Biocontrol Efficacy of Bacillus methylotrophicus TA-1 Against Meloidogyne incognita in Tomato.

Root-knot nematodes (RKNs) are harmful plant-parasitic nematodes of tomatoes which can cause significant yield losses. Therefore, there is increasing interest in exploring the application of bacterial nematicides. The bacterium Bacillus methylotrophicus TA-1 is a broad-spectrum biological control agent; however, its effect on RKNs control remains largely unclear. In this study, the toxicity of B. methylotrophicus TA-1 against Meloidogyne incognita was investigated in vitro, and the potential of B. methylotrophicus TA-1 to decrease infection of RKNs in tomato were evaluated in pot and field trials. Results showed that B. methylotrophicus TA-1 exhibited high nematicidal activity against second-stage juveniles (J2s) and eggs of M. incognita with 50% lethal concentration (LC50) values of 5.80 and 7.00 × 108 colony forming units (CFU)/ml, respectively. In the pot experiments and field trials conducted in 2020 and 2021, tomato plants treated with B. methylotrophicus TA-1 soil drench applied once at 3, 6, and 9 × 108 CFU/plant had significantly higher plant height and greater yield compared with the untreated control. Tomato yields of the treated plots with B. methylotrophicus TA-1 in 2 consecutive years' field trials were between 53.4 to 66.1 and 52.8 to 61.5 t/ha, while they were 49.7 and 48.2 t/ha in the untreated control for each year, respectively. The lowest population densities of M. incognita at 30 and 60 days after treatment were 119 and 135 J2s per 100 g soil in 2020 and 43 and 118 J2s in 2021 in TA-1-treated plots. The lowest gall index of 4.7 and 3.3 in 2020 and 2021, respectively, and the highest yield were all observed in the TA-1 at 9 × 108 CFU/plant treated plants, with no significant differences with the commercial control abamectin. These results provided a basis for further studies of B. methylotrophicus TA-1 formulations, application doses, frequencies, and mechanisms of action, which are necessary before it could be used as a component of integrated management programs to manage RKNs in tomato production.

Effects of Bacillus methylotrophicus SY200 Supplementation on Growth Performance, Antioxidant Status, Intestinal Morphology, and Immune Function in Broiler Chickens.

The present study was focused on evaluating the effects of Bacillus methylotrophicus SY200 in broiler production. A total of 120 healthy 7-day-old broiler chicks were randomly assigned to four dietary treatments, which included basal diet supplemented with 0%, 0.10%, 0.25%, or 0.50% (w/w) B. methylotrophicus SY200 preparation (1.0 × 109 cfu/g), regarded as negative control group (NC), low-dose group (BML), medium-dose group (BMM), and high-dose group (BMH), respectively. Each treatment was fed the corresponding experimental diet for 35 days. Results showed that dietary supplementation of B. methylotrophicus SY200 could improve broiler weight gain, especially the finisher phase. Further studies suggested that a certain amount of B. methylotrophicus SY200 enhanced the broiler antioxidant status and improved the morphological development of jejunum. Besides, dietary supplementation of B. methylotrophicus SY200 especially in 0.50% levels significantly increased the relative weight of immune organs and Newcastle disease virus antibody titer, similarly, increased mRNA expression levels of claudin-1, claudin-3, zonula occluden-1, and zonula occluden-2 were observed in the jejunum of BMM group. Moreover, B. methylotrophicus SY200 also showed beneficial effects in improving broilers microbiota homeostasis by increasing the number of beneficial bacteria. Conclusively, B. methylotrophicus SY200 could effectively improve the antioxidant status, modulate the intestinal structure, enhance the intestinal mucosal barrier function, and regulate the immune function of broilers, which finally improves the performance of the chicken in the finisher period.

RNA-Seq Provides Insights into the Mechanisms Underlying Ilyonectria robusta Responding to Secondary Metabolites of Bacillus methylotrophicus NJ13.

