Interface Engineering of PdPt Ultrafine Ethanol Electro-Oxidation Nanocatalysts by Bacterial Soluble Extracellular Polymeric Substances (s-EPS) to Break through Sabatier Principle.

Unsatisfactory performance of ethanol oxidation reaction (EOR) catalysts hinders the application of direct ethanol fuel cells (DEFCs), while traditional alloy catalysts (like PdPt) is cursed by Sabatier principle due to countable active site types. However, bacterial soluble extracellular polymeric substances (s-EPS) owning abundent functional groups may help breacking through it by contrusting different active sites on PdPt and inducing them to play synergy effect, which is called interface engineering. Using s-EPS to engineer catalysts is more green and consumes lower energy compared to chemical reagents. Herein, PdPt alloy nanoparticles (≈2.1 nm) are successfully in situ synthesized by/on s-EPS of Bacillus megaterium, an ex-holotype. Tryptophan residuals are proved as the main reductant. In EOR, PdPt@s-EPS shows higher activity (3.89 mA cm-2) than Pd@s-EPS, Pt@s-EPS, Pt/C and most reported akin catalysts. Its stability and durability are excellent, too. DFT modelling further demonstrates that, interface engineering by s-EPS breaks through Sabatier principle, by the synergy of diverse sites owning different degrees of d-p orbital hybridization. This work not only makes DEFCs closer to practice, but provides a facile and green strategy to design more catalysts.

Bacillus megaterium infection presenting as pulmonary alveolar proteinosis, a case report.

BACKGROUND: Pulmonary alveolar proteinosis (PAP) is a special clinical presentation mostly associated with autoimmune disorders. Here we report a rare case of PAP secondary to infection of Bacillus megaterium. CASE PRESENTATION: A 58-year-old woman presented with intermittent cough and dyspnea for half a year. Chest CT scan showed "crazy paving" pattern. B. megaterium was identified by percutaneous CT-guided needle biopsy. She continuously received antimicrobial treatment since the diagnosis and follow-up examination suggested great improvement. CONCLUSIONS: To our knowledge, this is the first case of B. megaterium infection presented with PAP pattern in healthy individuals. Attention should be paid on the secondary causes including rare pathogen infection when patients presented with PAP syndrome.

Heparosan biosynthesis in recombinant Bacillus megaterium: Influence of N-acetylglucosamine supplementation and kinetic modeling.

Heparosan, an unsulfated polysaccharide, plays a pivotal role as a primary precursor in the biosynthesis of heparin-an influential anticoagulant with diverse therapeutic applications. To enhance heparosan production, the utilization of metabolic engineering in nonpathogenic microbial strains is emerging as a secure and promising strategy. In the investigation of heparosan production by recombinant Bacillus megaterium, a kinetic modeling approach was employed to explore the impact of initial substrate concentration and the supplementation of precursor sugars. The adapted logistic model was utilized to thoroughly analyze three vital parameters: the B. megaterium growth dynamics, sucrose utilization, and heparosan formation. It was noted that at an initial sucrose concentration of 30 g L-1 (S1), it caused an inhibitory effect on both cell growth and substrate utilization. Intriguingly, the inclusion of N-acetylglucosamine (S2) resulted in a significant 1.6-fold enhancement in heparosan concentration. In addressing the complexities of the dual substrate system involving S1 and S2, a multi-substrate kinetic models, specifically the double Andrew's model was employed. This approach not only delved into the intricacies of dual substrate kinetics but also effectively described the relationships among the primary state variables. Consequently, these models not only provide a nuanced understanding of the system's behavior but also serve as a roadmap for optimizing the design and management of the heparosan production method.

Plant growth promoting bacteria and citric acid promote growth and cadmium phytoremediation in ryegrass.

