Antagonistic effect of Bacillus and Pseudomonas combinations against Fusarium oxysporum and their effect on disease resistance and growth promotion in watermelon.

AIMS: We aimed to develop an effective bacterial combination that can combat Fusarium oxysporum infection in watermelon using in vitro and pot experiments. METHODS AND RESULTS: In total, 53 strains of Bacillus and 4 strains of Pseudomonas were screened. Pseudomonas strains P3 and P4 and Bacillus strains XY-2-3, XY-13, and GJ-1-15 exhibited good antagonistic effects against F. oxysporum. P3 and P4 were identified as Pseudomonas chlororaphis and Pseudomonas fluorescens, respectively. XY-2-3 and GJ-1-15 were identified as B. velezensis, and XY-13 was identified as Bacillus amyloliquefaciens. The three Bacillus strains were antifungal, promoted the growth of watermelon seedlings and had genes to synthesize antagonistic metabolites such as bacilysin, surfactin, yndj, fengycin, iturin, and bacillomycin D. Combinations of Bacillus and Pseudomonas strains, namely, XY-2-3 + P4, GJ-1-15 + P4, XY-13 + P3, and XY-13 + P4, exhibited a good compatibility. These four combinations exhibited antagonistic effects against 11 pathogenic fungi, including various strains of F. oxysporum, Fusarium solani, and Rhizoctonia. Inoculation of these bacterial combinations significantly reduced the incidence of Fusarium wilt in watermelon, promoted plant growth, and improved soil nutrient availability. XY-13 + P4 was the most effective combination against Fusarium wilt in watermelon with the inhibition rate of 78.17%. The number of leaves; aboveground fresh and dry weights; chlorophyll, soil total nitrogen, and soil available phosphorus content increased by 26.8%, 72.12%, 60.47%, 16.97%, 20.16%, and 16.50%, respectively, after XY-13 + P4 inoculation compared with the uninoculated control. Moreover, total root length, root surface area, and root volume of watermelon seedlings were the highest after XY-13 + P3 inoculation, exhibiting increases by 265.83%, 316.79%, and 390.99%, respectively, compared with the uninoculated control. CONCLUSIONS: XY-13 + P4 was the best bacterial combination for controlling Fusarium wilt in watermelon, promoting the growth of watermelon seedlings, and improving soil nutrient availability.

Saprophytic Bacillus Accelerates the Release of Effective Components in Agarwood by Degrading Cellulose.

The value of Agarwood increases with time due to the gradual release of its major components, but the mechanism behind this remains unclear. Herein we reveal that the potential driving force of this process is the degradation of cellulose in Agarwood by its saprophytic Bacillus subtilis. We selected 10-year-old Agarwood from different places and then isolated the saprophytic bacteria. We confirmed these bacteria from different sources are all Bacillus and confirmed they can degrade cellulose, and the highest cellulase activity reached 0.22 U/mL. By co-cultivation of the bacterium and Agarwood powder, we found that three of the strains could release the effective components of Agarwood, while they had little effect in increasing the same components in living Aquilaria sinensis. Finally, we demonstrated that these saprophytic Bacillus subtilis have similar effects on Zanthoxylum bungeanum Maxim and Dalbergiaod orifera T. Chen, but not on Illicium verum Hook. f, Cinnamomum cassia Presl and Phellodendron chinense Schneid. In conclusion, our experiment revealed that the saprophytic Bacillus release the effective components of Agarwood by degrading cellulose, and we provide a promising way to accelerate this process by using this bacterial agent.

A Novel Rhizospheric Bacterium: Bacillus velezensis NKMV-3 as a Biocontrol Agent Against Alternaria Leaf Blight in Tomato.

A novel strain of Bacillus isolated from rhizosphere has shown to be an excellent biocontrol agent against various plant pathogens. In this study, a first report of a Bacillus strain NKMV-3 which effectively controls Alternaria solani, which cause the early blight disease in tomato. Based on the cultural and molecular sequencing of 16S rRNA gene sequence, the identity of the strain was confirmed as Bacillus velezensis NKMV-3. The presence of the lipopeptide which are antibiotic synthesis genes, namely iturin C, surfactin A and fengycin B and D, was confirmed through gene amplification. In addition, lipopeptides were also confirmed through liquid chromatography. The extract showed inhibitory effect against A. solani in vitro and detached tomato leaf assays. Bacillus velezensis strain NKMV-3-based formulations may provide an effective solution in controlling early blight disease in tomato and other crops.

