Alkalophilic Bacterium Research Articles

Allosteric mechanism of MntR transcription factor from alkalophilic bacterium Halalkalibacterium halodurans

Bacterium Halalkalibacterium halodurans is an industrially important alkalophilic bacteria. It is recognized as a producer of enzymes such as β-galactosidase, xylanase, amylase and protease which are able to function at higher pH values and thus can be used in textile, food, paper industry and more. This bacterium, as any other bacterium, requires a sensitive mechanism for regulation of homeostasis of manganese ions (Mn2+) in order to survive. The key protein regulating this mechanism in H. halodurans is MntR – a transcriptional factor that binds to DNA and regulates the transcription of genes for proteins involved in manganese homeostasis. Long range all-atom molecular dynamics (MD) simulations, from 500 ns up to 1.25 µs, were used to study different forms of H. halodurans MntR in order to investigate the differences in the protein’s structural and dynamical properties upon Mn2+ binding. Simulations revealed an allosteric mechanism which is activated by Mn2+ binding. The results of simulations show that Mn2+ binding alters the non-covalent interaction network of the protein structure which leads to a conformational change that primarily affects the positions of the DNA binding domains and, consequently, the DNA binding affinity of H. halodurans MntR. The key amino acid residues of the proposed mechanism were identified and their role in the proposed mechanism was computationally confirmed by MD simulations of in silico mutants. Communicated by Ramaswamy H. Sarma

Characterization of a recombinant endo-β-1,3-glucanase from Alkalihalobacillus Clausii KSM-K16 and its application in the production of curdlan oligosaccharides

Curdlan polysaccharides are hydrolyzed to provide bioactive β-1,3-gluco-oligosaccharides, which are finding increasingly used in biomedical applications. Alkalihalobacillus clausii KSM-K16 (AC-GA) is an alkalophilic bacterium endo-β-1,3-glucanase. It has a predicted molecular weight of 30.09 kDa and is made up of 257 amino acids. It was expressed in Escherichia coli and purified to homogeneity. This study analyses the properties of AC-GA, product distribution, and structural aspects of AC-GA that may be relevant to sugar molecule production. The optimum reaction condition of the enzyme is 70 °C, pH 7.0, and its catalytic activity increased by Mn2+. Meanwhile, Zn2+ and Cu2+ inhibited AC-GA activity. It is worth mentioning that the isolated enzyme demonstrated remarkable stability at both high temperature, and neutral environments, when curdlan was utilized as the substrate, the enzyme activity reached 1008.98 U/mg. Hydrolysates of curdlan polysaccharide were β-1,3-gluco-oligosaccharides with polymerization (DP) degrees ranging from 1 to 5. This implies that the AC-GA active site might identify up to five glucose units. High concentration of AC-GA mostly creates glucotriose (DP3), whilst low concentration of AC-GA produces oligosaccharides with varying DP (primarily DP1, DP3, and DP4). Additionally, the catalytic triad (Glu134, Asp136, and Glu139) plays a decisive role in the activity of AC-GA, and structural analysis revealed that Trp129, Trp118, Trp114 and Trp 232 are involved in hydrophobic stacking interactions. This study provides a theoretical basis for the acquisition of curdlan oligosaccharides with higher application value under high-temperature hydrolysis.

Efficient lignin biodegradation triggered by alkali-tolerant ligninolytic bacteria through improving lignin solubility in alkaline solution

Low lignin solubility in aqueous solution is one of the major bottlenecks for lignin biodegradation and bioconversion. Alkaline solution contributes to improving lignin solubility, whereas most microbes can not survive in alkaline conditions. Herein, lignin dissolution behaviors in different pH solutions were systematically investigated, which indicated that solution pH above 10.5 contributed to high solubility of alkali lignin. To match with alkaline lignin aqueous system, several alkali-tolerant ligninolytic bacteria were isolated, most of which are distinct to previously reported ones. Then, the ligninolytic capabilities of these isolates were assessed in different pH conditions by determining their assimilation on alkali lignin, lignin-derived monomers and dimers, their decolorization capabilities, and their lignin peroxidase activities. Thereafter, the underlying ligninolytic and alkali-tolerant mechanisms of Sutcliffiella sp. NC1, an alkalophilic bacterium, was analyzed on the basis of its genome information. The results not only provide valuable information for lignin biodegradation and lignin valorization, but also expand knowledge on alkali-tolerant bacteria.