Based on the growth-promoting effect of plant growth promoting bacteria on plants and the mobilization of Cd by citric acid, an experiment was designed in which the combined treatment of Bacillus megaterium and citric acid promoted ryegrass to repair Cd-contaminated soil. This study aimed to evaluate the effects of different treatments on the antioxidant enzyme activity, photosynthesis intensity, Cd accumulation, and rhizosphere cadmium migration under cadmium contamination conditions. And the soil morphology and structure changes were studied by infrared spectroscopy FourierTransformInfrared(FT-IR) and scanning electron microscope Energy Dispersive Spectrometer(SEM-EDS) before and after different treatments. The results show that the combined treatment of Bacillus megaterium and citric acid significantly improved the oxidative stress defense and plant photosynthesis and increased of rye biomass. rye biomass 1.28 times higher than CK treatment. Joint treatment significantly increased the amount of shoot accumulation of Cd, 2.31 times higher than CK treatment, increased the migration and accumulation of cadmium. FTIR and SEM-EDS also showed that the organic constituents such as O-H, C-O and C-N in soils as a major mechanism for mobilization of the heavy metal Cd. Thus, the combined treatment of Bacillus megaterium and citric acid can promote plant growth, improve the damage to ryegrass caused by single organic acid addition, and improve the plant extraction efficiency, which is a feasible way to repair Cd-contaminated soil through activated extraction system.

The novelty of this study is the combined application of bacteria and chelating agents to ryegrass to improve phytoremediation efficiency. Bacillus giganosus has a good role in promoting the growth of ryegrass. As citrate, a small molecule chelate, can activate heavy metal cadmium and detoxify heavy metals, so it was selected. This study revealed in detail the response of ryegrass to the heavy metal Cd after exogenous addition of Bacillus gigansus and citrate, which is important for the application of cadmium removal by phytoremediation.

Effects and mechanisms of microbial ecology and diversity on phytoremediation of cadmium-contaminated soil under the influence of biodegradable organic acids.

In recent years, heavy metal pollution has become a global environmental problem and poses a great threat to the health of people and ecosystems. Therefore, strategies for the effective remediation of Cd from contaminated soil are urgently needed. In this study, ryegrass was utilized as a remediation plant, and its remediation potential was enhanced through the application of Citric Acid (CA) in conjunction with Bacillus megaterium (B. megaterium). The P3 treatment (CA + Bacillus megaterium) exhibited a significantly higher efficiency in promoting cadmium extraction by ryegrass, resulting in a 1.79-fold increase in shoot cadmium accumulation compared to the control group (CK) with no Bacillus megaterium or CA. Moreover, the P3 treatment led to an increased abundance of Actinobacteriota, Acidobacteriota, and Patescibacteria in the rhizosphere. The concentration of amino derivatives (such as betaine, sulfolithocholylglycine, N-alpha-acetyl-lysine, glycocholic acid, arginyl-threonine) showed significant upregulation following the P3 treatment. In summary, this study proposes a viable approach for phytoremediation of soil contaminated with cadmium by harnessing the mobilizing abilities of soil bacteria.

Our aim was to gain a comprehensive understanding of the mechanisms involved in phytoremediation. These findings contribute to the existing knowledge by providing insights into the mechanism of phytoremediation in Cd-contaminated soil. They are expected to serve as a theoretical foundation for further elucidation of the phytoremediation mechanisms employed in Cd-contaminated soil.

Bacillus megaterium: Evaluation of Chemical Nature of Metabolites and Their Antioxidant and Agronomics Properties.

Bacillus megaterium is particularly known for its abundance in soils and its plant growth promotion. To characterize the metabolites excreted by this specie, we performed successive liquid/liquid extractions from bacteria culture medium with different polarity solvents (cyclohexane, dichloromethane, ethyl acetate and butanol) to separate the metabolites in different polarity groups. The extracts were characterized regarding their total phenolic content, the amount of reducing sugar, the concentration of primary amines and proteins, their chromatographic profile by HPLC-DAD-ELSD and their chemical identification by GC-MS. Among the 75 compounds which are produced by the bacteria, 19 identifications were for the first time found as metabolites of B. megaterium and 23 were described for the first time as metabolites in Bacillus genus. The different extracts containing B. megaterium metabolites showed interesting agronomic activity, with a global inhibition of seed germination rates of soya, sunflower, corn and ray grass, but not of corn, compared to culture medium alone. Our results suggest that B. megaterium can produce various metabolites, like butanediol, cyclic dipeptides, fatty acids, and hydrocarbons, with diverse effects and sometimes with opposite effects in order to modulate its response to plant growth and adapt to various environmental effects. These findings provide new insight into bioactive properties of this species for therapeutic uses on plants.

Preparation of an environment-friendly microbial limestone dust suppressant and its dust suppression mechanism.