(1) Background: Ilyonectria robusta can cause ginseng to suffer from rusty root rot. Secondary metabolites (SMs) produced by Bacillus methylotrophicus NJ13 can inhibit the mycelial growth of I. robusta. However, the molecular mechanism of the inhibition and response remains unclear. (2) Methods: Through an in vitro trial, the effect of B. methylotrophicus NJ13's SMs on the hyphae and conidia of I. robusta was determined. The change in the physiological function of I. robusta was evaluated in response to NJ13's SMs by measuring the electrical conductivity, malondialdehyde (MDA) content, and glucose content. The molecular interaction mechanism of I. robusta's response to NJ13's SMs was analyzed by using transcriptome sequencing. (3) Results: NJ13's SMs exhibited antifungal activity against I. robusta: namely, the hyphae swelled and branched abnormally, and their inclusions leaked out due to changes in the cell membrane permeability and the peroxidation level; the EC50 value was 1.21% (v/v). In transcripts at 4 dpi and 7 dpi, the number of differentially expressed genes (DEGs) (|log2(fold change)| > 1, p adj ≤ 0.05) was 1960 and 354, respectively. NJ13's SMs affected the glucose metabolism pathway, and the sugar-transporter-related genes were downregulated, which are utilized by I. robusta for energy production. The cell wall structure of I. robusta was disrupted, and chitin-synthase-related genes were downregulated. (4) Conclusions: A new dataset of functional responses of the ginseng pathogenic fungus I. robusta was obtained. The results will benefit the development of targeted biological fungicides for I. robusta and the study of the molecular mechanisms of interaction between biocontrol bacteria and phytopathogenic fungi.

Bacterial biosurfactant increases ex situ biodiesel bioremediation in clayey soil.

The contamination of soils by oily compounds has several environmental impacts, which can be reversed through bioremediation, using biosurfactants as auxiliaries in the biodegradation process. In this study, we aimed to perform ex situ bioremediation of biodiesel-contaminated soil using biosurfactants produced by Bacillus methylotrophicus. A crude biosurfactant was produced in a whey-based culture medium supplemented with nutrients and was later added to biodiesel-contaminated clayey soil. The produced lipopeptide biosurfactant could reduce the surface tension of the fermentation broth to 30.2 mN/m. An increase in the microbial population was observed in the contaminated soil; this finding can be corroborated by the finding of increased CO2 release over days of bioremediation. Compared with natural attenuation, the addition of a lower concentration of the biosurfactant (0.5% w/w in relation to the mass of diesel oil) to the soil increased biodiesel removal by about 16% after 90 days. The added biosurfactant did not affect the retention of the contaminant in the soil, which is an important factor to be considered when applying in situ bioremediation technologies.

Antagonistic microorganisms and nitrogen fertilization in control of tomato southern blight

The present study assessed the efficacy of formulated biocontrol agents and nitrogen fertilization on southern blight control. Antagonism test in vitro was performed to assess the inhibitory activity of Bacillus methylotrophicus and Trichoderma asperellum against the growth of Sclerotium rolfsii. Tomato seedlings were transplanted into the substrate added with ammonium nitrate doses and inoculated with the formulated biocontrol agents Ônix (B. methylotrophicus) or Quality (T. asperellum). Subsequently, seedlings were inoculated with S. rolfsii. Plant mortality, shoot and root weight were assessed 11 days after the last inoculation. Agents had effective inhibitory activity against S. rolfsii; thus, they could reduce southern blight severity when combined with ammonium nitrate. However, plant mortality was not reduced by them.(AU)

Antifungal Activity of Volatile Organic Compounds Produced by Bacillus methylotrophicus and Bacillus thuringiensis against Five Common Spoilage Fungi on Loquats.

Loquat fruit is one of the most perishable fruits in China, and has a very limited shelf life because of mechanical injury and microbial decay. Due to an increasing concern about human health and environmental security, antagonistic microorganisms have been a potential alternative for fungicides to control postharvest diseases. In this work, the antifungal effect of volatile organic compounds (VOCs) produced by Bacillus methylotrophicus BCN2 and Bacillus thuringiensis BCN10 against five postharvest pathogens isolated from loquat fruit, Fusarium oxysporum, Botryosphaeria sp., Trichoderma atroviride, Colletotrichum gloeosporioides, and Penicillium expansum were evaluated by in vitro and in vivo experiments. As a result, the VOCs released by BCN2 and BCN10 were able to suppress the mycelial growth of all targeted pathogens according to inhibition ratio in the double petri-dish assay as well as disease incidence and disease diameter on loquat fruits. The main volatile compounds were identified by solid-phase microextraction (SPME)-gas chromatography. These VOCs produced by the two strains played complementary roles in controlling these five molds and enabled loquat fruits to keep fresh for ten days, significantly. This research will provide a theoretic foundation and technical support for exploring the functional components of VOCs applicable in loquat fruit preservation.