High-Performance Capacitive Deionization and Killing Microorganism in Surface-Water by ZIF-9 Derived Carbon Composites.

The protection and regeneration of the water environment is currently one of the most critical concerns for the sustainable development of human society. To solve the water crisis, the use of capacitive deionization (CDI) technology to extract fresh-water that is suitable for human consumption from abundant surface-water is a feasible solution. In this work, a cobalt benzimidazole frameworks (ZIF-9) derived carbon composites with a unique quasi-microcubic morphology is synthesized and used the as-prepared materials as an electrode material for the CDI. Interestingly, the ZIF-9 derived carbon composites exhibit an impressive desalination capacity of 55.4 mg g-1 and can be reused. Measurements in surface-water (Beijing-Hangzhou Grand Canal, Slender West Lake, Initial rainwater, Rain water) show that this CDI technology based on ZIF-9 derived carbon composites not only has a strong adsorption effect on metal ions but also can remarkably kill microorganisms. The results show that the technology can effectively kill bacteria (Escherichia coli and Bacillus) and algae with 95% and 91.7% inhibition rates, respectively. This work provides a valuable example for the use of metal-organic framework-derived carbon composites as high-performance electrode materials of CDI and opens a new direction for promoting the application of CDI in surface-water.

Lipopeptide Interplay Mediates Molecular Interactions between Soil Bacilli and Pseudomonads.

Some Bacillus species, such as B. velezensis, are important members of the plant-associated microbiome, conferring protection against phytopathogens. However, our knowledge about multitrophic interactions determining the ecological fitness of these biocontrol bacteria in the competitive rhizosphere niche is still limited. Here, we investigated molecular mechanisms underlying interactions between B. velezensis and Pseudomonas as a soil-dwelling competitor. Upon their contact-independent in vitro confrontation, a multifaceted macroscopic outcome was observed and characterized by Bacillus growth inhibition, white line formation in the interaction zone, and enhanced motility. We correlated these phenotypes with the production of bioactive secondary metabolites and identified specific lipopeptides as key compounds involved in the interference interaction and motile response. Bacillus mobilizes its lipopeptide surfactin not only to enhance motility but also to act as a chemical trap to reduce the toxicity of lipopeptides formed by Pseudomonas. We demonstrated the relevance of these unsuspected roles of lipopeptides in the context of competitive tomato root colonization by the two bacterial genera. IMPORTANCE Plant-associated Bacillus velezensis and Pseudomonas spp. represent excellent model species as strong producers of bioactive metabolites involved in phytopathogen inhibition and the elicitation of plant immunity. However, the ecological role of these metabolites during microbial interspecies interactions and the way their expression may be modulated under naturally competitive soil conditions has been poorly investigated. Through this work, we report various phenotypic outcomes from the interactions between B. velezensis and 10 Pseudomonas strains used as competitors and correlate them with the production of specific metabolites called lipopeptides from both species. More precisely, Bacillus overproduces surfactin to enhance motility, which also, by acting as a chemical trap, reduces the toxicity of other lipopeptides formed by Pseudomonas. Based on data from interspecies competition on plant roots, we assume this would allow Bacillus to gain fitness and persistence in its natural rhizosphere niche. The discovery of new ecological functions for Bacillus and Pseudomonas secondary metabolites is crucial to rationally design compatible consortia, more efficient than single-species inoculants, to promote plant health and growth by fighting economically important pathogens in sustainable agriculture.

Novel Genetic Dysregulations and Oxidative Damage in Fusarium graminearum Induced by Plant Defense Eliciting Psychrophilic Bacillus atrophaeus TS1.