Persistent Dysbiosis, Parasite Rise and Growth Impairment in Aquacultured European Seabass after Oxytetracycline Treatment.

The use of antibiotics in open-water aquaculture is often unavoidable when faced with pathogens with high mortality rates. In addition, seasonal pathogen surges have become more common and more intense over the years. Apart from the apparent cost of antibiotic treatment, it has been observed that, in aquaculture practice, the surviving fish often display measurable growth impairment. To understand the role of gut microbiota on the observed growth impairment, in this study, we follow the incidence of Photobacterium damselae subsp. piscicida in a seabass commercial open-water aquaculture setting in Galaxidi (Greece). Fish around 10 months of age were fed with feed containing oxytetracycline (120 mg/kg/day) for twelve days, followed by a twelve-day withdrawal period, and another eighteen days of treatment. The fish were sampled 19 days before the start of the first treatment and one month after the end of the second treatment cycle. Sequencing of the 16S rRNA gene was used to measure changes in the gut microbiome. Overall, the gut microbiota community, even a month after treatment, was highly dysbiotic and characterized by very low alpha diversity. High abundances of alkalophilic bacteria in the post-antibiotic-treated fish indicated a rise in pH that was coupled with a significant increase in gut parasites. This study's results indicate that oxytetracycline (OTC) treatment causes persistent dysbiosis even one month after withdrawal and provides a more suitable environment for an increase in parasites. These findings highlight the need for interventions to restore a healthy and protective gut microbiome.

Bio-deposition approaches for sustainable execution of recycled aggregates in concretes

Our study offers a quantitative framework for microbial-induced calcium carbonate precipitation (MICP) to uplift the properties of recycled aggregate concrete (RAC). In this regard, a marine alkalophilic bacterium Bacillus sp. B6 was employed, and its growth and mineralization efficiency under seawater conditions was investigated. Optimization of MICP was achieved with different nutrient sources and bacterial introduction methods (dip and spray). The efficiency of treated recycled aggregates (RA) was determined by using scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) in combination with basic technologies of density improvement and water absorption. The results show that the optimal nutrient sources of carbon and nitrogen are glucose (7 g L−1) and tryptone (5 gL−1), respectively. Spray treated recycled aggregate (RA) is reported to be efficient with a reduction in water absorption (∼19%), and improvement in bulk (∼2.4%) and apparent density (∼1.7%). The effectiveness of the MICP treatment process is confirmed with SEM and EDX observations, indicating the filling of microcracks with calcium carbonates. As such, the mechanical properties of RAC with spray treated RA significantly increase by up to ∼24% in compressive strength as compared to the control treatment. Current findings will help revitalize the construction industry by utilizing the MICP-treated RA and minimizing the load on natural aggregates.

Bacillus Swezeyi B2 Strain: A Novel Alkaliphilic Bacterium Producer of Alkaline-, Thermal, Oxidant-, and Surfactant-Stable Protease, Extremely Efficient in Detergency.