Isolation and evaluation of the plant growth promoting rhizobacterium Bacillus methylotrophicus (DD-1) for growth enhancement of rice seedling.

Bacillus methylotrophicus has been demonstrated to promote growth of various plants, whereas the promoting effect of B. methylotrophicus on rice growth has been rarely reported. In this study, B. methylotrophicus DD-1, capable of efficiently promoting the growth of rice, was isolated from the root soil of rice plants. The isolate exhibited potassium-solubilizing (1.18 mg/L), Indole-3-acetic acid (IAA) (87.26 mg/L), Gibberellic acid (GA) (25.91 mg/L) and Siderophore production activity (52.32%). As indicated from the result, plant growth parameters (e.g., dry weight, tiller number, root and shoot length) of rice seedlings treated with the isolate DD-1 were more effective than those of the control group in pot and soilless culture experiments. Moreover, the adsorption capacity of rice roots which were soaked in the bacterial suspension of isolate increased with the increase in concentration and absorption time. In sterilized and unsterilized soil, conformation of root colonization activity by bacterial isolate established by its nearer existence to the rice root. Thus, the B. methylotrophicus DD-1 enhances plant growth promotion by multifarious growth promoting and root colonization traits, thereby augmenting potassium level in soil. Henceforth, the potential bacterium could be exploited for the development of biological fertilizer, leading towards sustainable agronomy.

Effects of Bacillus methylotrophicus M4-1 on physiological and biochemical traits of wheat under salinity stress.

AIM: The aim of this study was to evaluate the ability of Bacillus methylotrophicus M4-1 to protect winter wheat from the harmful effects of soil salinity and alkalinity. METHODS AND RESULTS: We isolated the halotolerant B. methylotrophicus M4-1. Two representative soils with different salt contents (S1, 213 µs cm-1 ; S2, 786 µs cm-1 ) in the Yellow River delta region of China were selected for experiments. The effects of the M4-1 strain on the typical wheat variety (Jimai 21) in this environment were proven. In S1 soil, the M4-1 strain reduced the wheat rhizosphere soil pH (1·61%) and electrical conductivity (EC) (8·01%) and increased the exchangeable K content (11·14%). The uptake of Mg2+ (20·73%) by wheat roots and K+ (8·84%) by leaves was increased, and the content of Na+ (23·62%) in leaves was reduced. In S2 soil, the M4-1 strain was able to reduce soil EC (2·56%) and increase exchangeable K (11·20%) content. The absorption of K+ (13·28%) in wheat leaves was increased, and the content of Na+ (12·41%) in roots was decreased. Total N and organic matter contents in rhizosphere soil were significantly positively correlated with wheat growth and salt tolerance, whereas EC showed a significant negative correlation. CONCLUSIONS: M4-1 attenuates salt stress injury in wheat under both low and high salt stress. SIGNIFICANCE AND IMPACT OF THE STUDY: We demonstrated the efficacy and value of plant growth-promoting rhizobacteria addition to protect winter wheat against salt stress and improve crop yield. We also elucidated the physicochemical and biochemical interactions among M4-1, the rhizosphere and the host plant.

Efficacy of Bacillus methylotrophicus SY200 strain as feed additive against experimental Salmonella typhimurium infection in mice.

To investigate the effects of Bacillus methylotrophicus SY200 on Salmonella typhimurium (STM) infection in mice, a total of 36 three-week-old male mice were selected and randomly divided into 3 equal groups (N = 12). Group A and group B were fed with basal diet while group C was fed the basal diet supplemented with 0.1% (w/w) B. methylotrophicus SY200 during the 21 days experimental period. On the 14th day of the experiment, mice of group A were intragastrically administered with 0.5 ml of normal saline, group B and C were orally administered with 0.5 ml of STM suspension. On the first day and seventh day after STM challenge, the number of total white blood cells (WBCs) and neutrophils, relative weight of visceral organs, the number of Salmonella spp., Escherichia coli, Lactobacillus spp. and Bifidobacterium spp. in ileum and cecum, and diversity of cecal microflora were measured. The results showed that: on the first day and seventh day after STM challenge, the number of WBCs and neutrophils in the blood of the mice was the highest in group B, then followed by group C, and group A. On the first day after STM challenge, the relative weight of spleen in group C was significantly higher than that in group B (p < 0.05), moreover, compared with group B, B. methylotrophicus SY200 significantly reduced the number of Salmonella spp. and E. coli (p < 0.05), and increased the number of Lactobacillus spp. and Bifidobacterium spp. (p < 0.05) in the intestines of mice, and improved the Shannon-Wiener diversity (H), Simpson (E) and richness (S) indices of cecal flora of mice (p < 0.05). The results indicated that B. methylotrophicus SY200 could alleviate the inflammatory reaction after STM infection and resist the adverse effects of STM infection on mice intestinal flora.