This study elaborates inter-kingdom signaling mechanisms, presenting a sustainable and eco-friendly approach to combat biotic as well as abiotic stress in wheat. Fusarium graminearum is a devastating pathogen causing head and seedling blight in wheat, leading to huge yield and economic losses. Psychrophilic Bacillus atrophaeus strain TS1 was found as a potential biocontrol agent for suppression of F. graminearum under low temperature by carrying out extensive biochemical and molecular studies in comparison with a temperate biocontrol model strain Bacillus amyloliquefaciens FZB42 at 15 and 25 °C. TS1 was able to produce hydrolytic extracellular enzymes as well as antimicrobial lipopeptides, i.e., surfactin, bacillomycin, and fengycin, efficiently at low temperatures. The Bacillus strain-induced oxidative cellular damage, ultrastructural deformities, and novel genetic dysregulations in the fungal pathogen as the bacterial treatment at low temperature were able to downregulate the expression of newly predicted novel fungal genes potentially belonging to necrosis inducing protein families (fgHCE and fgNPP1). The wheat pot experiments conducted at 15 and 25 °C revealed the potential of TS1 to elicit sudden induction of plant defense, namely, H2O2 and callose enhanced activity of plant defense-related enzymes and induced over-expression of defense-related genes which accumulatively lead to the suppression of F. graminearum and decreased diseased leaf area.

Plant immune system activation is necessary for efficient root colonization by auxin-secreting beneficial bacteria.

Although plant roots encounter a plethora of microorganisms in the surrounding soil, at the rhizosphere, plants exert selective forces on their bacterial colonizers. Unlike immune recognition of pathogenic bacteria, the mechanisms by which beneficial bacteria are selected and how they interact with the plant immune system are not well understood. To better understand this process, we studied the interaction of auxin-producing Bacillus velezensis FZB42 with Arabidopsis roots and found that activation of the plant immune system is necessary for efficient bacterial colonization and auxin secretion. A feedback loop is established in which bacterial colonization triggers an immune reaction and production of reactive oxygen species, which, in turn, stimulate auxin production by the bacteria. Auxin promotes bacterial survival and efficient root colonization, allowing the bacteria to inhibit fungal infection and promote plant health. Thus, a feedback loop between bacteria and the plant immune system promotes the fitness of both partners.

Up-Regulated Salivary Proteins of Brown Marmorated Stink Bug Halyomorpha halys on Plant Growth-Promoting Rhizobacteria-Treated Plants.

Plant Growth-Promoting Rhizobacteria (PGPR) induce systemic resistance (SR) in plants, decreasing the development of phytopathogens. The FZB42 strain of Bacillus velezensis is known to induce an SR against pathogens in various plant species. Previous studies suggested that it could also influence the interactions between plants and associated pests. However, insects have developed several strategies to counteract plant defenses, including salivary proteins that allow the insect escaping detection, manipulating defensive pathways to its advantage, deactivating early signaling processes, or detoxifying secondary metabolites. Because Brown Marmorated Stink Bug (BMSB) Halyomorpha halys is highly invasive and polyphagous, we hypothesized that it could detect the PGPR-induced systemic defenses in the plant, and efficiently adapt its salivary compounds to counteract them. Therefore, we inoculated a beneficial rhizobacterium on Vicia faba roots and soil, previous to plant infestation with BMSB. Salivary gland proteome of BMSB was analyzed by LC-MS/MS and a label-free quantitative proteomic method. Among the differentially expressed proteins, most were up-regulated in salivary glands of insects exposed to PGPR-treated plants for 24 h. We could confirm that BMSB was confronted with a stress during feeding on PGPR-treated plants. The to-be-confirmed defensive state of the plant would have been rapidly detected by the invasive H. halys pest, which consequently modified its salivary proteins. Among the up-regulated proteins, many could be associated with a role in plant defense counteraction, and more especially in allelochemicals detoxification or sequestration.

Co-Cultivation of Two Bacillus Strains for Improved Cell Growth and Enzyme Production to Enhance the Degradation of Aflatoxin B1.