Proteolytic enzymes that are currently used to meet industrial demand are usually derived from Bacillus species. They find multiple technical applications, particularly they have been increasingly used as a key bio-additive in detergents. In this study, a novel alkalophilic bacterium was isolated from contaminated soil, exhibiting 1400U/ml proteolytic activity, and identified as Bacillus swezeyi B2. The crude enzyme likely contained a single extracellular protease. This enzyme revealed optimum activity at pH 10 and 70°C and was highly alkaline thermostable (7-12.5) and up to 70°C. The protease activity was completely inhibited by Phenylmethylsulfonyl fluoride (PMSF) suggesting that it belongs to the serine protease group. It was highly stable in the presence of the strong anionic surfactant (SDS) and oxidizing agents (H2O2). The supernatant was lyophilized and showed high storage stability retaining 100% of its original activity after one year of conservation at 4°C. The lyophilized product was evaluated for its detergent efficacy, it revealed excellent compatibility with various laundry detergents keeping its full original activity after incubation for 1h with seven solid and liquid commercial detergents and it effectively removed chocolate stains at low washing temperature (40°C) and low supplementation level (125U/ml). The features of this single alkaline and thermotolerant protease, stable toward surfactants, oxidizing agents, and commercial detergents with stain removal efficacy support its ideal choice for supplementation in detergent formulations.

The Eastern Nebraska Salt Marsh Microbiome Is Well Adapted to an Alkaline and Extreme Saline Environment.

The Eastern Nebraska Salt Marshes contain a unique, alkaline, and saline wetland area that is a remnant of prehistoric oceans that once covered this area. The microbial composition of these salt marshes, identified by metagenomic sequencing, appears to be different from well-studied coastal salt marshes as it contains bacterial genera that have only been found in cold-adapted, alkaline, saline environments. For example, Rubribacterium was only isolated before from an Eastern Siberian soda lake, but appears to be one of the most abundant bacteria present at the time of sampling of the Eastern Nebraska Salt Marshes. Further enrichment, followed by genome sequencing and metagenomic binning, revealed the presence of several halophilic, alkalophilic bacteria that play important roles in sulfur and carbon cycling, as well as in nitrogen fixation within this ecosystem. Photosynthetic sulfur bacteria, belonging to Prosthecochloris and Marichromatium, and chemotrophic sulfur bacteria of the genera Sulfurimonas, Arcobacter, and Thiomicrospira produce valuable oxidized sulfur compounds for algal and plant growth, while alkaliphilic, sulfur-reducing bacteria belonging to Sulfurospirillum help balance the sulfur cycle. This metagenome-based study provides a baseline to understand the complex, but balanced, syntrophic microbial interactions that occur in this unique inland salt marsh environment.

Production, Purification, and Characterization of an Industrially Important Enzyme Alkaline Protease Produced from Locally Isolated <i>Bacillus</i> Bacteria

The worldwide demand for industrially essential enzymes is growing due to their eco-friendly and sustainable applications in broad industrial sectors. The main objective of this investigation is intended to isolate potent alkalophilic native bacteria from local habitats for the cost-effective production of an enzyme alkaline protease. For screening of alkalophilic bacteria, bacterial samples were taken from various natural alkaline habitats, and bacterial cultures were screened on agar plate having skimmed milk as protein substrate by using the protein hydrolysis method. The bacteria showing maximum proteolytic potential were identified by microscopic, biochemical, and 16S rDNA analysis. Additionally, the culture components and other medium parameters have been optimized for higher enzyme production. High yield alkaline protease was obtained with conditions of 1% inoculum, pH 9.0, and at 37°C temperature, with 1% sugarcane molasses as the best suitable carbon substrate. Partial pur¬ification of the enzyme was performed and characterized for their optimum activity on various parameters (temperatures and pH range). The approx. molecular weight of the enzyme was estimated to be ~35 kDa. This study demonstrates the production of an industrially important enzyme (alkaline protease) from a newly isolated native Bacillus spp. Economical production of this enzyme can be commercially applied in vast industrial sectors like agriculture, textile, food, detergent, and leather industries.

Isothermal Environmental Heat Energy Utilization by Transmembrane Electrostatically Localized Protons at the Liquid-Membrane Interface.