Screening of cellulolytic bacteria from rotten wood of Qinling (China) for biomass degradation and cloning of cellulases from Bacillus methylotrophicus.

BACKGROUND: Cellulosic biomass degradation still needs to be paid more attentions as bioenergy is the most likely to replace fossil energy in the future, and more evaluable cellulolytic bacteria isolation will lay a foundation for this filed. Qinling Mountains have unique biodiversity, acting as promising source of cellulose-degrading bacteria exhibiting noteworthy properties. Therefore, the aim of this work was to find potential cellulolytic bacteria and verify the possibility of the cloning of cellulases from the selected powerful bacteria. RESULTS: In present study, 55 potential cellulolytic bacteria were screened and identified from the rotten wood of Qinling Mountains. Based on the investigation of cellulase activities and degradation effect on different cellulose substrates, Bacillus methylotrophicus 1EJ7, Bacillus subtilis 1AJ3 and Bacillus subtilis 3BJ4 were further applied to hydrolyze wheat straw, corn stover and switchgrass, and the results suggested that B. methylotrophicus 1EJ7 was the most preponderant bacterium, and which also indicated that Bacillus was the main cellulolytic bacteria in rotten wood. Furthermore, scanning electron microscopy (SEM) and X-ray diffraction analysis of micromorphology and crystallinity of wheat straw also verified the significant hydrolyzation. With ascertaining the target sequence of cellulase ß-glucosidase (243 aa) and endoglucanase (499 aa) were successfully heterogeneously cloned and expressed from B. methylotrophicus 1EJ7, and which performed a good effect on cellulose degradation with enzyme activity of 1670.15 ± 18.94 U/mL and 0.130 ± 0.002 U/mL, respectively. In addition, based on analysis of amino acid sequence, it found that ß-glucosidase were belonged to GH16 family, and endoglucanase was composed of GH5 family catalytic domain and a carbohydrate-binding module of CBM3 family. CONCLUSIONS: Based on the screening, identification and cellulose degradation effect evaluation of cellulolytic bacteria from rotten wood of Qinling Mountains, it found that Bacillus were the predominant species among the isolated strains, and B. methylotrophicus 1EJ7 performed best on cellulose degradation. Meanwhile, the ß-glucosidase and endoglucanase were successfully cloned and expressed from B. methylotrophicus for the first time, which provided new materials of both strain and the recombinant enzymes for the study of cellulose degradation and its application in industry.

Purification and Characterization of a Novel Antifungal Flagellin Protein from Endophyte Bacillus methylotrophicus NJ13 Against Ilyonectria robusta.

Endophyte Bacillus methylotrophicus NJ13 was isolated from Panax ginseng. Its sterile fermentation liquid showed a significant inhibitory effect against Ilyonectria robusta, causing the rusty root rot of P. ginseng and P. quinquefolius. The antifungal protein was obtained after precipitation by 20% saturated ammonium sulfate, desalted by Sephadex G-25, weak anion exchange chromatography, and gel filtration chromatography. SDS-PAGE showed that the purified protein was approximately 29 KDa. The antifungal protein after desalting was not resistant to temperatures higher than 100 °C, resistant to acid conditions, and did not tolerate organic solvents and protease K. The amino acid sequence of purified antifungal protein had an identity of 76% to flagellin from Bacillus velezensis. The isoelectric point of the protein was 4.97 and its molecular mass was 27 KDa. Therefore, a specific primer G1 was designed based on the flagellin gene sequence, and a 770 bp gene sequence was cloned in NJ13 genomic DNA, which shared the same size of flagellin. There were ten base differences between the gene sequences of flagellin and the cloned gene, however, the amino acid sequence encoded by the cloned gene was identical to the flagellin. In conclusion, the antifungal protein produced by biocontrol agent NJ13 contained a flagellin protein.