Bacillus sp. H16v8 and Bacillus sp. HGD9229 were identified as Aflatoxin B1 (AFB1) degrader in nutrient broth after a 12 h incubation at 37 °C. The degradation efficiency of the two-strain supernatant on 100 µg/L AFB1 was higher than the bacterial cells and cell lysate. Moreover, degradations of AFB1 were strongly affected by the metal ions in which Cu2+ stimulated the degradation and Zn2+ inhibited the degradation. The extracellular detoxifying enzymes produced by co-cultivation of two strains were isolated and purified by ultrafiltration. The molecular weight range of the detoxifying enzymes was 20-25 kDa by SDS-PAGE. The co-culture of two strains improved the total cell growth with the enhancement of the total protein content and detoxifying enzyme production. The degradation efficiency of the supernatant from mixed cultures increased by 87.7% and 55.3% compared to Bacillus sp. H16v8 and HGD9229, individually. Moreover, after the degradation of AFB1, the four products of the lower toxicity were identified by LC-Triple TOF-MS with the two proposed hypothetical degradation pathways.

Simple stain-free screening method for pectinolytic microorganisms under alkalophilic conditions.

OBJECTIVES: To develop a simple pectin-degrading microorganism screening method. RESULTS: We developed a method utilizing the phenomenon whereby cooling an alkaline agar medium containing pectin causes the agar to become cloudy. This highly simplified method involves culturing the microorganisms on pectin-containing agar medium until colony formation is observed, and subsequent overnight cooling of the agar medium to 4 °C. Using this simple procedure, we successfully identified pectin-degrading microorganisms by observing colonies with halos on the clouded agar medium. We used alkaline pectinase and Bacillus halodurans, which is known to secrete alkaline pectinase, to establish the screening method. We demonstrated the screening of pectin-degrading microorganisms using the developed method and successfully isolated pectin-degrading microorganisms (Paenibacillus sp., Bacillus clausii, and Bacillus halodurans) from a soil sample. CONCLUSIONS: The developed method is useful for identifying pectin-degrading microorganisms.

Residual biomass from surfactin production is a source of arginase and adsorbed surfactin that is useful for environmental remediation.

Lipopeptides are important secondary metabolites produced by microbes. They find applications in environmental decontamination and in the chemical, pharmaceutical and food industries. However, their production is expensive. In the present work we propose three strategies to lower the production costs of surfactin. First, the coproduction of surfactin and arginase in a single growth. Second, extract the fraction of surfactin that adsorbs to the biomass and is removed from the growth medium through centrifugation. Third, use microbial biomass for the remediation of organic and inorganic contaminants. The coproduction of surfactin and arginase was evaluated by factorial design experiments using the LB medium supplemented with arginine. The best conditions for surfactin production were 22 h of growth at 37 °C using LB supplemented with arginine 7.3 g/L. Almost similar conditions were found to produce highest levels of arginase, 24 h and 6.45 g/L arginine. Decontamination of phenol and copper from artificial samples was attained by treatment with residues from lipopeptide production. Thus, cell suspensions and wash-waters used to extract surfactin from the biomass. Cell suspensions were used to successfully remove hydroquinone. Cell suspensions and wash-waters containing surfactin were successfully used to recover copper from solution. Specific monitoring methods were used for phenol and metal solutions, respectively a biosensor based on tyrosinase and either atomic absorption flame ionization spectrometry or absorbance coupled to the Arduino™ platform. Therefore, we report three alternative strategies to lower the production costs in lipopeptide production, which include the effective recovery of copper and phenol from contaminated waters using residues from surfactin production. Sustainable and profitable production of surfactin can be achieved by a coproduction strategy of lipopeptides and enzymes. Lipopeptides are collected in the supernatant and enzymes in the biomass. In addition, lipopeptides that coprecipitate with biomass can be recovered by washing. Lipopeptide wash-waters find applications in remediation and cells can also be used for environmental decontamination.

Statistical Based Bioprocess Design for Improved Production of Amylase from Halophilic Bacillus sp. H7 Isolated from Marine Water.

Amylase (EC 3.2.1.1) enzyme has gained tremendous demand in various industries, including wastewater treatment, bioremediation and nano-biotechnology. This compels the availability of enzyme in greater yields that can be achieved by employing potential amylase-producing cultures and statistical optimization. The use of Plackett-Burman design (PBD) that evaluates various medium components and having two-level factorial designs help to determine the factor and its level to increase the yield of product. In the present work, we are reporting the screening of amylase-producing marine bacterial strain identified as Bacillus sp. H7 by 16S rRNA. The use of two-stage statistical optimization, i.e., PBD and response surface methodology (RSM), using central composite design (CCD) further improved the production of amylase. A 1.31-fold increase in amylase production was evident using a 5.0 L laboratory-scale bioreactor. Statistical optimization gives the exact idea of variables that influence the production of enzymes, and hence, the statistical approach offers the best way to optimize the bioprocess. The high catalytic efficiency (kcat/Km) of amylase from Bacillus sp. H7 on soluble starch was estimated to be 13.73 mL/s/mg.