The development of an efficient and environmentally friendly dust suppressant is crucial to address the issue of dust pollution in limestone mines. Leveraging the synergistic microbial-induced calcium carbonate precipitation (MICP) technology involving NaHCO3 and dodecyl glucoside (APG), the optimal ratio of the dust suppressant was determined through single-factor and response surface tests. The dust suppression efficacy and mechanisms were analyzed through performance testing and microscopic imaging techniques, indicating that the optimal ratio of the new microbial dust suppressant was 20% mineralized bacteria cultured for 72 h, 0.647 mol L-1 cementing solution, 3.142% NaHCO3, and 0.149% APG. Under these conditions, the yield of calcium carbonate increased by 24.89% as compared to when no NaHCO3 was added. The dust suppressant demonstrated excellent wind, moisture, and rain resistance, as well as curing ability. More calcite was formed in the dust samples after treatment, and the stable form of the dust suppressant contributed to consolidating the limestone dust into a cohesive mass. These findings indicate that the synergistic effect of NaHCO3 and APG significantly enhanced the dust suppression capabilities of the designed microbial dust suppressant.

Optimization of a medium composition for the heterologous production of Alcaligenes faecalis penicillin G acylase in Bacillus megaterium.

Penicillin G acylase (PGA) is a strategic enzyme in the production processes of beta-lactam antibiotics. High demand for ß-lactam semisynthetic antibiotics explain the genetic and biochemical engineering strategies devoted towards novel ways for PGA production and application. This work presents a fermentation process for the heterologous production of PGA from Alcaligenes faecalis in Bacillus megaterium with optimization. The thermal stability from A. faecalis PGA is considerably higher than other described PGA and the recombinant enzyme is secreted to the culture medium by B. megaterium, which facilitates the separation and purification steps. Media optimization using fractional factorial design experiments was used to identify factors related to PGA activity detection in supernatant and cell lysates. The optimized medium resulted in almost 6-fold increased activity in the supernatant samples when compared with the basal medium. Maximum enzyme activity in optimized medium composition achieves values between 135 and 140 IU/ml. The results suggest a promising model for recombinant production of PGA in B. megaterium with possible extracellular expression of the active enzyme.

Influence of redox potential on the accumulation of poly(3-hydroxybutyrate) by Bacillus megaterium.

The main goal of the present study was to evaluate the oxidation-reduction potential (ORP) on the production of poly(3-hydroxybutyrate) (P(3HB)) by Bacillus megaterium. Each microorganism has an optimal ORP range, and changes to the culture medium's ORP may redistribute the cell's metabolic flux, as such, the measurement and control of the ORP profile allows one to, in a way, manipulate the microbial metabolism, affecting the expression of certain enzymes and allowing for better control over the fermentative process. The ORP tests were carried out in a fermentation vessel coupled with an ORP probe, containing 1 L of mineral medium added with agroindustry byproducts (60% v/v of confectionery wastewater, and 40% v/v of rice parboiling water). The system's temperature was kept at 30 °C, with an agitation speed of 500 rpm. The vessel's airflow rate was controlled via a solenoid pump based on the ORP probe's data. Different ORP values were evaluated to verify their impact on biomass and polymer production. Cultures using OPR levels of 0 mV displayed the highest amounts of total biomass (5.00 g L-1) when compared to - 20 mV and - 40 mV (2.90 g L-1 and 0.53 g L-1, respectively). Similar results were also found for P(3HB)-to-biomass ratio, with polymer concentration being reduced when using ORP levels below 0 mV and with a maximum amount of polymer-to-biomass ratio of 69.87% after 48 h of culture. Furthermore, it was possible to observe that the culture's pH can also affect total biomass and polymer concentration, albeit to a lesser extent. Thus, when considering the data found during this study, it is possible to observe that ORP values can greatly impact B. megaterium cell's metabolism. Furthermore, the measurement and control of ORP levels may be an invaluable asset when trying to maximize polymer production under different culture conditions.

Plant growth promoting Bacillus species elicit defense against Meloidogyne incognita infecting tomato in polyhouse.

