Safety evaluation of an extension of use of the food enzyme ß-amylase from the non-genetically modified Bacillus flexus strain AE-BAF.

The food enzyme ß-amylase (4-α-d-glucan maltohydrolase, EC 3.2.1.2) is produced with the non-genetically modified Bacillus flexus strain AE-BAF by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in three food manufacturing processes. Subsequently, the applicant requested to extend its use to four additional processes and revised the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme for use in a total of seven food manufacturing processes. As the food enzyme-total organic solids (TOS) are removed from the final foods in one food manufacturing process, the dietary exposure to the food enzyme-TOS was estimated only for the remaining six processes. The dietary exposure was estimated to be up to 0.247 mg TOS/kg body weight per day in European populations. Based on the data provided for the previous evaluation and the dietary exposure revised in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Evaluating Bacillus flexus as bioremediators for ammonia removal in shrimp culture water and wastewater and characterizing microbial communities in shrimp pond sludge.

The accumulation of nitrogen compounds in shrimp farming water and effluent presents a major challenge. Ammonia is a form of nitrogen that limits shrimp growth due to its potential toxicity and effects on shrimp health and water quality. This study is aimed at identifying promising bioremediators from shrimp pond sludge to mitigate ammonia levels in both culture water and wastewater and at determining major bacterial communities in sludge using metagenomic analysis. A sludge sample was collected from a shrimp pond in Selangor, Malaysia, to isolate potential ammonia-removing bacteria. Out of 64 isolated strains, Bacillus flexus SS2 showed the highest growth in synthetic basal media (SBM) containing ammonium sulfate at a concentration of 70 mg/L as the sole nitrogen source. The strain was then incubated in SBM with varying pH levels and showed optimal growth at pH 6.5-7. After 24 h of incubation, B. flexus SS2 reduced the ammonia concentration from an initial concentration of 5 to 0.01 mg/L, indicating a 99.61% reduction rate, which was highest in SBM at pH 7. Moreover, the strain showed ammonia removal ability at concentrations ranging from 5 to 70 mg/L. Metagenomic analysis revealed that Proteobacteria was the most abundant phylum in the sludge, followed by Cyanobacteria, Actinobacteria, Chloraflexi, Firmicutes, and Campilobacterota. Bacillus flexus SS2 belongs to the Bacillota phylum and has the potential to serve as a bioremediator for removing ammonia from shrimp culture water and wastewater.

Oscillatoria sancta Cultivation Using Fruit and Vegetable Waste Formulated Media and Its Potential as a Functional Food: Assessment of Cultivation Optimization.

The most cost-effective technique to cultivate microalgae is with low-cost resources, like fruit and vegetable peels. This study examined the viability of culturing microalgae (Oscillatoria sancta PCC 7515) isolated from a waterlogged region of Punjab, India, in a low-cost medium (fruit and vegetable waste peels) for pharmaceutical use. 16S rRNA sequencing discovered O. sancta PCC 7515. Fruit and vegetable peels were mineralized and chemically analyzed. At a 5% Bacillus flexus concentration, fruit and vegetable peels were liquefied at room temperature for 24 h. Response Surface Methodology (RSM) was used to assess and improve important cultural variables. The RSM predicted the best results at 10 pH, 30 days of incubation, 5% inoculum concentration, and 5% fruit and vegetable waste liquid leachate. The optimum conditions yielded more biomass than the basal conditions (0.8001 g/L). O. sancta PCC 7515 produced more lipids, proteins, Chl a, and Chl b in a formulated alternate medium than standard media. This study shows that O. sancta PCC 7515 may thrive on fruit and vegetable peel media. Fruit and vegetable waste (FVW) media assure low-cost microalgae-based functional foods.

Characterization and Process Optimization for Enhanced Production of Polyhydroxybutyrate (PHB)-Based Biodegradable Polymer from Bacillus flexus Isolated from Municipal Solid Waste Landfill Site.