Biopreservation of Fresh Strawberries by CarboxymethylCellulose Edible Coatings Enriched with a Bacteriocin from Bacillus methylotrophicus BM47.

Bacteriocins are a large group of antimicrobial compounds that are synthesized by representatives of the genus Bacillus and lactic acid bacteria. They are used extensively in the food industry as biopreservatives. Incorporated in the composition of edible coatings, bacteriocins can reduce microbial growth and decay incidence in perishable fruits, thus improving product shelf-life and commercial appearance. The present study aims to investigate the effect of edible coatings of 0.5% carboxymethyl cellulose (CMC) enriched with a purified bacteriocin from Bacillus methylotrophicus BM47 on the shelf-life extension of fresh strawberries. During storage at 4 °C and 75% relative humidity for 16 days, the measurements of mass loss, decay percentage, total soluble solids (TSS), titratable acidity (TA), pH, organic acids, total phenolic and anthocyanin contents and antioxidant activity were made. The results demonstrate that the application of edible coatings with 0.5% CMC and 0.5% CMC with bacteriocin (CMC+B) led to a significant decrease of mass loss in the treated strawberries compared to the uncoated fruit. After the 8th day of storage, significant reductions in decay percentage along with the absence of fungal growth in CMC+B-coated fruit were observed in comparison with the CMC-coated and control strawberries. During the second half of the storage period, CMC and CMC+B treatments reduced TSS amount in the coated fruit compared to the control, but did not affect the increase of TA and decrease of pH values that are normally associated with postharvest changes. The CMC and CMC+B coatings did not prevent the decrease of ascorbic acid, and total phenolic and anthocyanin contents during cold storage. The application of CMC and CMC+B coatings had a significant inhibitory effect on decreasing the antioxidant activity throughout the storage period and maintained the antioxidant levels in both treatments close to the initial value of 76.8 mmol Trolox equivalents per 100 g of fresh mass.

[Inhibition effect of biocontrol bacteria NJ13 and its mixture with chemical fungicides against ginseng root rot caused by Fusarium solani].

This study was aimed to clarify the toxicity indoor and inhibition effect of biocontrol strain NJ13 and its mixture with chemical fungicides against Fusarium solani causing ginseng root rot. The method of mycelial growth rate and Sun Yunpei method were used to determine the indoor toxicity and co-toxicity coefficient of strain NJ13 and their mixture with chemical pesticides against F. solani. The dual culture assay method,mixed culture method and microscopic observation were used to determine the sporulation and germination of spores and mycelial growth and morphological change of hyphae of F. solani treated by strain NJ13. The results of toxicity indoor showed that strain NJ13 had the best inhibitory effect on pathogen,and its EC_(50) value was 0. 071 mg·L~(-1). It was all synergistic for antifungal effect that strain NJ13 was mixed with propiconazole and difenoconazole respectively with a range from 1 ∶4 to 4 ∶1( volume ratio). Both of optimal ratios were 1 ∶1,and the co-toxicity coefficients were 848. 70 and 859. 73,respectively. The strain NJ13 could inhibit the sporulation,germination and mycelial growth of F. solani. The biocontrol strain NJ13 had an inhibition effect on F. solani,and the optimal antifungal ratio of strain NJ13 mixed with propiconazole and difenoconazole was obtained.

Screening of Endophytic Antagonistic Bacterium from Phellodendron amurense and Their Biocontrol Effects against Canker Rot.

Thirty-four strains of bacteria were isolated from Phellodendron amurense. Using Nectria haematococca as an indicator strain, the best strain, B18, was obtained by the growth rate method. The morphological, physiological and biochemical characteristics of strain B18 and its 16S DNA gene sequence were identified, and the biocontrol effect of strain B18 was assessed in pot and field tests, as well as in a field-control test. Drilling methods were used to determine the antibacterial activity of metabolites from strain B18 and their effects on the growth of pathogen mycelia and spores. The best bacteriostatic rate was 85.4%. B18 can hydrolyse starch and oxidize glucose but does not produce gas; a positive result was obtained in a gelatine liquefaction test. According to 16S DNA gene sequencing, strain B18 is Bacillus methylotrophicus (GenBank accession number: MG457759). The results of pot and field-control trials showed 98% disease control when inoculating 108 cfu/ml of the strain. The disease control effect of the B18 culture liquid (concentrations of 108, 2 × 106, 106, 5 × 105 and 2.5 × 105 cfu/ml) in the field-control test was higher than 80%, and the cure rate of the original delivery solution was 96%. Therefore, in the practical forestry production, a 2.5 × 105 cfu/ml culture liquidshould be applied in advance to achieve good control effects.

