Microbial Interactions in a Vitamin C Industrial Fermentation System: Novel Insights and Perspectives.

In industrial production, the precursor of l-ascorbic acid (L-AA, also referred to as vitamin C), 2-keto-l-gulonic acid (2-KLG), is mainly produced using a classic two-step fermentation process performed by Gluconobacter oxydans, Bacillus megaterium, and Ketogulonicigenium vulgare. In the second step of the two-step fermentation process, the microbial consortium of K. vulgare and B. megaterium is used to achieve 2-KLG production. K. vulgare can transform l-sorbose to 2-KLG, but the yield of 2-KLG is much lower in the monoculture than in the coculture fermentation system. The relationship between the two strains is too diverse to analyze and has been a hot topic in the field of vitamin C fermentation. With the development of omics technology, the relationships between the two strains are well explained; nevertheless, the cell-cell communication is unclear. In this review, based on current omics results, the interactions between the two strains are summarized, and the potential cell-cell communications between the two strains are discussed, which will shed a light on the further understanding of synthetic consortia.

Exopolysaccharides production from marine Bacillus strains and their antioxidant and bio-flocculant capacities.

Microbial exopolysaccharides (EPS) have gained high scientific concern due to their exceptional physicochemical features and high industrial applicability. Owing to their biotechnological importance, the present study was designed to screen and isolate the EPS-producing Bacillus strains based on their growth potential on specific media and colony morphologies. The bacterial isolates Bacillus subtilis Bs1-01, Bacillus licheniformis Bl1-02, and Bacillus brevis Bb1-04 showed excellent EPS production due to their shortened lag phase and abundant biomass production. Shake-flask fermentation valued the maximum production yield of 50.19 ± 1.14 g/L by Bl1-02 after 72 h incubation (about 3.40 times higher than that of Bacillus thuringiensis Bt1-05). The basic component analysis revealed the improved amount of total carbohydrate, reducing sugar ends, and protein contents by Bl1-02 strain. Structural characteristics and functional groups of the EPS characterized by Fourier transform infrared spectroscopy demonstrated that all EPS were in close agreement to each other due to the presence of similar chemical bonds and functional groups. EPS from Bl1-02 strain showed stronger and more stable bio-emulsifying and hygroscopicity activities (12.23%). The crude EPS exhibited potent antioxidant properties which were examined against reducing potential (H2O2 scavenging) and total antioxidant tests. Among bio-flocculation activities of EPS at different concentrations, Bs1-01 strain produced EPS at a concentration of 60 mg/mL was observed to show the maximum value of 79.20%. In conclusion, the EPS from marine Bacillus strains showed excellent functional properties suggesting potential industrial applications that demand separate investigations.

Effect of metal ions on lipopeptide secretion from Bacillus subtilis strain FJAT-4: Negative regulation by Ca2.

AIMS: This study aimed to investigate the effect of metal ions on lipopeptide production by Bacillus subtilis strain FJAT-4 and the mechanism of negative regulation by Ca2+ . METHODS AND RESULTS: The quantitative measurement of lipopeptides in response to K+ , Na+ , Mg2+ and Ca2+ addition was carried out by LC-MS. The contents of fengycin and surfactin varied within the range of 116.24-129.80 mg/L and 34.03-63.11 mg/L in the culture media containing K+ , Na+ and Mg2+ , while the levels were 0.86 and 0.63 mg/L in the media containing Ca2+ . Ca2+ at a high concentration (45 mM) did not adversely affect the growth of strain FJAT-4, but caused significant downregulation of lipopeptide synthesis-related gene expression, corresponding to a decrease in lipopeptide production. This inhibition by Ca2+ was further investigated by proteomic analysis. In total, 112 proteins were upregulated and 524 proteins were downregulated in the presence of additional Ca2+ (45 mM). Among these differentially expressed proteins (DEPs), 28 were related to phosphotransferase activity, and 42 were related to kinase activity. The proteomics results suggested that altered levels of three two-component signal-transduction systems (ResD/ResE, PhoP/PhoR and DegU/DegS) might be involved in the control of expression of the fen and srfA operons of FJAT-4 under high calcium stress. CONCLUSIONS: The Ca2+ at the high concentration (45 mM) triggers a decrease in lipopeptide production, which might be attributed to the regulation of three two-component signal-transduction systems ResD/ResE, PhoP/PhoR and DegU/DegS. SIGNIFICANCE AND IMPACT OF THE STUDY: The regulatory effect of calcium on the expression of genes encoding lipopeptide synthetases can be applied to optimize the production of lipopeptides.