In recent years, there has been a growing interest in bio-based degradable plastics as an alternative to synthetic plastic. Polyhyroxybutyrate (PHB) is a macromolecule produced by bacteria as a part of their metabolism. Bacteria accumulate them as reserve materials when growing under different stress conditions. PHBs can be selected as alternatives for the production of biodegradable plastics because of their fast degradation properties when exposed to natural environmental conditions. Hence, the present study was undertaken in order to isolate the potential PHB-producing bacteria isolated from the municipal solid waste landfill site soil samples collected from the Ha'il region of Saudi Arabia to assess the production of PHB using agro-residues as a carbon source and to evaluate the growth of PHB production. In order to screen the isolates for producing PHB, a dye-based procedure was initially employed. Based on the 16S rRNA analysis of the isolates, Bacillus flexus (B. flexus) accumulated the highest amount of PHB of all the isolates. By using a UV-Vis spectrophotometer and Fourier-transform infrared spectrophotometer (FT-IR), in which a sharp absorption band at 1721.93 cm-1 (C=O stretching of ester), 1273.23 cm-1 (-CH group), multiple bands between 1000 and 1300 cm-1 (stretching of the C-O bond), 2939.53 cm-1 (-CH3 stretching), 2880.39 cm-1 (-CH2 stretching) and 3510.02 cm-1 (terminal -OH group), the extracted polymer was characterized and confirmed its structure as PHB. The highest PHB production by B. flexus was obtained after 48 h of incubation (3.9 g/L) at pH 7.0 (3.7 g/L), 35 °C (3.5 g/L) with glucose (4.1 g/L) and peptone (3.4 g/L) as carbon and nitrogen sources, respectively. As a result of the use of various cheap agricultural wastes, such as rice bran, barley bran, wheat bran, orange peel and banana peel as carbon sources, the strain was found to be capable of accumulating PHB. Using response surface methodology (RSM) for optimization of PHB synthesis using a Box-Behnken design (BBD) proved to be highly effective in increasing the polymer yield of the synthesis. With the optimum conditions obtained from RSM, PHB content can be increased by approximately 1.3-fold when compared to an unoptimized medium, resulting in a significant reduction in production costs. Thus, isolate B. flexus is a highly promising candidate for the production of industrial-size quantities of PHB from agricultural wastes and is capable of removing the environmental concerns associated with synthetic plastics from the industrial production process. Moreover, the successful production of bioplastics using a microbial culture provides a promising avenue for the large-scale production of biodegradable and renewable plastics with potential applications in various industries, including packaging, agriculture and medicine.

Production, purification, characterization, and applications of α-galactosidase from Bacillus flexus JS27 isolated from Manikaran hot springs.

α-Galactosidase hydrolyzes the α-1,6-linkage present at the non-reducing end of the sugars and results in the release of galactosyl residue from oligosaccharides like melibiose, raffinose, stachyose, etc. In the present study we report, α-galactosidase from Bacillus flexus isolated from Manikaran hot springs (India). Maximum enzyme production was obtained in guar gum and soybean meal after 72 h at 150 rpm. While, the temperature/pH of production was optimized at 50 °C and 7.0, respectively. Isoenzymes (α-gal I and II) were obtained and characterized based on temperature/pH optima along with their stability profile. JS27 α-Gal II was purified with a final purification fold of 11.54. Native and SDS-PAGE were used to determine the molecular weight of the enzyme as 86 and 41 kDa, respectively, indicating its homodimeric form. JS27 α-Gal II showed optimum enzyme activity at 55 °C and pH 7 (10 min). The enzyme displayed Km value of 2.3809 mM and Vmax of 2.0 × 104 µmol/min/ml with pNPG as substrate. JS27 α-Gal II demonstrated substrate hydrolysis and simultaneous formation of transgalactosylation products (α-GOS) with numerous substrates (sugar/sugar alcohols, oligosaccharides, and complex carbohydrates) which were verified by TLC and HPLC analysis. α-GOS are significant functional food ingredients and can be explored as prebiotics.

Haloalkaline Lipase from Bacillus flexus PU2 Efficiently Inhibits Biofilm Formation of Aquatic Pathogen Vibrio parahaemolyticus.