Biological characterization of Bacillus flexus strain SSAI1 transforming highly toxic arsenite to less toxic arsenate mediated by periplasmic arsenite oxidase enzyme encoded by aioAB genes.

Bacillus flexus strain SSAI1 isolated from agro-industry waste, Tuem, Goa, India displayed high arsenite resistance as minimal inhibitory concentration was 25 mM in mineral salts medium. This bacterial strain exposed to 10 mM arsenite demonstrated rapid arsenite oxidation and internalization of 7 mM arsenate within 24 h. The Fourier transformed infrared (FTIR) spectroscopy of cells exposed to arsenite revealed important functional groups on the cell surface interacting with arsenite. Furthermore, scanning electron microscopy combined with electron dispersive X-ray spectroscopy (SEM-EDAX) of cells exposed to arsenite revealed clumping of cells with no surface adsorption of arsenite. Transmission electron microscopy coupled with electron dispersive X-ray spectroscopic (TEM-EDAX) analysis of arsenite exposed cells clearly demonstrated ultra-structural changes and intracellular accumulation of arsenic. Whole-genome sequence analysis of this bacterial strain interestingly revealed the presence of large number of metal(loid) resistance genes, including aioAB genes encoding arsenite oxidase responsible for the oxidation of highly toxic arsenite to less toxic arsenate. Enzyme assay further confirmed that arsenite oxidase is a periplasmic enzyme. The genome of strain SSAI1 also carried glpF, aioS and aioE genes conferring resistance to arsenite. Therefore, multi-metal(loid) resistant arsenite oxidizing Bacillus flexus strain SSAI1 has potential to bioremediate arsenite contaminated environmental sites and is the first report of its kind.

Enhanced cercosporin production by co-culturing Cercospora sp. JNU001 with leaf-spot-disease-related endophytic bacteria.

BACKGROUND: Owing to the excellent properties of photosensitization, cercosporin, one of naturally occurring perylenequinonoid pigments, has been widely used in photodynamic therapy, or as an antimicrobial agent and an organophotocatalyst. However, because of low efficiency of total chemical synthesis and low yield of current microbial fermentation, the limited production restricts its broad applications. Thus, the strategies to improve the production of cercosporin were highly desired. Besides traditional optimization methods, here we screened leaf-spot-disease-related endophytic bacteria to co-culture with our previous identified Cercospora sp. JNU001 to increase cercosporin production. RESULTS: Bacillus velezensis B04 and Lysinibacillus sp. B15 isolated from leaves with leaf spot diseases were found to facilitate cercosporin secretion into the broth and then enhance the production of cercosporin. After 4 days of co-culture, Bacillus velezensis B04 allowed to increase the production of cercosporin from 128.2 mg/L to 984.4 mg/L, which was 7.68-fold of the previously reported one. Lysinibacillus sp. B15 could also enhance the production of cercosporin with a yield of 626.3 mg/L, which was 4.89-fold higher than the starting condition. More importantly, we found that bacteria B04 and B15 employed two different mechanisms to improve the production of cercosporin, in which B04 facilitated cercosporin secretion into the broth by loosening and damaging the hyphae surface of Cercospora sp. JNU001 while B15 could adsorb cercosporin to improve its secretion. CONCLUSIONS: We here established a novel and effective co-culture method to improve the production of cercosporin by increasing its secretion ability from Cercospora sp. JNU001, allowing to develop more potential applications of cercosporin.

Incubation temperature and culture medium formulation impact the accuracy of pour-plate techniques for the enumeration of industrial Bacillus assemblages.