Effective bioremediation of petroleum-contaminated saline soil using halotolerant Bacillus strains isolated from the Persian Gulf.

A consortium isolated from the Persian Gulf is evaluated for its ability to bioremediate petroleum-contaminated soils. The soil sample was collected from oil fields of South Western Iran. The crude oil concentrations were set to 1000-10,000 mg/kg, and the sodium chloride concentration was set to 0.5%, 1%, 1.5%, 2%, and 2.5%. Operational parameters including volume (2-20 ml) and soil moisture (25%, 50%, and 100%) were studied consecutively according to one factor at the time of experimental design. A total number of eight different isolates capable of degrading crude oil were isolated from hydrocarbon-contaminated sites (KL1-KL8). The removal efficiency of Total petroleom hydrocarbons (TPH) with an initial concentration of 1000 mg/kg for numbers of bacterial cells per gram soil of 2, 10, and 20 CFU/g was 20.9%, 45%, and 60%, respectively. The removal efficiency of TPHs (initial concentration of 1000 mg/kg) at the end of fifth week for salinity amounts of 0.5%, 1%, 1.5%, 2%, and 2.5% was 10.87%, 22.4%, 25.7%, 68.6%, and 60.5%, respectively. The TPHs biodegradation efficiencies at different soil/water ratios of 25%, 50%, and 100% (slurry) were 12%, 28.7%, and 60.8%, respectively. In sunflowers, there was no statistically significant difference in seed germination for different levels of soil pollution (p > 0.05). The results of the current work suggest that this process is a viable and efficient method for remediating contaminated sites. To enhance the removal results in real soil, a scale-up study should also be conducted.

Effects of different probiotic combinations on the components and bioactivity of Spirulina.

Spirulina acts as a good dietary nutritional supplement. However, few research studies have been conducted on its fermentation. Three groups of probiotic combinations, lactic acid bacteria, Bacillus strains, and their mixture, were used to investigate Spirulina fermentation. The results showed that lactic acid bacteria significantly increased the content of amino acids and the ratio of essential amino acids to total amino acids in the fermented Spirulina, compared with the unfermented Spirulina, and this trend was enhanced by the strains' mixture. However, compared to unfermented Spirulina, the amino acid levels were significantly decreased after fermentation with Bacillus strains and so was the total free amino acid and essential amino acid content. Fermentation significantly reduced the contents of the offensive components of Spirulina, with significant differences among the three mixed bacterial treatments. Moreover, Bacillus strain fermentation increased the contents of flavonoids and polyphenols compared to the unfermented Spirulina, and significantly enhanced 1,1-diphenyl-2-trinitrophenylhydrazine free-radical scavenging ability and total antioxidant ability. On the contrary, treatments with lactic acid bacteria and the mixture of lactic acid bacteria and Bacillus strains endowed the fermented supernatants with good antibacterial ability. The results showed that probiotic fermentation has a good effect on Spirulina and can serve as a new procedure for developing new Spirulina-containing food items.

Lipopeptide biodiversity in antifungal Bacillus strains isolated from Algeria.

Several Bacillus strains have been well studied for their ability to control soil-borne plant diseases. This property is linked to the production of several families of lipopeptides. Depending of their structure, these compounds show antifungal and/or plant systemic resistance inducing activities. In this work, the biodiversity of lipopeptides produced by different antifungal Bacillus strains isolated from seeds, rhizospheric, and non-rhizospheric soils in Algeria was analyzed. Sixteen active strains were characterized by PCR for their content in genes involved in lipopeptide biosynthesis and by MALDI-ToF for their lipopeptide production, revealing a high biodiversity of products. The difficulty to detect kurstakin genes led us to design two new sets of specific primers. An interesting potential of antifungal activity and the synthesis of two forms of fengycins differing in the eighth amino acid (Gln/Glu) were found from the strain 8. Investigation of its genome led to the finding of an adenylation domain of the fengycin synthetase predicted to activate the glutamate residue instead of the glutamine one. According to the comparison of both the results of MALDI-ToF-MS and genome analysis, it was concluded that this adenylation domain could activate both residues at the same time. This study highlighted that the richness of the Algerian ecosystems in Bacillus strains is able to produce: surfactin, pumilacidin, lichenysin, kurstakin, and different types of fengycins.