A gene encoding lipase enzyme from Bacillus flexus PU2 was cloned and expressed in E. coli BL21 (DE3) pLysS and purified protein having the molecular weight of 34 kDa. This lipase was found to be alkaline (pH 9) and slightly thermophilic. This lipase was observed to retain its activity in the presence of methanol, ethanol, DMSO, and acetone. Ferrous sulfate, copper sulfate, and manganese sulfate highly enhanced the lipase activity. All the surfactants and detergents were found to inhibit the enzyme activity, whereas the bleaching agent hydrogen peroxide was found to increase the activity. This lipase was observed as a metalloenzyme, and its activity was highly inhibited by EDTA. Also, it is moderately halophilic and can retain the activity between 0.2 and 0.8 M NaCl. Biofilm inhibitory potential of purified lipase was tested against pathogenic Vibrio parahaemolyticus, and the minimal inhibitory concentration observed was 350 U. Different concentration of this enzyme significantly changed the morphology and biofilm density of V. parahaemolyticus and was evinced by SEM and CLSM imaging. The transcriptome levels of genes responsible for biofilm formation, motility, and virulence such as, motX, fliG, and trh were significantly downregulated with lipase treatment.

Structural characterization and active site prediction of keratinase from Bacillus flexus.

Sixteen keratinolytic bacteria were isolated from poultry farm soil samples. The highest keratinlytic enzyme producers of Bacillus flexus was confirmed with 16S rRNA sequence analysis. It is of interest to understand the binding efficiency of the modelled keratinase from Bacillus flexus with different substrates using molecular docking studies. Data provides insights for the identification of substrate recognition patterns, and the development of suitable enzymes to improve their use in keratin degradation.

Structural assessment of the bioplastic (poly-3-hydroxybutyrate) produced by Bacillus flexus Azu-A2 through cheese whey valorization.

The demand for the production of biodegradable plastics has significantly increased. Bioplastics have become an essential alternative to the threats of the daily consumable plastics, sourced from fossil fuels, to the environment. Polyhydroxyalkonates (PHAs) are a ubiquitous group of bioderived and biodegradable plastics, however their production is limited by the costs associated mainly with the carbon sources. Herein, this study aims to reduce the PHAs production cost by using a by-product from the dairy industry, i.e., cheese whey (CW), as a sole carbon source. The developed process recruits an aquatic isolate, Bacillus flexus Azu-A2, and is optimized via studying various parameters using the shaking flasks technique. The results showed that the maximum PHA production (0.95 g L-1) and PHA content (20.96%, w/w), were obtained after incubation period 72 h at 45 °C, 100 rpm agitation rate, 50% CWS concentration, pH 8.5, and 1.0 g L-1 ammonium chloride. Physiochemically, Fourier transform infrared spectroscopy (FTIR), gas chromatography-mass spectroscopy (GC-MS), nuclear magnetic resonance (NMR), and energy-dispersive X-ray (EDX) techniques, emphasized the type of the extracted PHA as polyhydroxybutyrate (PHB). The thermal properties of PHB were measured using differential scanning calorimetry (DSC), recording melting transition temperature (Tm) at 170.96 °C. Furthermore, a scanning electron microscope (SEM) visualized a homogenous microporous structure for the thin PHB biofilm. In essence, this study highlights the ability of Bacillus flexus Azu-A2 to produce a good yield of highly purified PHB at reduced production cost from dairy CW. Consequently, the current study paves the way for an improved whey management strategy.

Enzymatic characterization of the saliva of the eriophyid mite, Aceria pongamiae Keifer1966 (Acari: Eriophyidae) and the bacterial endobiome of the galls induced on Pongamia pinnata (L.) Pierre (Fabaceae).

Galls, like other regular plant organs, possess their own histological and physiological features. A high degree of specificity is maintained between the host and the inducer, and hence gall morphogenesis is highly conserved and would help trace gall lineages and cell fate. The present study highlights the induction and subsequent development of leaf galls on the Indian Beech tree, Pongamia pinnata (L) Pierre (Fabaceae), mediated through the active participation of a gall-inducing species of eriophyid mite, Aceria pongamiae Keifer and gall-associated bacterial endobiome. The saliva of A. pongamiae and selected strains of gall-associated bacterial endobiome were characterized in part during the study. Three strains of Staphylococcus arlettae (PGP1-3) and one strain of Bacillus flexus (PGP4) were identified from the leaf galls through 16S rDNA sequencing. The mite saliva displayed tryptophanase activity, and the bacterial strains showed differential enzyme activities (protease, amylase, cellulase, DNAse, pectinase, tryptophanase, and catalase). All four strains of bacterial endobiome exhibited unique metal tolerance as well as pH and temperature regulating activity. Evaluation of the potential role of the mite saliva and the gall associated bacterial endobiome in gallogenesis was done by monitoring the plant growth-promoting activity of the salivary extract and the isolated bacterial strains through in vitro seed (Vigna radiata) germination assay. Salivary extract of the mite showed the highest rate of plant growth-promoting activity compared with that of the isolated strains of bacterial endobiome. The present study forms the first attempt that illustrates the characteristic features of the saliva of the gall inducer and the gall associated bacterial endobiome. Based on the results of the current study, we suggest that eriophyid mite saliva and the gall-associated microbes play significant roles in the induction of cecidia.