This study employing the latest theory on transmembrane electrostatic proton localization has now, for the first time, consistently elucidated a decades-longstanding bioenergetic conundrum in alkalophilic bacteria and more importantly discovered an entirely new feature: isothermal environmental heat utilization by electrostatically localized protons at the liquid–membrane interface. It was surprisingly revealed that the protonic motive force (equivalent to Gibbs free energy) from the isothermal environmental heat energy utilization through the electrostatically localized protons is not constrained by the overall energetics of the redox-driven proton pump system because of the following: (a) the transmembrane electrostatically localized protons are not free to move away from the membrane surface as a protonic capacitor feature; (b) the proton pumps embedded in the cell membrane extend beyond the localized proton layer apparently as an asymmetric property of the biological membrane; and (c) the protonic inlet mouth of the ATP synthase that accepts protons is located within this layer as another natural property of the asymmetric biological membrane. This work has now, for the first time, shown a novel thermotrophic feature where biological systems can isothermally utilize environmental heat energy through transmembrane electrostatically localized protons to help drive ATP synthesis.

Functional characterisation and product specificity of Endo-β-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1

Endo-β-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1 (Blg32) composed of 284 amino acids with a predicted molecular mass of 31.6 kDa is expressed in Escherichia coli and purified to homogeneity. Herein, Blg32 characteristics, substrates and product specificity as well as structural traits that might be involved in the production of sugar molecules are analysed. This enzyme functions optimally at the temperature of 70 °C, pH value of 8.0 with its catalytic activity strongly enhanced by Mn2+. Remarkably, the purified enzyme is highly stable in high temperature and alkaline conditions. It exhibits the highest activity on laminarin (376.73 U/mg) followed by curdlan and yeast β-glucan. Blg32 activity increased by 62% towards soluble substrate (laminarin) compared to insoluble substrate (curdlan). Hydrolytic products of laminarin were oligosaccharides with degree of polymerisation (DP) of 1 to 5 with the main product being laminaritriose (DP3). This suggests that the active site of Blg32 could recognise up to five glucose units. High concentration of Blg32 mainly produces glucose whilst low concentration of Blg32 yields oligosaccharides with different DP (predominantly DP3). A theoretical structural model of Blg32 was constructed and structural analysis revealed that Trp156 is involved in multiple hydrophobic stacking interactions. The amino acid was predicted to participate in substrate recognition and binding. It was also exhibited that catalytic groove of Blg32 has a narrow angle, thus limiting the substrate binding reaction. All these properties and knowledge of the subsites are suggested to be related to the possible mode of action of how Blg32 produces glucooligosaccharides.

Bioreduction of hexavalent chromium by chromium resistant alkalophilic bacteria isolated from tannery effluent

Two chromium resistant alkalophilic bacterial strains (CRAB), namely Bacillus cohnii SR2 and Bacillus licheniformis SR3 were isolated and identified from tannery effluent. Bioreduction of chromium (VI) into chromium (III) by CRAB was investigated in the present study. Optimization of process parameters with respect to bioreduction percentage was studied by Response Surface Methodology (RSM) software, v12. Both the strains were found to achieve highest chromium (VI) bioreduction of 94 and 95% in 100 mg/L at the end of 25 h at pH 9.0. At an optimized concentration of 550 mg/L, a maximum bioreduction of 82 and 90% by SR2 and SR3 was noted within 25 h. In addition, both the strains showed tolerance to chromium until 1000 mg/L. Scale-up studies using Air lift bioreactor system by CRAB with real tannery effluent showed 100% bioreduction within 8 h. Therefore, this study signified the astonishing bioreduction capability of CRAB isolates that was quite evident in scale-up studies too.