The abundance and mineral-weathering effectiveness of Bacillus strains in the altered rocks and the soil.

In this study, we collected different levels of altered rocks of a rocky mountain and the adjacent soil and characterized the abundance and weathering effectiveness of Bacillus strains. Based on qPCR and culture-dependent approaches, the gene copies or the numbers of Bacillus strains were significantly higher in the soil than in the altered rocks, while the ratio of the gene copies or the numbers of Bacillus strains to those of total bacteria was higher in the less altered rock, followed by the more altered rock and the soil. The relative abundance of the highly active Al-solubilizing Bacillus strains was higher in the more altered rock, followed by the less altered rock and the soil. Among the Al-solubilizing Bacillus species, 30-36% of them were different between the altered rocks and the soil, however, similar Al-solubilizing Bacillus species were found in the less altered rocks and the more altered ones. The results showed the alteration-related changes in the abundance and mineral weathering effectiveness of Bacillus strains and suggested the ecological adaptation of the mineral-weathering Bacillus populations and their role in mineral weathering in the rock and soil environments.

New application of Bacillus strains for optically pure L-lactic acid production: general overview and future prospects.

Members of the genus Bacillus are considered to be both, among the best studied and most commonly used bacteria as well as the most still unexplored and the most wide-applicable potent bacteria because novel Bacillus strains are continuously being isolated and used in various areas. Production of optically pure l-lactic acid (l-LA), a feedstock for bioplastic synthesis, from renewable resources has recently attracted attention as a valuable application of Bacillus strains. l-LA fermentation by other producers, including lactic acid bacteria and Rhizopus strains (fungi) has already been addressed in several reviews. However, despite the advantages of l-LA fermentation by Bacillus strains, including its high growth rate, utilization of various carbon sources, tolerance to high temperature, and growth in simple nutritional conditions, it has not been reviewed. This review article discusses new findings on LA-producing Bacillus strains and compares them to other producers. The future prospects for LA-producing Bacillus strains are also discussed.

Enhancing Water Status and Nutrient Uptake in Drought-Stressed Lettuce Plants (Lactuca sativa L.) via Inoculation with Different Bacillus spp. Isolated from the Atacama Desert.

Drought is a major challenge for agriculture worldwide, being one of the main causes of losses in plant production. Various studies reported that some soil's bacteria can improve plant tolerance to environmental stresses by the enhancement of water and nutrient uptake by plants. The Atacama Desert in Chile, the driest place on earth, harbors a largely unexplored microbial richness. This study aimed to evaluate the ability of various Bacillus sp. from the hyper arid Atacama Desert in the improvement in tolerance to drought stress in lettuce (Lactuca sativa L. var. capitata, cv. "Super Milanesa") plants. Seven strains of Bacillus spp. were isolated from the rhizosphere of the Chilean endemic plants Metharme lanata and Nolana jaffuelii, and then identified using the 16s rRNA gene. Indole acetic acid (IAA) production, phosphate solubilization, nitrogen fixation, and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity were assessed. Lettuce plants were inoculated with Bacillus spp. strains and subjected to two different irrigation conditions (95% and 45% of field capacity) and their biomass, net photosynthesis, relative water content, photosynthetic pigments, nitrogen and phosphorus uptake, oxidative damage, proline production, and phenolic compounds were evaluated. The results indicated that plants inoculated with B. atrophaeus, B. ginsengihumi, and B. tequilensis demonstrated the highest growth under drought conditions compared to non-inoculated plants. Treatments increased biomass production and were strongly associated with enhanced N-uptake, water status, chlorophyll content, and photosynthetic activity. Our results show that specific Bacillus species from the Atacama Desert enhance drought stress tolerance in lettuce plants by promoting several beneficial plant traits that facilitate water absorption and nutrient uptake, which support the use of this unexplored and unexploited natural resource as potent bioinoculants to improve plant production under increasing drought conditions.