Safety evaluation of the food enzyme ß-amylase from Bacillus flexus strain AE-BAF.

The food enzyme ß-amylase (4-α-d-glucan maltohydrolase, EC 3.2.1.2) is produced with the non-genetically modified Bacillus flexus strain AE-BAF by Amano Enzyme Inc. The production strain has been shown to qualify for Qualified Presumption of Safety (QPS) status. The food enzyme is intended to be used in baking and brewing processes, and in starch processing for the production of glucose syrups and other starch hydrolysates. Since residual amounts of total organic solids (TOS) are removed by the purification steps applied during the production of glucose syrups, dietary exposure was not calculated for this food process. Based on the maximum use levels recommended by the applicant for the baking and brewing processes and individual data from the EFSA Comprehensive European Food Database, dietary exposure was estimated to be up to 2 mg TOS/kg body weight (bw) per day in European populations. Toxicological studies were not considered necessary given the QPS status of the production strain and the nature of the manufacturing process. Similarity of the amino acid sequence to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the data provided, in particular, the QPS status of the production strain and that no issues of concern arose from the production process, the Panel concluded that the food enzyme ß-amylase produced with B. flexus strain AE-BAF does not give rise to safety concerns under the intended conditions of use.

Bioremediation of mortar made from Ordinary Portland Cement degraded by Thiobacillus thioparus using Bacillus flexus.

Cement is widely used as a construction material in the construction industry. However, there are challenges affecting its durability efficacy. Cement mortar/concrete is subject to degradation by aggressive ions such as sulphates and chlorides. Sulphates can be introduced into the concrete or mortar by Sulphur producing bacteria of the species Thiobacilli. Microbiologically induced CaCO3 precipitation (MICP) has found its application in bioremediating cement based materials. It has been found to be environmental friendly. However, no work has been reported on bioremediation of biodegraded cement based materials. This paper presents findings of possible bioremediation of mortars after undergoing biodegradation. Bacillus flexus, a beneficial bacterium was used. The control mortars were prepared using Ordinary Portland Cement (OPC). The test mortars were prepared and cured in a solution of Thiobacillus thioparus, a Sulphur oxidizing bacteria, deleterious bacterium for 14, 28, 56 and 90 days. Compressive strength analysis was conducted on the 14th, 28th, 56th and 90th day of curing. Results showed that the lowest compressive strength was recorded on the 90th day as 31.02 MPa. This was a 34.17 % loss in compressive strength. Another category of mortar cured in Thiobacillus thioparus for 28 days was bioremediated for 28 days using Bacillus flexus solution. Compressive strength and Scanning Electron Microscopy (SEM) analyses were then done. The results show a compressive strength of 45.83 MPa at the 56th day. This represents a 99.91 % strength recovery from biodeterioration. The SEM analysis results revealed a denser material. This was due to massive precipitation of calcium carbonate in the mortar matrix and pores/voids for bioremediated mortars as opposed to the biodegraded mortars. The results further revealed reduced ettringite crystals on the bioremediated mortars. Bacillus flexus could perhaps be used in restoring lost compressive strength as well as in sealing voids in degraded concrete in sewer lines and other cement based materials. This could improve on its efficacy with minimal repair.

Root exudates-driven rhizosphere recruitment of the plant growth-promoting rhizobacterium Bacillus flexus KLBMP 4941 and its growth-promoting effect on the coastal halophyte Limonium sinense under salt stress.