The effects of four nematicidal rhizobacterial isolates; Bacillus subtilis, Bacillus pumilus, Bacillus megaterium, and Bacillus cereus on infection and multiplication of root-knot nematode, Meloidogyne incognita on tomato were compared with the application of a chemical nematicide, fluopyram 34.48% SC (Velum Prime). The bio-efficacy trial conducted in pots preinoculated with the above isolates followed by M. incognita inoculation resulted in a significant reduction in percent root galling viz. 91.95 in B. subtilis, 84.21 in B. pumilus, 83.70 in B. megaterium, and 81.8 in B. cereus, at 75 days after inoculation (DAI). The reproduction factor of the nematode was the lowest (15.83) in B. subtilis, followed by B. pumilus (21.00), compared with 48.16 in control, with enhanced photosynthetic and transpiration rates. The mechanism of induced resistance was assessed using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) for quantification of three key defense genes (PR-1b, JERF3, and CAT) at 0,2,4,8 and16 days DAI. The defence genes, PR-1b, JERF3, and CAT were expressed at 2.5-7.5-folds in rhizobacterialtreated plants, but not in nematicide treatment. The defense enzymes viz., super oxide dismutase (SOD), polyphenol oxidase (PPO), peroxidase (PO), and phenylalanine ammonia lyase (PAL) when quantified (µmol/mg protein) showed an increase from 1.5 to 17.5 for SOD, 2.1 to 7.8 in PPO, 1.8 to 10.2 in PO, and 1.8 to 8.7 in PAL during 0 to 16 DAI, in rhizobacteria-treated plants.

Characterization of a novel detergent-resistant type I pullulanase from Bacillus megaterium Y103 and its application in laundry detergent.

This study aims to find a moderate pullulanase for detergent industry. The pulY103B gene (2217 bp) from Bacillus megaterium Y103 was cloned and expressed in Escherichia coli. PulY103B contained four conserved regions of glycoside hydrolase family (GH) 13 and the typical sequence of type I pullulanase. The optimal reaction conditions of PulY103B were pH 6.5 and 40 °C. In addition, it remained stable below 40 °C and over 80% of activity was retained at pH ranging from 6.0 to 8.5. The best substrate for the enzyme was pullulan. Furthermore, it exhibited activity toward wheat starch (36.5%) and soluble starch (33.4%) but had no activity toward amylose and glycogen. Maltotriose and maltohexaose were major pullulan hydrolysis products. Soluble starch and amylopectin were mainly hydrolyzed into maltotetraose. These results indicated that PulY103B is a novel type I pullulanase with transglycosylation activity via formation of α-1,4-glucosidic linkages. Moreover, PulY103B was strongly stimulated by nonionic detergents [viz, Tween 20 (10%), Tween 80 (1%), Triton X-100 (20%)] and commercial liquid detergents (3.0 g/L). Wash performance tests demonstrated that the mixture of PulY103B and detergent removed starch-based stains better than using detergent alone (p < 0.05). Therefore, this pullulanase has big potential as a detergent additive.

Plant Growth Promoting Bacterial Consortium Induces Shifts in Indigenous Soil Bacterial Communities and Controls Listeria monocytogenes in Rhizospheres of Cajanus cajan and Festuca arundinacea.

The rhizosphere is a dynamic and complex interface between plant roots and microorganisms. Owing to exudates, a web of interactions establishes among the microbial members of this micro-environment. The present study explored the impact of a bacterial consortium (Azotobacter chroococcum, Bacillus megaterium and Pseudomonas fluorescens, ABP), on the fate of a human pathogen, Listeria monocytogenes EGD-e, in soil and in the rhizospheres of Cajanus cajan and Festuca arundinacea, in addition to its plant growth promoting effect. The study further assessed the impact these bioinoculants exert on the autochthonous soil bacterial communities. Experiments in sterilised soil inoculated with bioinoculants and L. monocytogenes revealed the inhibition of L. monocytogenes by approximately 80-fold compared to that without the consortium. Subsequently, experiments were conducted in non-sterile soil microcosms planted with C. cajan and F. arundinacea, and in bulk soil. The consortium led to a significant increase in plant growth in both plants and prevented growth of L. monocytogenes. However, the presence of resident soil bacterial communities overshadowed this inhibitory effect, and a sharp decline in L. monocytogenes populations (5-6 log reduction) was recorded under non-sterile soil conditions. A shift in the soil resident bacterial communities was observed upon amendment with the bioinoculants. A significant increase of potential Plant Growth Promoting Rhizobacteria (PGPR) and biocontrol agents was observed, while the abundance of potential phytopathogens dropped. The present study opens up new avenues for the application of such a consortium given their dual benefits of plant growth promotion and restricting phytopathogens as well as human pathogen.