Plant-Microbes Interaction: Exploring the Impact of Cold-Tolerant Bacillus Strains RJGP41 and GBAC46 Volatiles on Tomato Growth Promotion through Different Mechanisms.

The interaction between plant and bacterial VOCs has been extensively studied, but the role of VOCs in growth promotion still needs to be explored. In the current study, we aim to explore the growth promotion mechanisms of cold-tolerant Bacillus strains GBAC46 and RJGP41 and the well-known PGPR strain FZB42 and their VOCs on tomato plants. The result showed that the activity of phytohormone (IAA) production was greatly improved in GBAC46 and RJGP41 as compared to FZB42 strains. The in vitro and in-pot experiment results showed that the Bacillus VOCs improved plant growth traits in terms of physiological parameters as compared to the CK. The VOCs identified through gas chromatography-mass spectrometry (GC-MS) analysis, namely 2 pentanone, 3-ethyl (2P3E) from GBAC46, 1,3-cyclobutanediol,2,2,4,4-tetramethyl (CBDO) from RJGP41, and benzaldehyde (BDH) from FZB42, were used for plant growth promotion. The results of the partition plate (I-plate) and in-pot experiments showed that all the selected VOCs (2P3E, CBDO, and BDH) promoted plant growth parameters as compared to CK. Furthermore, the root morphological factors also revealed that the selected VOCs improved the root physiological traits in tomato plants. The plant defense enzymes (POD, APX, SOD, and CAT) and total protein contents were studied, and the results showed that the antioxidant enzymes and protein contents significantly increased as compared to CK. Similarly, plant growth promotion expression genes (IAA4, ARF10A, GA2OX2, CKX2, and EXP1) were significantly upregulated and the ERF gene was downregulated as compared to CK. The overall findings suggest that both Bacillus isolates and their pure VOCs positively improved plant growth promotion activities by triggering the antioxidant enzyme activity, protein contents, and relative gene expressions in tomato plants.

Enhancement of the enzymatic hydrolysis efficiency of wheat bran using the Bacillus strains and their consortium.

In the downstream process, the bioconversion of lignocellulosic biomass can be improved by applying a biological pretreatment procedure using microorganisms to produce hydrolytic enzymes to modify the recalcitrant structure of lignocellulose. In this study, various Bacillus strains (B. subtilis B.01162 and B.01212, B. coagulans B.01123 and B.01139, B. cereus B.00076 and B.01718, B. licheniformis B.01223 and B.01231) were evaluated for the degrading capacity of wheat bran in the submerged medium using enzymatic activities, reducing sugars and weight loss as indicators. The obtained results revealed that the B. subtilis B.01162, B. coagulans B.01123 and B. cereus B.00076 could be promising degraders for the wheat bran pretreatment. Besides, the application of their consortium (the combination of 2-3 Bacillus species) showed the positive effects on cellulose bioconversion compared with monocultures. Among them, the mixture of B. subtilis B.01162 and B. coagulans B.01123 increased significantly the cellulase, endo-glucanase, and xylanase enzyme activity resulting in accelerating the lignocellulose degradation. Our results served a very good base for the development of microbial consortium for biological pretreatment of lignocellulosic raw materials.

Solubilization of rock phosphate by phosphate solubilizing bacteria isolated from effluent treatment plant sludge of a fertilizer plant.