Halophytes play an important role in the bioremediation of saline soils. Increased evidence has revealed that plant growth-promoting rhizobacteria (PGPR) have colonized the halophytic rhizosphere, and they have evolved the capacity to reduce salt stress damage to the host. However, the mechanism by which halophytes attract and recruit beneficial PGPR has rarely been reported. This study reports the interaction between the halophyte Limonium sinense and its rhizosphere PGPR strain Bacillus flexus KLBMP 4941, as well as the mechanism by which KLBMP 4941 promotes host plant growth under salt stress. After salt stress treatment, we collected the root exudates (REs) of L. sinense and found that the REs could promote the growth and chemotaxis of the bacterium KLBMP 4941. In addition, the components of the REs under salt stress were analyzed, and some organic acids (2-methylbutyric acid, stearic acid, palmitic acid, palmitoleic acid, and oleic acid) were detected as the major components. Further assessment showed that each of these components had positive effects on the growth, motility, chemotaxis, and root colonization of strain KLBMP 4941. Further pot experiments revealed the potential PGP mechanisms induced by strain KLBMP 4941 on the host plant under salt stress. Inoculation with KLBMP 4941 promoted the accumulation of chlorophyll to enhance photosynthesis, increased osmotic regulator contents, enhanced flavonoid and antioxidant enzymes, and regulated Na+/K+ homeostasis to help the host ameliorate salinity stress damage. Our findings indicate that the halophyte L. sinense can attract and recruit beneficial rhizosphere bacteria by REs under salt stress, and the recruited B. flexus KLBMP 4941 elicited PGP effects under salinity stress through complex plant physiological regulatory mechanisms. This study provides a foundation for the enhancement of the rhizosphere colonization ability of the PGP strain KLBMP 4941, which shows potential applications in phytoremediation of saline soils.

Rhizosphere mediated biodegradation of benzo(A)pyrene by surfactin producing soil bacilli applied through Melia azadirachta rhizosphere.

Benzo(a)pyrene is a high-molecular-weight polycyclic aromatic hydrocarbon highly persistent in the environment as a biohazard. The present research emphasizes on rhizodegradation of BaP using bacterial isolates, Bacillus flexus S1I26 (NCBI accession no- KX692271), and Paenibacillus sp. S1I8 (KX602663) with plant Melia azadirachta. The isolates produced surfactin type bio-surfactant with high emulsification index that could solubilize BaP efficiently. The extracted crude bio-surfactants could solubilize BaP up to 24.41%, which was higher than the efficiency of synthetic surfactant SDS (9.7%) but less than other synthetic surfactant, tweens 80 (42.79%). The isolates showed excellent degradation of BaP after 21 days in laboratory conditions where B. flexus S2I26 showed degradation of BaP up to 70.7% and isolates Paenibacillus sp. S1I8 showed degradation rate of 76.76% in a liquid medium. Pot trial experiment showed efficient rhizodegradation of BaP in the soil after 60 days in the rhizosphere of plant Melia azadirachta. After application of S1I8 and S1I26, the rate of degradation was found to be much higher (87.42 and 86.08%) than in bulk (68.22%). Therefore, the results suggest that the bio-surfactant producing isolates could be a promising biodegradation tool for benzo(a)pyrene in soil and may be used for bioremediation of hydrocarbon contaminated sites.

Response surface methodology mediated optimization of Indanthrene Blue RS by a novel isolated bacterial strain Bacillus flexus TS8.

The enhanced decolorization and detoxification of Indanthrene Blue RS dye, under aerobic conditions, by a novel isolated anthraquinone-degrading bacterium, Bacillus flexus TS8, has been presented in this paper. The optimal decolorization conditions were determined by response surface methodology based on Box-Behnken design. The results indicated that the strain TS8 possessed the highest decolorization efficacy at pH 10.26, temperature 30.97 ºC and an inoculum size of 10.48% (v/v). It also revealed that about 98.01% of 100 mg/L of Indanthrene Blue RS could be decolorized within 24 hr under these optimized conditions. The subsequent degradation of the dye and the formation of metabolites were studied using analytical techniques such as UV-Vis spectroscopy, FTIR, and ESI/LC-MS analysis. The UV-Vis analysis of the colorless bacterial cells demonstrated that Bacillus sp. TS8 possessed this decolorizing activity through biodegradation. The degraded products obtained from ESI/LC-MS analysis were identified as 1-hydroxyanthracene-9, 10-dione (m/z-224), 1, 4-di-hydroxyanthracene-9, 10-dione (m/z-240), and phthalic acid (m/z-168). This study investigated the highest decolorization efficacy of strain TS8 to be utilized in the biological treatment of wastewaters containing anthraquinone dyes. PRACTITIONER POINTS: Enhanced decolorization of anthraquinone dye wastewater. Ninety-eight percentage of dye decolorization was obtained within 24 hr. Optimization of process parameters through the response surface methodology. ESI/LC-MS analysis identified phthalic acid as the end product of Indanthrene Blue RS degradation. Degradation pathway for Indanthrene Blue RS is outlined.