Novel mechanism and degradation kinetics of allethrin using Bacillus megaterium strain HLJ7 in contaminated soil/water environments.

As a common pyrethroid insecticide, allethrin is widely used for various purposes in agriculture and home applications. At present, allethrin residues have been frequently detected worldwide, yet little is known about the kinetics and degradation mechanisms of this insecticide. In this study, a highly efficient allethrin-degrading bacterium, Bacillus megaterium strain HLJ7, was obtained through enrichment culture technology. Strain HLJ7 can remove 96.5% of 50 mg L-1 allethrin in minimal medium within 11 days. The first-order kinetic analysis of degradation demonstrated that the half-life of allethrin degradation by strain HLJ7 was 3.56 days, which was significantly shorter than the 55.89 days of the control. The Box-Behnken design of the response surface method optimized the degradation conditions for strain HLJ7: temperature 32.18 °C, pH value 7.52, and inoculation amount 1.31 × 107 CFU mL-1. Using Andrews equation, the optimal concentration of strain HLJ7 to metabolize allethrin was determined to be 21.15 mg L-1, and the maximum specific degradation rate (qmax), half-rate constant (Ks) and inhibition coefficient (Ki) were calculated to be 1.80 d-1, 1.85 mg L-1 and 68.13 mg L-1, respectively. Gas chromatography-mass spectrometry identified five intermediate metabolites, suggesting that allethrin could be degraded firstly by cleavage of its carboxylester bond, followed by degradation of the five-carbon ring and subsequent metabolism. The results of soil remediation experiments showed that strain HLJ7 has excellent bioremediation potential in the soils. After 15 days of treatment, about 70.8% of the initial allethrin (50 mg kg-1) was removed and converted into nontoxic intermediate metabolites, and its half-life was significantly reduced in the soils. Taken together, these findings shed light on the degradation mechanisms of allethrin and also highlight the promising potentials of B. megaterium HLJ7 in bioremediation of allethrin-comtaminated environment.

Engineering precursor and co-factor supply to enhance D-pantothenic acid production in Bacillus megaterium.

High-yielding chemical and chemo-enzymatic methods of D-pantothenic acid (DPA) synthesis are limited by using poisonous chemicals and DL-pantolactone racemic mixture formation. Alternatively, the safe microbial fermentative route of DPA production was found promising but suffered from low productivity and precursor supplementation. In this study, Bacillus megaterium was metabolically engineered to produce DPA without precursor supplementation. In order to provide a higher supply of precursor D-pantoic acid, key genes involved in its synthesis are overexpressed, resulting strain was produced 0.53 ± 0.08 g/L DPA was attained in shake flasks. Cofactor CH2-THF was found to be vital for DPA biosynthesis and was regenerated through the serine-glycine degradation pathway. Enhanced supply of another precursor, ß-alanine was achieved by codon optimization and dosing of the limiting L-asparate-1-decarboxylase (ADC). Co-expression of Pantoate-ß-alanine ligase, ADC, phosphoenolpyruvate carboxylase, aspartate aminotransferase and aspartate ammonia-lyase enhanced DPA concentration to 2.56 ± 0.05 g/L at shake flasks level. Fed-batch fermentation in a bioreactor with and without the supplementation of ß-alanine increased DPA concentration to 19.52 ± 0.26 and 4.78 ± 0.53 g/L, respectively. This present study successfully demonstrated a rational approach combining precursor supply engineering with cofactor regeneration for the enhancement of DPA titer in recombinant B. megaterium.

Process optimization of vanillin production by conversion of ferulic acid by Bacillus megaterium.