Evaluation of the ability of alkalophilic bacteria to form a biofilm on the surface of Portland cement-based mortars

This paper investigates bacteria colonisation through biofilm formation, based on the premise that biofilm helps bacteria to have a better development. The aim is to homogenize bacterial growth on recycled concrete aggregates (RCA) to obtain a homogeneous precipitation of calcium carbonate (CaCO3). In previous studies, Bacillus halodurans C-125 was selected to perform biocarbonation on RCA to generate a coat of CaCO3 and diminish water absorption. Contrary to expectations, its poor development led to an heterogeneous CaCO3 precipitation, resulting in an inefficient treatment. Within the framework of this criterion the genetic information of B. halodurans C-125 was compared with a homologous specie “Bacillus subtilis str. 168” to know if it possessed the genes to encode Tas A and Tap A proteins. These proteins consolidate a robust biofilm in Bacillus subtilis str. 168, which promotes bacterial development and adhesion to a surface. Remarkably, B. halodurans C-125 lacks the genes to produce Tas A and Tap A. B. halodurans C-125 was also compared with a group of bacteria isolated from RCA to produce biofilm on MSgg media. Curiously, B. halodurans C-125 did not form a robust biofilm while the bacteria isolated from RCA did. Because of the capacity of the isolated bacteria to form biofilm, they were inoculated on a mortar disk with nutrient and MSgg broth. The results showed traces of bacterial development and precipitation of CaCO3 in form of calcite.

New Finding in Oxidative Phosphorylation: Isothermal Utilization of Latent Heat Energy by Electrostatically Localized Protons at Liquid‐Membrane Interface

Upon application of a newly formulated equation for the proton motive force (pmf) across a biological membrane which takes electrostatically localized protons into account in elucidating the bioenergetics of oxidative phosphorylation in alkalophilic bacteria, it was shown that the localized protons at the liquid‐membrane interface dominate the total pmf. The decreasing trend of the pmf with higher pH values is due to the increasing exchange of cations with the localized protons and this trend matches excellently with the observed rapid increase in the bacteria population growth cell doubling times. It was revealed that the pmf from the isothermal latent heat utilization through the electrostatically localized protons is not constrained by the overall energetics of the redox‐driven proton pump system because (i) the localized protons are not free to move away from the membrane surface, (ii) the proton pumps embedded in the cell membrane extend beyond the localized proton layer, and (iii) the protonic inlet mouth of the ATP synthase that accepts protons is located within this layer. Remarkably, this new understanding agrees excellently with the true structures of mitochondrial respiratory protein complexes. It therefore appears that the oxidative phosphorylation system is able to isothermally utilize the latent heat energy effectively through electrostatically localized protons to help driving the synthesis of ATP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Elucidating the 30-year-Longstanding Bioenergetic Mystery in Alkalophilic Bacteria

Elucidating the 30-year-Longstanding Bioenergetic Mystery in Alkalophilic Bacteria

Elucidating the 30-Year-Longstanding Bioenergetic Mystery in Alkalophilic Bacteria

The decades-longstanding energetic mystery of alkalophilic bacteria as to how they are able to synthesize ATP has now, for the first time, been clearly solved using the proton-electrostatics localization hypothesis [Lee JW (2015) Proton-Electrostatic Localization: Explaining the Bioenergetic Conundrum in Alkalophilic Bacteria. Bioenergetics 4:121. doi:10.4172/2167-7662.1000121; Lee JW (2012) Proton-Electrostatics Hypothesis for Localized Proton Coupling Bioenergetics. Bioenergetics 1:104. doi:10.4172/2167-7662.1000104]. This is a major breakthrough advance in understanding proton-coupling bioenergetics over the Nobel-prize work of Peter Mitchell's chemiosmotic theory. The widespread textbook Mitchellian proton motive force (pmf) equation could significantly underestimate the true pmf value and thus must be revised. Use of our newly derived bioenergetics equation results in an overall pmf value (215∼233 mV) that is more than 4 times larger than that (44.3 mV) calculated from the Mitchellian equation for the alkalophilic bacteria growing at pH 10.5. This newly calculated pmf value is sufficient to overcome the observed phosphorylation potential ∆Gp of −478 mV to synthesize ATP in the bacteria, which can now explain the 30-year-longstanding bioenergetics conundrum. This finding may have fundamental implications not only in the science of bioenergetics but also in understanding the importance of water to life not only as a solvent and substrate but also as a proton conductor for proton coupling energy transduction. In this presentation, the results of our latest bioenergetics calculation and analysis will be presented and discussed in greater details.