This study evaluates the efficiency of Phosphate solubilizing bacteria isolated from Effluent Treatment Plant sludge of Paradeep Phosphate Limited, Odisha, India, to solubilize rock phosphate (RP) and the mechanism and structural changes during solubilization investigated by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR). Out of 13 bacterial isolates, Bacillus cereus S0B4, Solibacillus isronensis S0B8, and Bacillus amyloliquefaciens S0B17 strains were found to be the best RP solubilizers. This is the first report on S. isronensis S0B8 with the ability to solubilize RP. In particular, the potent strain B. cereus S0B4 showed maximum soluble P (338.5 mg/L) on the 7th day. Negative correlations (r = -0.70; p ≤ 0.01) were observed between soluble P concentration and pH, whereas positive correlation exists with the growth of B. cereus S0B4 (r = 0.91, p ≤ 0.01), S. isronensis S0B8 (r = 0.75, p ≤ 0.01), and B. amyloliquefaciens S0B17 (r = 0.77, p ≤ 0.01) respectively. The P release kinetics followed the 1st order model well (R2 = 0.8001-0.8503). It seems that H+ ions and organic anions released from the organic acids are major factors responsible for RP solubilization. The SEM observations demonstrate that B. cereus S0B4 corroded the RP surface significantly due to the proton attack. The XRD analysis confirms that the intensity of all mineral peaks decreases after treatment with B. cereus S0B4. The FTIR analysis indicated a significant decrease of calcite and fluorapatite's vibrational bands with the disappearance of quartz.

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.

Decolorization of textile azo dye Novacron Red using bacterial monoculture and consortium: Response surface methodology optimization.

The present study was intended toward the optimization of a textile dye Novacron Red decolorization by single and mixed culture of Bacillus strains namely, B. firmus, B. filamentosus and B. subterraneus. Optimization of dye decolorization using Bacillus monocultures was conducted using central composite design. The maximum dye decolorization achieved under optimized conditions for B. firmus, B. filamentosus and B. subterraneus was 89.24%, 88.28% and 88.45%, respectively. The effect of various consortia of selected Bacillus strains on dye removal was evaluated by applying a mixture design. The best dye (100 mg/L) decolorization yield (84%) was achieved using the consortium of B. filamentosus and B. subetrraneus.The Fourier Transform Infrared Spectroscopy analyses confirmed biodegradation potential of the two Bacillus strains. The results highlighted the potential of mono- and co-cultures of Bacillus strains for application in textile wastewater treatment. PRACTITIONER POINTS: Novel dye-decolorizing Bacillus strains were isolated from marine sediment. Optimization of decolorization was conducted using response surface methodology. Efficient decolorization of textile dye by Bacillus strains on mono- and co-cultures. The efficiency of the consortium B. filamentosus and B. subetrraneus on dye removal.

Antimicrobial Potential of Bioactive Metabolites and Silver Nanoparticles from Bacillus spp. and of Some Antibiotics Against Multidrug Resistant Salmonella spp.

OBJECTIVES: The synthesis of nanoparticles using microorganisms and their metabolites is of increasing interest because they are potential producers of biocompatible and environmental friendly nanoparticles. Their nanoparticles can serve as potent alternatives to antibiotics against multidrug resistant (MDR) bacteria. The antibacterial potential of Bacillus spp. metabolites, their silver nanoparticles (SNPs), and some antibiotics against MDR Salmonella spp. was evaluated. MATERIALS AND METHODS: The antimicrobial potential of metabolites and SNPs biosynthesized from Bacillus spp. was characterized, the effect of physicochemical parameters on SNP biosynthesis, the antimicrobial activity of the SNPs, and combination of SNPs and antibiotics against MDR Salmonella strains were evaluated. RESULTS: The bioactive metabolites of the Bacillus spp. exhibited varied antimicrobial potential against the tested MDR Salmonella spp. The metabolites were able to bioreduce silver nitrate (AgNO3) to Ag+ for SNP biosynthesis. Change in color from whitish to darkish brown and a surface plasma resonance peak of 600-800 nm were observed. The SNPs were aggregated, rods, and crystalline in shape and their sizes were 15 µm, 16 µm, and 13 µm. Carboxylic acid, amino acid, alcohol, esters, and aldehydes were the functional groups found in the biosynthesized SNPs. The antibacterial activity of BAC1-SNPs, BAC7-SNPs, and BAC20-SNPs against MDR Staphylococcus aureus 9 (MDRSA9) and MDRSA18 was 6.0-22 mm and 11-20 mm. SNPs biosynthesized at pH 7 and 10 mM AgNO3 had the highest antagonistic activity. Combination of SNPs and antibiotics exhibited the best antagonistic potential. CONCLUSION: The metabolites and SNPs from Bacillus spp. exhibited antagonistic effects against MDR Salmonella spp. The combined SNPs and antibiotics had better antimicrobial activity.