BACKGROUND: Vanillin is an important flavoring and aromatic ingredient found mainly in the pods of the tropical plant vanilla and is widely used in the food industry. Attempts have been made to produce vanillin from ferulic acid esters in agricultural residues of wheat bran. RESULTS: The results showed that a strain with high tolerance to the substrate ferulic acid was isolated and screened from soil and identified as belonging to the genus Bacillus (Bacillus megaterium). The concentration of vanillin produced by this strain was 0.048 g L-1 , and the molar conversion of vanillin was 12.25%. The production of vanillin was optimized by orthogonal experiments. Beef pastes 6.0 g L-1 , soybean meal 5.0 g L-1 , magnesium sulfate heptahydrate 1.0 g L-1 , iron(II) sulfate heptahydrate 1.0 g L-1 , calcium chloride 1.0 g L-1 , dipotassium hydrogen phosphate trihydrate 1.0 g L-1 ; fermentation culture conditions were pH 7.0, inoculum level 5%, loading volume 20%, ferulic acid 1.0 g L-1 , fermentation culture temperature 35 °C. The concentration of vanillin obtained was 0.218 g L-1 . Finally, transcriptomic analysis of the strain samples before and after the optimization of the fermentation conditions was carried out to study the effect of the optimization of the fermentation conditions on the concentration of vanillin produced by the strain. CONCLUSION: This study provides a theoretical basis for further improving the yield of vanillin and gradually realizing efficient industrial production. © 2022 Society of Chemical Industry.

High-level expression and optimization of pantoate-ß-alanine ligase in Bacillus megaterium for the enhanced biocatalytic production of D-pantothenic acid.

D-Pantothenic acid (DPA), also known as vitamin B5 is associated with several biological functions and its deficiency causes metabolic and energetic disorders in humans. Fortification of foods with DPA is the viable option to address this risk. DPA biological production route employs pantoate-ß-alanine ligase (PBL) as the key enzyme, which avoids the tedious and time-consuming optical resolution process. The selection of an efficient PBL enzyme is vital for the biological production of DPA. In this study, the panC gene encoding PBL from Escherichia coli, Bacillus megaterium, Corynebacterium glutamicum and Bacillus subtilis was expressed in B. megaterium. B. subtilis derived panC exhibited high PBL activity 61.62 ± 2.15 U/mL. Co-expression of phosphoenolpyruvate carboxykinase (pckA) did not improve the DPA production in B. megaterium. Biocatalytic fed-batch fermentation with externally supplemented precursor substrates (D-pantoic acid and ß-alanine) improved DPA titer to 45.56 ± 0.53 g/L. Daily dietary requirements of DPA for different age groups (including babies, small children, athletes and elderly people) is steadily increasing and the improved DPA production addressed in this study offers advantage for its application in fortification of food products meeting the emerging nutritional demand. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13197-021-05093-6.

Inoculation of Bacillus megaterium strain A14 alleviates cadmium accumulation in peanut: effects and underlying mechanisms.

AIMS: A cadmium (Cd)-tolerant Bacillus megaterium strain A14 was used to investigate the effects and mechanisms of bacterial inoculation on peanut growth, Cd accumulation in grains and Cd fixation in Cd-contaminated soil. METHODS AND RESULTS: Spectroscopic analysis showed that A14 has many functional groups (-OH, -NH2 and -COO et al.) distributed on its surface. The pot experiment indicated that compared to the Cd-contaminated soil alone treatment, inoculation with strain A14 increased shoot and root biomass by 59·93 and 58·31% respectively. The accumulation of Cd in grains decreased by 48·14%, while the proportion of exchangeable Cd in soil decreased from 40 to 26% in A14 inoculated soil. CONCLUSIONS: Inoculation with B. megaterium A14 improved peanut plant growth via (i) adsorbing Cd2+ through functional groups on cell surface, (ii) immobilization of Cd in soil through extracellular secretions, (iii) scavenging the reactive oxygen species through production of antioxidant enzymes, and (iv) by reducing the phytoavailable Cd through regulation of Cd transport gene expression. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided a new sight on microbial approach for the chemical composition transformation of soil Cd and associated food safety production, which pointed out an efficient way to improve peanut cultivation.

Microbial effect on water sorptivity and sulphate ingress by Bacillus megaterium on mortars prepared using Portland Pozzolana cement.