Simultaneous production of alkaline amylase and biosurfactant by Bacillus methylotrophicus DCS1: application as detergent additive.

This study investigated the coproduction of alkaline amylase and lipopeptides by Bacillus methylotrophicus DCS1 strain, as well as their biochemical characterisation. The best production of both amylase and biosurfactant was obtained when potato starch (10 g/L) and glutamic acid (5 g/L) were used as carbon and nitrogen sources, respectively. The bacterial strain was incubated for 48 h at 25 °C and 150 rpm. This strain produced a unique amylase as showed by zymography technique. The optima pH and temperature were 60-65 °C and 8.0, respectively. Amylase activity was partially inhibited by EDTA (5 mM). The main hydrolysis products of potato starch were maltose and maltotriose. The alkaline amylase showed excellent stability and compatibility with various solid and liquid detergents. Furthermore, the biosurfactant, produced simultaneously with alkaline amylase, demonstrated high stability at different ranges of salinity, pH, and temperature. Considering its promising properties, B. methylotrophicus DCS1 crude extract containing both biosurfactants and amylase activity may be considered as a potential candidate for future use in detergent processing industries and environmental remediation processes.

Inhibition effect of biocontrol bacteria NJ13 and its mixture with chemical fungicides against ginseng root rot caused by Fusarium solani / 中国中药杂志

This study was aimed to clarify the toxicity indoor and inhibition effect of biocontrol strain NJ13 and its mixture with chemical fungicides against Fusarium solani causing ginseng root rot. The method of mycelial growth rate and Sun Yunpei method were used to determine the indoor toxicity and co-toxicity coefficient of strain NJ13 and their mixture with chemical pesticides against F. solani. The dual culture assay method,mixed culture method and microscopic observation were used to determine the sporulation and germination of spores and mycelial growth and morphological change of hyphae of F. solani treated by strain NJ13. The results of toxicity indoor showed that strain NJ13 had the best inhibitory effect on pathogen,and its EC_(50) value was 0. 071 mg·L~(-1). It was all synergistic for antifungal effect that strain NJ13 was mixed with propiconazole and difenoconazole respectively with a range from 1 ∶4 to 4 ∶1( volume ratio). Both of optimal ratios were 1 ∶1,and the co-toxicity coefficients were 848. 70 and 859. 73,respectively. The strain NJ13 could inhibit the sporulation,germination and mycelial growth of F. solani. The biocontrol strain NJ13 had an inhibition effect on F. solani,and the optimal antifungal ratio of strain NJ13 mixed with propiconazole and difenoconazole was obtained.

Effects of antagonistic bacteria B6 against the pathogens of apple replant disease on the biomass of Malus hupehensis Rehd seedlings and soil environment under replanting.

We isolated strains from the rhizosphere soil of apple trees with replanting disease and evaluated the biological control potential for the pathogens Fusarium proliferatum, F. moniliforme, F. oxysporum, and F. solani. The morphological, physiological and biochemical character, and 16S rDNA sequence of the strain with the highest inhibitory rate were analyzed. The effect of strain biofertilizer on the biomass of Malus hupehensis Rehd. seedlings and soil environment under replanting disease was evaluated in a pot experiment. The results showed that the strain B6 had the strongest antagonistic activity. The inhibitory rate of B6 for F. proliferatum, F. moniliforme, F. oxysporum, Fusarium solani reached 71.8%, 70.1%, 72.6% and 91.5%, respectively. The strain B6 was identified as Bacillus methylotrophicus according to the results of morphological, physiological and biochemical character and 16S rDNA sequence analysis. Compared with the control, the bacterial manure made from the strain B6 enhanced the biomass of Malus hupehensis Rehd. seedlings in replanting soil to different extent. The ground diameter, fresh and dry mass were significantly increased by 18.3%, 49.6% and 51.2%, respectively. The strain B6 dramatically increased the number of cultivable bacteria and actinomyces in replanting soil and reduced the abundance of fungus to 37.7%, which accelerated the conversion of fungal soil to bacterial soil. It also dramatically increased the activities of sucrase, phosphatase, ureaseandcatalase in soil by 37.3%, 24.0%, 42.9% and 49.4%, respectively. In conclusion, the B6 fertilizer could improve the structure of cultivable microbial communities in the continuous cropping soil of apple trees, increase the soil enzyme activity, and enhance the growth of Malus hupehensis seedlings.