Isolation of cyclodextrin glucanotransferase producing alkalophilic bacteria and optimization of culture Conditions for its production

Isolation of cyclodextrin glucanotransferase producing alkalophilic bacteria and optimization of culture Conditions for its production

Isolation and Identification of a New Cold and Alkaline Tolerant Antibiotic Producing Streptomyces sp. From Soil

Background: The ever-increasing resistance of microorganisms to pathogens and our inability to control infectious diseases indicate the importance of conducting research to find new antibiotics. Objectives: The aim of the present research was to isolate, identify, and optimize a cold and pH resistant strain of Streptomyces with high antibiotic production capability. Methods: Soil samples were taken from the Telar forests of northern Iran, and the ACS52 strain was identified as a Streptomyces strain based on the morphological, biochemical, and 16S rRNA gene analysis. Antibacterial features of the obtained strain were studied against a wide range of Gram-positive and Gram-negative bacteria using the cross-culture method. In order to evaluate the antibacterial activity of culture product, two seed cultures and three fermentation media were examined. Finally, the effects of various factors such as the type of carbon and nitrogen sources, temperature, pH, and inoculation rate were investigated. Results: Streptomyces sp. ACS52 is a cold-resistant alkalophilic bacterium strain with antibacterial activity against a broad spectrum of pathogenic bacteria. The fermentation medium containing glycerol and ethyl acetate was suitable for the production and extraction of antibacterial compound, respectively. Glycerol and starch as the carbon source, soybean extract as the nitrogen source, temperature of 25°C, pH of 7, and inoculation rate of 10% were found to provide the optimal conditions for the antibacterial activity. Conclusions: The crude extract of Streptomyces sp. ACS52 had high antibacterial activity against a wide spectrum of bacteria and could be of interest due to its features such as ability to grow at low temperatures and alkaline pH levels.

성·복토용으로 사용된 전로제강슬래그가 주변 토양환경에 미치는 영향

The effect of blast oxygen furnace (BOF) slag used as filling materials on the soil environment was studied using column tests that simulated the flow of the BOF slag leachate through the soil layer. The Cu, Mn, Zn, Ni, and F contents of the leachate affected soil were similar to that of the controls (i.e., soils that were not affected by the leachate). The As, Cd, and Pb contents were lower in the leachate affected soils than the controls. The changes in these contaminants contents can be attributed to the interactions between anions such as alkalinity generating anions (e.g., CO₃^2−, HCO₃^−, OH^−) or calcium ions with heavy metals or F, which consequently affected the fate of heavy metals and F in the leachate affected soils. The germination and growth of Spinapis alba in the soils affected by the leachate and the controls were also similar. However, the proportion of alkalophilic bacteria in the soils affected by the leachate significantly increased, and this can be explained by the increased soil pH due to the alkaline leachate. Overall, this study shows that the alkalinity of the BOF slag leachate, rather than the presence of heavy metals and F in the leachate, needs to be considered when the BOF slag is to be reused as structural filling materials.

Identification of an Alkalophilic Bacterium Producing an Alkaline Protease from Soil

Identification of an Alkalophilic Bacterium Producing an Alkaline Protease from Soil

Newly isolated alkalophilic Advenella species bioaugmented in activated sludge for high p-cresol removal

In this work, an alkalophilic bacterium (LVX-4) capable of using p-cresol as sole source of carbon and energy was screened and isolated from soil polluted by used oil. Phylogenetic (16S rRNA) and phenotypic characterization using Biolog GN microplates and API 20NE strips indicated that LVX-4 strain is a new Advenella species. It showed both the capability to degrade of p-cresol at high concentrations (750 mg/L) and to use p-cresol for growth in a pH from 7 to 10, although the optimum pH was 9. Moreover bioaugmentation of activated sludge with this strain lead to the complete removal of p-cresol in less than 100 h. This is the first study that shows the potential of Advenella sp. to be bioaugmented in activated sludge system for p-cresol biodegradation.