AIMS: To determine the effect of direct embedment of Bacillus megaterium into Portland pozzolana cement mortars on water sorptivity and diffusivity coefficient of sulphate ions. METHODS AND RESULTS: Prisms with a water/cement ratio of 0·5 were prepared by blending Portland Pozzolana cement with the requisite volume of a B. megaterium (microbial) solution whose concentration was 1·0 × 107 cells per ml. Mortar prisms of 160 mm × 40 mm × 40 mm were fabricated for this study. Mortars cured for 28 days were exposed to 0·2465 mol l-1 Na2 SO4 solution using accelerated ion migration test method for 36-h session using a 12V DC power source. Sulphate ion concentration was then determined through the ingressed mortar at 10 mm interval. A minimum water sorption gain of 0·61% was observed on the prism prepared with and cured in microbial solution. A maximum of 0·0289 and a minimum of 0·0093 water sorptivity coefficients were exhibited by the control prism and microbial prisms, respectively. The microbial prisms exhibited the lowest apparent diffusion coefficient (Dapp ) of 4·5179 × 10-11  m2  s-1 . CONCLUSIONS: Direct incorporation of B. megaterium in mortar preparation, curing or both regimes significantly retarded water sorption and lowered sulphate ion ingress. The inclusion of this bacterial in the mortar further complements the pozzolana pore structure benefits. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel B. megaterium bacteria which can survive and cause biocementation within hydrating cement mortar when not encapsulated would result in a green innovation. Once adopted and applied in real-life scenario, it would promote construction of durable, safe, resilient and affordable shelter.

Characterization of a Bacillus megaterium strain with metal bioremediation potential and in silico discovery of novel cadmium binding motifs in the regulator, CadC.

Bioremediation of toxic metal ions using bacterial strains is a promising tool. Metal binding motifs in microbial proteins are involved in the regulation and transport of such toxic metals for metal detoxification. A bacterial strain designated TWSL_4 with metal (Cu, Cd, and Pb) resistance and removal ability was isolated and identified as a Bacillus megaterium strain using 16S rRNA gene analysis. An operon with 2 open reading frames (ORFs) was identified, cloned, and sequenced. ORF1 and ORF2 were identical to the cadmium efflux system accessory protein (CadC) and cadmium-translocating P-type ATPases (CadA) of B. megaterium strain YC4-R4 respectively. A protein homology search using Swiss model retrieved no crystal structures for CadC and CadA of Bacillus sp.. CadC of TWSL_4 had a sequence identity of 53% to the CadC (121aa) protein and 51.69% to the CadC crystal structure (1U2W.1.B; GMQE=0.75) of Staphylococcus sp. pI258. Molecular dynamic simulation studies revealed the presence of three metal binding regions in CadC of TWSL_4, [ASP7-TYR9], [ASP100-HIS102], and [LYS113-ASP116]. This is the first report showing evidence for the presence of Cd2+ and Zn2+ metal binding motifs in the CadC regulator of the Bacillus megaterium cad operon. The bacterial strain TWSL_4 was also found to contain two different P type ATPases encoding genes, cadA and zosA involved in metal resistance. Furthermore, the metal bioremediation potential of strain TWSL_4 was confirmed using an industrial effluent. KEY POINTS: • Isolation of a metal-resistant bacterial strain with potential for industrial bioremediation. • Discovery of novel Cd binding sites in CadC of the cad operon from B. megaterium. • Involvement of aspartic acid in the coordination of metal ions (Cd2+).

The "beauty in the beast"-the multiple uses of Priestia megaterium in biotechnology.

Over 30 years, the Gram-positive bacterium Priestia megaterium (previously known as Bacillus megaterium) was systematically developed for biotechnological applications ranging from the production of small molecules like vitamin B12, over polymers like polyhydroxybutyrate (PHB) up to the in vivo and in vitro synthesis of multiple proteins and finally whole-cell applications. Here we describe the use of the natural vitamin B12 (cobalamin) producer P. megaterium for the elucidation of the biosynthetic pathway and the subsequent systematic knowledge-based development for production purposes. The formation of PHB, a natural product of P. megaterium and potential petro-plastic substitute, is covered and discussed. Further important biotechnological characteristics of P. megaterium for recombinant protein production including high protein secretion capacity and simple cultivation on value-added carbon sources are outlined. This includes the advanced system with almost 30 commercially available expression vectors for the intracellular and extracellular production of recombinant proteins at the g/L scale. We also revealed a novel P. megaterium transcription-translation system as a complementary and versatile biotechnological tool kit. As an impressive biotechnology application, the formation of various cytochrome P450 is also critically highlighted. Finally, whole cellular applications in plant protection are completing the overall picture of P. megaterium as a versatile giant cell factory. KEY POINTS: • The use of Priestia megaterium for the biosynthesis of small molecules and recombinant proteins through to whole-cell applications is reviewed. • P. megaterium can act as a promising alternative host in biotechnological production processes.