Formulation and application of a biosurfactant from Bacillus methylotrophicus as collector in the flotation of oily water in industrial environment.

The present study describes the formulation of a biosurfactant produced by Bacillus methylotrophicus UCP1616 and investigates its long-term stability for application as a collector in a bench-scale dissolved air flotation (DAF) prototype. For formulation, the conservative potassium sorbate was added to the biosurfactant with or without prior heat treatment at 80 °C for 30 min. After formulation, the biosurfactant samples were stored at room temperature for 180 days and the tensioactive properties of the biomolecule were determined with different pH values, temperatures and concentrations of salt. Then, a central composite rotatable design was used to evaluate the influence of the independent variables (effluent flow rate and formulated biosurfactant flow rate) on the oil removal efficiency in the DAF prototype. The formulated biosurfactant demonstrated good stability in both conservation methods, with tolerance to a wide pH range, salinity and high temperatures, enabling its use in environments with extreme conditions. The efficiency of the formulated biomolecule through heating and addition of sorbate was demonstrated by the 92% oil removal rate in the DAF prototype. The findings demonstrate that the biosurfactant from Bacillus methylotrophicus enhances the efficiency of the DAF process, making this technology cleaner. This biosurfactant can assist in the mitigation and management of industrial effluents, contributing toward a reduction in environmental pollution caused by petroleum-based hydrocarbons.

Bacillus methylotrophicus ASWU-C2, a strain inhabiting hot desert soil, a new source for antibacterial bacillopyrone, pyrophen, and cyclopeptides.

A strain of Bacillus methylotrophicus was isolated from a soil sample collected in Aswan eastern desert, which is known for its extremely arid climate. After fermentation of the strain in liquid culture and subsequent extraction, a bioassay-guided isolation procedure yielded five compounds: 2-benzyl-4H-pyran-4-one, named bacillopyrone (1), pyrophen (2), macrolactin A (3) and the cyclopeptides malformin A1 (4), and bacillopeptin A (5). The structures were determined by interpretation of nuclear magnetic resonance (NMR) spectroscopy and high resolution mass spectrometry (HR-MS) data. This is the first report on the isolation of compounds 1 and 2 from Bacillus species; compound 1 was reported previously as synthetic product. Bacillopyrone (1) exhibited moderate activity against the Gram-negative Chromobacterium violaceum with minimum inhibitory concentration 266.6 µg/mL, while macrolactin A (3) and malformin A1 (4) inhibited Staphylococcus aureus (minimum inhibitory concentrations 13.3 and 133.3 µg/mL, respectively).

Isolation, Characterization and Amino Acid Composition of a Bacteriocin Produced by Bacillus methylotrophicus Strain BM47.

Members of the bacterial genus Bacillus are known as producers of a broad spectrum of antibiotic compounds of proteinaceous nature that possess inhibitory activity against different saprophytic and pathogenic microorganisms. In the current research, a peptide synthesized by Bacillus methylotrophicus strain BM47, previously isolated from a natural thermal spring in Bulgaria, was identified and characterized as a bacteriocin. In vitro antimicrobial screening of the crude bacteriocin substance of B. methylotrophicus BM47 showed activity against the plant pathogenic fungi Fusarium moniliforme, Aspergillus awamori, Penicillium sp., Aspergillus niger and Gram-negative bacterium Pseudomonas aeruginosa. The antimicrobial activity of the crude bacteriocin substance was partially inhibited by the enzymes trypsin, Alcalase®, Savinase®, proteinase K, papain and Esperase®, while catalase was not effective. The crude bacteriocin substance was relatively pH resistant, but sensitive to the action of heat and most organic solvents and detergents tested. To obtain the active protein fractions, crude bacteriocin substance was purified by fast protein liquid chromatography (FPLC) using a strong anion exchange column. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that the purified bacteriocin had molecular mass of 19 578 Da. The amino acid analysis performed by high-performance liquid chromatography (HPLC) revealed that the isolated bacteriocin consisted of 17 types of amino acids, with the highest mol fraction expressed as percent of serine (29.3), valine (10.3), alanine (9.8) and tyrosine (7.1).