Probiotic Properties of Bacillus Strains Isolated from Stingless Bee (Heterotrigona itama) Honey Collected across Malaysia.

This study aimed to isolate, identify, and evaluate the probiotic properties of Bacillus species from honey of the stingless bee Heterotrigona itama. Bacillus spp. were isolated from five different H. itama meliponicultures, and the isolates were characterized through Gram-staining and a catalase test. Tolerance to acidic conditions and bile salt (0.3%), hydrophobicity, and autoaggregation tests were performed to assess the probiotic properties of the selected isolates, B. amyloliquefaciens HTI-19 and B. subtilis HTI-23. Both Bacillus isolates exhibited excellent antimicrobial activity against both Gram-positive and Gram-negative bacteria and possessed significantly high survival rates in 0.3% bile solution for 3 h. Their survival rates in acidic conditions were also comparable to a commercial probiotic strain, Lactobacillus rhamnosus GG. Interestingly, the hydrophobicity and autoaggregation percentage showed no significant difference from L. rhamnosus GG, a commercial probiotic strain. The results from this study suggest that B. amyloliquefaciens HTI-19 and B. subtilis HTI-23 isolated from stingless bee honey have considerably good probiotic properties. Therefore, more studies should be done to investigate the effects of these bacteria cultures on gastrointestinal health.

Biocontrol activity of surfactin A purified from Bacillus NH-100 and NH-217 against rice bakanae disease.

The potential of the Bacillus genus to antagonize phytopathogens is associated with the production of cyclic lipopeptides. Depending upon the type of lipopeptide, they may serve as biocontrol agents that are eco-friendly alternatives to chemical fertilizers. This study evaluates the biocontrol activity of surfactin-producing Bacillus (SPB) strains NH-100 and NH-217 and purified surfactin A from these strains against rice bakanae disease. Biologically active surfactin fractions were purified by HPLC, and surfactin A variants with chain lengths from C12 to C16 were confirmed by LCMS-ESI. In hemolytic assays, a positive correlation between surfactin A production and halo zone formation was observed. The purified surfactin A had strong antifungal activity against Fusarium oxysporum, F. moniliforme, F. solani, Trichoderma atroviride and T. reesei. Maximum fungal growth suppression (84%) was recorded at 2000 ppm against F. moniliforme. Surfactin A retained antifungal activity at different pH levels (5-9) and temperatures (20, 50 and 121 °C). Hydroponic and pot experiments were conducted to determine the biocontrol activity of SPB strains and the purified surfactin A from these strains on Super Basmati rice. Surfactin production in the rice rhizosphere was detected by LCMS-ESI at early growth stages in hydroponics experiments inoculated with SPB strains. However, the maximum yield was observed with a consortium of SPB strains (T4) and purified surfactin A (T5) treatments in the pot experiment. The outcomes of the present study revealed that surfactin A significantly reduced rice bakanae disease by up to 80%. These findings suggest that purified surfactin A could be an effective biocontrol agent against bakanae disease in rice and should be incorporated into strategies for disease management.

Mini review: Recombinant production of tailored bio-pharmaceuticals in different Bacillus strains and future perspectives.

Bio-pharmaceuticals like antibodies, hormones and growth factors represent about one-fifth of commercial pharmaceuticals. Host candidates of growing interest for recombinant production of these proteins are strains of the genus Bacillus, long being established for biotechnological production of homologous and heterologous proteins. Bacillus strains benefit from development of efficient expression systems in the last decades and emerge as major industrial workhorses for recombinant proteins due to easy cultivation, non-pathogenicity and their ability to secrete recombinant proteins directly into extracellular medium allowing cost-effective downstream processing. Their broad product portfolio of pharmaceutically relevant recombinant proteins described in research include antibody fragments, growth factors, interferons and interleukins, insulin, penicillin G acylase, streptavidin and different kinases produced in various cultivation systems like microtiter plates, shake flasks and bioreactor systems in batch, fed-batch and continuous mode. To further improve production and secretion performance of Bacillus, bottlenecks and limiting factors concerning proteases, chaperones, secretion machinery or feedback mechanisms can be identified on different cell levels from genomics and transcriptomics via proteomics to metabolomics and fluxomics. For systematical identification of recurring patterns characteristic of given regulatory systems and key genetic targets, systems biology and omics-technology provide suitable and promising approaches, pushing Bacillus further towards industrial application for recombinant pharmaceutical protein production.