Scale Up Studies for Polyhydroxyalkanoate Production by a Bacillus flexus Strain with Industrial Potential.

Polyhydroxyalkanoates (PHA) are synthesized by bacteria under unfavourable growth conditions like excess of carbon over nitrogen, coupled with oxygen limitation. The PHA polymers of microbial origin are diverse in chemical composition and material properties. A bioprocess for PHA production by indigenously isolated Bacillus flexus MTCC 12841 was devised and optimized at a laboratory fermentor scale. Fermentation strategies that involved modifications in some parameters like aeration, agitation, temperature, nutrient feeding or changes in C:N ratio led to substantial improvement of 59% in PHA production reaching highest concentration of 9.73 g/L. Biomass too was enhanced to 15.70 g/L equivalent to 126% increase over the optimized shake flask runs. PHA (Yp/s) and biomass (Yx/s) yields were found to be 0.32 and 0.51 g/g respectively, indicating good carbon utilization efficiency. The characterization of polymer by GC-MS revealed that the culture produced poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) as a co-polymer. The novelty of the research findings lies in the demonstration of increased production of PHA at lab fermentor level coupled with the identification of the natural ability of the strain to also produce PHBV without any need for exogenous addition of precursors. The fermentation process as well as the strain may be subjected to further optimization to increase the PHA production as well as to increase the % of HV content in the co-polymer.

Polyvinyl chloride biodegradation by Pseudomonas citronellolis and Bacillus flexus.

The accumulation of high amounts of petroleum-derived plastics in the environment has raised ecological and health concerns. The aim of this work was to study the biodegradative abilities of five bacterial strains, namely Pseudomonas chlororaphis, Pseudomonas citronellolis, Bacillus subtilis, Bacillus flexus and Chelatococcus daeguensis, towards polyethylene, polypropylene, polystyrene and polyvinyl chloride films under aerobic conditions. Preliminary screening resulted in the selection of P. citronellolis and B. flexus as potential PVC film degraders. Both strains were able to form a biofilm on the plastic film surface and to cause some modifications to the FTIR spectra of biomass-free PVC films. The two strains were then used to set up a PVC film biodegradation assay in 2-liter flasks. After 45 days incubation, fragmentation of the film was observed, suggesting that PVC biodegradative activity took place. Gel permeation chromatography analysis showed a reduction in average molecular weight of 10% for PVC incubated with P. citronellolis, with PVC polymer chains apparently attacked. Based on these results, the P. citronellolis strain was selected for biodegradation assays of two waste PVC films, used either nonsterile or subjected to ethanol sterilization. Chemical analyses on the incubated films confirmed the biodegradation of waste PVC plastics as shown by a gravimetric weight loss of up to about 19% after 30 days incubation. In summary, this work reports the biodegradation of PVC films by P. citronellolis and B. flexus. Both strains were shown to act mainly against PVC additives, exhibiting a low biodegradation rate of PVC polymer.

Complete genome sequence of endophyte Bacillus flexus KLBMP 4941 reveals its plant growth promotion mechanism and genetic basis for salt tolerance.

Bacillus flexus KLBMP 4941 is a halotolerant endophyte isolated from the halophyte Limonium sinense. This strain can improve host seedling growth under salt stress conditions. We here report the complete genome information of endophyte KLBMP 4941. It has a circular chromosome and two plasmids for a total genome 4,104,242 bp in size with a G+C content of 38.09%. Genes related to plant growth promotion (PGP), such as those associated with nitrogen fixation, siderophore, spermidine, and acetoin synthesis were found in the KLBMP 4941 genome. Some genes responsible for high salinity tolerance, like genes associated with the Na+/H+ antiporter, glycine betaine transporter, and betaine-aldehyde dehydrogenase were also found in the KLBMP 4941 genome. The genome analysis will provide better understanding of the mechanisms underlying the promotion of plant growth in strain KLBMP 4941 under salt stress conditions and its ability to adapt to coastal salt marsh habitats, and provide a basis for its further biotechnological applications in agriculture.