Aerobic plate counting assays based on the pour-plate technique are frequently used to enumerate microbial products; however, colony swarming and merging at the agar surface can reduce the accuracy of these assays. Some plating methods mitigate this risk through the inclusion of strategies including agar overlays; however, these interventions may be inadequate to mitigate swarming and merging of certain Bacillus colonies. In the present study, we assessed the accuracy of several pour-plate techniques for the enumeration of a mixed-species Bacillus assemblage. Tested modifications included a customized culture medium formulation, agar overlays, decreased incubation times and increased incubation temperature. Methods which produced countable plates were assessed for agreement with a Bacillus-specific plate counting assay and with total cell counts rendered by flow cytometry. While all tested pour-plate methods underestimated Bacillus endospore concentrations relative to flow cytometry and customized spread-plating, our results suggest that increasing incubation temperature and the inclusion of bile salts into culture medium formulations can improve the accuracy of pour-plate techniques when used to enumerate Bacillus assemblages by decreasing the incidence of spreading colonies. As Bacillus endospore preparations become more ubiquitous in the market, familiar enumeration methods such as the pour-plate technique may require methodological modifications to ensure that the cGMP compliance of Bacillus-based microbial products is assessed accurately.

In Vitro Preformed Biofilms of Bacillus safensis Inhibit the Adhesion and Subsequent Development of Listeria monocytogenes on Stainless-Steel Surfaces.

Listeria monocytogenes continues to be one of the most important public health challenges for the meat sector. Many attempts have been made to establish the most efficient cleaning and disinfection protocols, but there is still the need for the sector to develop plans with different lines of action. In this regard, an interesting strategy could be based on the control of this type of foodborne pathogen through the resident microbiota naturally established on the surfaces. A potential inhibitor, Bacillus safensis, was found in a previous study that screened the interaction between the resident microbiota and L. monocytogenes in an Iberian pig processing plant. The aim of the present study was to evaluate the effect of preformed biofilms of Bacillus safensis on the adhesion and implantation of 22 strains of L. monocytogenes. Mature preformed B. safensis biofilms can inhibit adhesion and the biofilm formation of multiple L. monocytogenes strains, eliminating the pathogen by a currently unidentified mechanism. Due to the non-enterotoxigenic properties of B. safensis, its presence on certain meat industry surfaces should be favored and it could represent a new way to fight against the persistence of L. monocytogenes in accordance with other bacterial inhibitors and hygiene operations.

Lipopeptide production by Bacillus atrophaeus strain B44 and its biocontrol efficacy against cotton rhizoctoniosis.

OBJECTIVES: An assay was conducted to show the comparisons the effects of nine metal ions on antagonistic metabolites (lipopeptides, siderophores and gibberellins) by Bacillus atrophaeus strain B44 using well-diffusion assays, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, chrome azurol S plus mannitol salt agar (CAS-MSA) tests, and reversed-phase high-performance liquid chromatography (RP-HPLC) analysis. This assay is also designed to demonstrate the biocontrol efficacy of B44 against cotton rhizoctoniosis using pot culture tests. RESULTS: Both the lipopeptide yield and the antimicrobial activity of B44 increase with the MnSO4, MgSO4, CaCO3, and CuSO4 treatments and either have no effect or decreased lipopeptide yield and antimicrobial activity with the FeSO4, K2HPO4, KCl, KH2PO4 and ZnSO4 treatments. The medium containing MgSO4 has no significant effect on either the lipopeptide yield or antimicrobial activity. MALDI-TOF-MS analysis shows a broad range of m/z peaks, indicating that strain B44 produces a complex mixture of iturin, surfactin, and fengycin lipopeptides. Gibberellin production by strain B44 varies greatly depending on the culture medium, and the siderophore production is not significantly affected by the culture medium. Pot tests show that lipopeptide production affects the disease control efficacy of strain B44. CONCLUSION: The biocontrol efficacy of B. atrophaeus strain B44 is related to the lipopeptide yield. Moreover, B. atrophaeus strain B44 significantly increases the size of cotton seedlings, which is related to the GA3 concentration.

Dynamic consolidated bioprocessing for innovative lab-scale production of bacterial alkaline phosphatase from Bacillus paralicheniformis strain APSO.

