tannery effluent–contaminated sites and identified as Bacillus and Arthrobacter. The objective of the present study was to evaluate their potential for future application in soil bioremediation. The results showed that both Bacillus sp. FY1 and Arthrobacter sp. WZ2 were tolerant to 1000 mg L^?1 Cr(VI) and capable of reducing 78–85% and 75–82% of Cr(VI) (100–200 mg L^?1) within 24 h, respectively. The Cr(VI) reduction rate decreased with increasing levels of Cr(VI) concentration (200–1000 mg L^?1). The optimum pH, temperature, and inoculum concentration for Cr(VI) reduction were found to be between pH 7.0 and 8.0; 30 and 35°C; and 1 × 10⁸ cells ml^?1, respectively. Further evidence for the bioremediation potential of Bacillus sp. FY1 and Arthrobacter sp. WZ2 was provided by the high capacity to reduce 100, 200, and 500 mg kg^?1 Cr(VI) in contaminated soil by 83–91%, 78–85%, and 71–78% within 7 days, respectively. These findings demonstrated the high potential of Bacillus sp. FY1 and Arthrobacter sp. WZ2 for application in future soil bioremediation.     

Cloning, purification, and characterization of chitosanase from Bacillus sp. DAU101

A chitosanase-producing Bacillus sp. DAU101 was isolated from Korean traditional food. This strain was identified on the basis of phylogenetic analysis of the 16S rDNA sequence, gyrA gene, and phenotypic analysis. The gene encoding chitosanase (csn) was cloned and sequenced. The csn gene consisted of an open reading frame of 837 nucleotides and encodes 279 amino acids with a deduced molecular weight of 31,420 Da. The deduced amino acid sequence of the chitosanase from Bacillus sp. DAU101 exhibits 88 and 30 % similarity to those from Bacillus subtilis and Pseudomonas sp., respectively. The chitosanase was purified by glutathione S-transferase fusion purification system. The molecular weight of purified enzyme was about 27 kDa, which suggests the deletion of a signal peptide by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The pH and temperature optima of the enzyme were 7.5 and 50 °C, respectively. The enzyme activity was increased by about 1.6-fold by the addition of 5 or 10 mM Ca²⁺. However, Hg²⁺ and Ni⁺ ions strongly inhibited the enzyme. The enzyme produced, GlcN_2–4, were the major products from a soluble chitosan.