Adaptation of primocane fruiting raspberry plants to environmental factors under the influence of Bacillus strains in Western Siberia.

In geographical locations with a short vegetative season and continental climate that include Western Siberia, growing primocane fruiting raspberry varieties becomes very important. However, it is necessary to help the plants to overcome the environmental stress factors. This study aimed to evaluate the impact of the pre-planting treatment of primocane fruiting raspberry root system with Bacillus strains on the following plant development under variable environmental conditions. In 2012, Bacillus subtilis RCAM В-10641, Bacillus amyloliquefaciens RCAM В-10642, and Bacillus licheniformis RCAM В-10562 were used for inoculating the root system of primocane fruiting raspberry cultivar Nedosyagaemaya before planting. The test suspensions were 105 CFU/ml for each bacterial strains. The effects of this treatment on plant growth and crop productivity were estimated in 2012-2015 growing seasons differed by environmental conditions. The pre-planting treatment by the bacterial strains increased the number of new raspberry canes and the number of plant generative organs as well as crop productivity compared to control. In addition, these bacilli acted as the standard humic fertilizer. Variable environmental factors such as air temperature, relative humidity, and winter and spring frosts seriously influenced the plant biological parameters and crop productivity of control plants. At the same time, the pre-planting primocane fruiting root treatment by Bacillus strains decreased the negative effects of abiotic stresses on plants in all years of the research. Of the three strains studied, B. subtilis was shown to reveal the best results in adaptation of primocane fruiting raspberry plants to environmental factors in Western Siberia. For the first time, the role of Bacillus strains in enhancing frost resistance in primocane fruiting raspberry plants was shown. These bacilli are capable of being the basis of multifunctional biological formulations for effective plant and environmental health management in growing primocane fruiting raspberry.

Bacillus amyloliquefaciens SB14 from rhizosphere alleviates Rhizoctonia damping-off disease on sugar beet.

The use of biocontrol strains recently has become a popular alternative to conventional chemical treatments. A set of bacteria isolated from sugar beet rhizosphere and from roots and shoots of apple and walnut were evaluated for their potential to control sugar beet seedling damping-off caused by R. solani AG-4 and AG2-2.The results of in vitro assays concluded that three isolates, SB6, SB14, SB15, obtained from rhizosphere of sugar beet and five isolates, AP2, AP4, AP6, AP7, AP8, obtained from shoots and roots of apple were the most effective antagonists that inhibited the mycelial growth of both R. solani isolates. Combination of several biochemical tests and partial sequencing of 16S rRNA and gyrBgenes revealed that eight efficient bacterial isolates could be assigned to the genus Bacillus and all could tolerate high temperatures and salt concentrations in their vegetative growth. The potential biocontrol activity of the eight bacterial antagonists were tested in greenhouse condition. The results indicated that four strains,B. amyloliquefaciens SB14, B. pumilus SB6,B. siamensis AP2 and B. siamensisAP8 exerted a significant influence on controlling of seedling damping-off and performed significantly better than others.However, the treatment of the seeds with bacteria was most effective when the isolate SB14 was used, which significantly controlled damping-off disease by 58% caused by R. solani AG-4 and by 52.5% caused by R. solani AG-2-2. This indicates that the use of beneficial bacterial native to the host plant may increase the success rate in screening biocontrols, because these microbes are likely to be better adapted to their host and its associated environmental conditions than are strains isolated from other plant species grown in different environmental conditions. We can infer from the results reported here that sugar beet plantsmay recruitbeneficial microbes to the rhizosphere to help them solve context-specific challenges.