To meet the present and forecasted market demand, bacterial alkaline phosphatase (ALP) production must be increased through innovative and efficient production strategies. Using sugarcane molasses and biogenic apatite as low-cost and easily available raw materials, this work demonstrates the scalability of ALP production from a newfound Bacillus paralicheniformis strain APSO isolated from a black liquor sample. Mathematical experimental designs including sequential Plackett-Burman followed by rotatable central composite designs were employed to select and optimize the concentrations of the statistically significant media components, which were determined to be molasses, (NH4)2NO3, and KCl. Batch cultivation in a 7-L stirred-tank bioreactor under uncontrolled pH conditions using the optimized medium resulted in a significant increase in both the volumetric and specific productivities of ALP; the alkaline phosphatase throughput 6650.9 U L-1, and µ = 0.0943 h-1; respectively, were obtained after 8 h that, ameliorated more than 20.96, 70.12 and 94 folds compared to basal media, PBD, and RCCD; respectively. However, neither the increased cell growth nor enhanced productivity of ALP was present under the pH-controlled batch cultivation. Overall, this work presents novel strategies for the statistical optimization and scaling up of bacterial ALP production using biogenic apatite.

Imaging high-performance thin-layer chromatography as powerful tool to visualize metabolite profiles of eight Bacillus candidates upon cultivation and growth behavior.

Imaging high-performance thin-layer chromatography (HPTLC) was explored with regard to its ability to visualize changes in the metabolite profile of bacteria. Bacillus subtilis has become a model organism in many fields. The increasing interest in these bacteria is driven by their attributed probiotic activity. However, growth behavior and metabolism of Bacillus species have a considerable influence on their activity and secondary metabolite profile. On the HPTLC plate, cultivation broths of Bacillus species (B. subtilis, B. licheniformis, B. pumilus and B. amyloliquefaciens) and some B. subtilis strains of high genetic similarity up to 99.5% were applied directly and compared with their respective liquid-liquid extracts. The latter as well as the cultivation in a minimal medium reduced the matrix load and improved the zone resolution. Cultivation parameters such as nutrient supply, cultivation temperature, cultivation time and rotational speed (oxygen level) as well as medium change were shown to have a considerable influence on the growth behavior and resulting metabolite profiles. Imaging HPTLC turned out to be an efficient and affordable tool to visualize such influences of cultivation parameters on the metabolite profiles. It converts the complexity of reaction processes occurring during cell cultivation in easy-to-understand images, which are helpful to figure out factors of influence and understand activity changes. The results highlighted that optimal cultivation conditions need to be found for the intended bacterial application, and in particular, these conditions have to be kept constant. It must be ensured that small variations in cultivation parameters of bacteria do not change the specified (probiotic) effect on the health of animals and humans. The HPTLC metabolite profiles represented the cultivation conditions of specific bacteria and were found to be a proof of the activity of distinct bacteria. In addition, HPTLC can also be used to optimize and streamline the culture media. The quality control of cultivation or fermentation processes can benefit from such a powerful tool, as a picture is worth a thousand words.

Evidences for antioxidant response and biosorption potential of Bacillus simplex strain 115 against lead.

In this study, we investigated effects of lead on growth response and antioxidant defense protection in a new identified strain isolated from a soil, in the rhizosphere of Sainfoin Hedysarum coronarium L. Different concentrations of lead (0, 0.2, 1.5 and 3 g L-1) added to Bacillus simplex strain 115 cultures surprisingly did not inhibit its growth. However, a resulting oxidative stress as attested by overproduction of H2O2 (+ 6.2 fold) and malondialdehyde (+ 2.3 fold) concomitantly to the enhancement of proteins carbonylation (+ 221%) and lipoxygenase activity (+ 59%) was observed in presence of 3 g L-1 of lead. Intrinsic antioxidant defenses were revealed by the coupled up-regulation of catalase (+ 416%) and superoxide dismutase (+ 4 fold) activities, with a more important Fe-SOD increase in comparison to the other isoforms. Bioaccumulation assays showed both intracellular and extracellular lead accumulation. Biosorption was confirmed as a particularly lead resistance mechanism for Bacillus simplex strain 115 as the metal sequestration in cell wall accounted for 88.5% to 98.5% of the total endogenous metal accumulation. Potentiality of this new isolated microorganism as a biotechnological tool for agricultural soil lead bioremediation was thus proposed.