Purification and properties of beta-cyclomaltodextrin glucanotransferase from Bacillus flexus SV 1.

Cyclomaltodextrin glucanotransferase is a unique enzyme that degrades starch into cyclic oligosaccharides called cyclodextrins, which have numerous applications in various industries such as pharmaceutical, textile, agricultural, cosmetics etc. Due to its wide applications, microorganism producing one type of cyclodextrin is of interest as it simplifies the down streaming process of separating mixture of cyclodextrins. In the present study, ß-CGTase was isolated from Bacillus flexus SV 1 and biochemically characterized. Enzyme was purified by starch adsorption followed by DEAE cellulose column chromatography which resulted in a fold purification of 6.1, with a yield of 44.07%. Molecular weight of the purified enzyme was found to be 96.68 kDa, enzyme was monomeric in nature with a Km and Vmax of 0.08976 µmol mL-1 and 585.1 µmol/ml/min, respectively. Optimum pH and temperature of the purified enzyme was found to be 8.0 and 60 °C. Ca2+ showed significant increase in enzyme activity. The inhibition of enzyme by EDTA indicates that CGTase is a metalloenzyme. CGTase produced majorly ß-CD and was alkalotolarent and active at high temperatures which is a promising candidate for various industries such as textile, food, agriculture, and pharmaceuticals.

Aerobic degradation of fenvalerate by a Gram-positive bacterium, Bacillus flexus strain XJU-4.

Synthetic pyrethroid-fenvalerate-is one of the most widespread toxic pollutants and has adverse effect on living systems. However, little is known about its biotransformation mechanism in different microorganisms. To elucidate the pathway that might be involved in the catabolism of fenvalerate, we used Bacillus flexus strain XJU-4 (3-nitrobenzoate degrading organism) as an ideal fenvalerate degrading bacterium. Thin layer chromatography, high performance liquid chromatography and gas chromatography-mass spectrometry analysis results revealed that 3-phenoxybenzoate, protocatechuate, and catechol are the three main by-products of fenvalerate metabolism. Additionally, the bacterial cell-free enzymes showed the activities of fenvalerate hydrolyzing esterase, 3-phenoxybenzaldehyde dehydrogenase, 3-phenoxybenzoate dioxygenase, phenol hydroxylase, protocatechuate 2,3-dioxygenase and catechol-2,3-dioxygenase. Thus, in strain XJU-4, protocatechuate and catechol were further metabolized through meta-cleavage pathway. Moreover, laboratory-scale soil experiments results suggest that B. flexus strain XJU-4 is a suitable contender for bioremediation of pyrethroid fenvalerate-contaminated sites.

Bacillus flexus strain As-12, a new arsenic transformer bacterium isolated from contaminated water resources.

A total of 14 arsenic-resistant bacteria were isolated from an arsenic-contaminated travertine spring water in the central district of Qorveh county, Kurdistan Province, Iran. One of strains designated As-12 was selected for further investigation because of its ability to transform arsenic. The strain was identified by cultural, morphological and biochemical tests, and 16S rRNA gene sequencing. Finally, the growth characteristics of the isolate were investigated in a chemically defined medium which included varied ranges of environmental factors such as pH, temperature and salinity. Moreover, the resistance of this strain to some heavy metals was evaluated. The bacterium was a Gram-positive, endospore-forming with all other characteristics of the genus Bacillus. It revealed maximum similarity at the 16S rRNA gene level with Bacillus flexus. The optimum growth of the strain was observed at 38 °C, pH 9 and 2% salinity. This strain was resistant to heavy metals such as zinc, chromium, lead, nickel, copper, mercuric and cadmium at concentrations of 15 mM, 15.5 mM, 11.5 mM, 12 mM, 11 mM, 5.5 mM, and 1 mM, respectively. The isolated bacterium was able to reduce As (V) to As (III) (about 28%) and oxidize As (III) to As (V) (about 45%) after 48 h of incubation at 37 °C. In conclusion, Bacillus flexus strain As-12, was identified as an arsenic transformer, for the first time.