Unique behavioral patterns of wandering colonies of Brevibacillus thermoruber on agar plates.

Brevibacillus thermoruber strain Nabari cells grow as widely spreading dendritic colonies on reasoner's 2A-agar (1.5%) plates at around 55°C but as small motile colonies at 37°C. Motile colonies can be divided into colonies that move in straight or curved lines over long distances (wandering colonies), and colonies that rotate at a fixed location (rotating colonies). The addition of surfactant to the agar medium greatly increased the frequency of wandering colonies and facilitated the study of such colonies. The morphology of the wandering colonies varied: circular at the tip and pointed at the back, lemon-shaped with pointed ends, crescent-shaped, bullet-shaped, fish-like, and so on. A single colony may split into multiple colonies as it moves, or multiple colonies may merge into a single colony. The most surprising aspect of the movement of wandering colonies was that when a moving colony collides with another colony, it sometimes does not make a U-turn, but instead retreats straight back, as if bouncing back. The migration mechanisms of wandering colonies are discussed based on optical microscopic observations of swimming patterns of single cells in water and scanning electron microscopy of the arrangement of bacterial cells in wandering colonies.

Rapid polymerase chain reaction assays for Brevibacillus laterosporus detection.

The identification of the ubiquitous spore-forming bacterium Brevibacillus laterosporus, whose interest in pharma, agriculture, and other industrial sectors is raising, mostly relies on 16S ribosomal RNA gene sequence analysis. However, due to bacterial gene homology, this method appears insufficient for a proper discrimination of this species, so that the availability of other target genes is necessary. Leveraging the morphological and genetic feature uniqueness of B. laterosporus, a sensitive and reliable detection and quantification method based on polymerase chain reaction (PCR) and quantitative PCR assays, respectively, was developed. Targeting a highly conserved spore surface protein-related gene, B. laterosporus could be easily found in different matrices including soil, food, and insect body. Primer set selectivity was confirmed to be very specific and no false positives or negatives were observed using DNA of different bacterial species as a template. The method developed is also suitable for the rapid identification of newly isolated B. laterosporus strains.

Tolerance of triazole-based fungicides by biocontrol agents used to control Fusarium head blight in wheat in Argentina.

Fusarium head blight (FHB) caused by Fusarium graminearum species complex is a devastating disease that causes extensive yield and quality losses to wheat around the world. Fungicide application and breeding for resistance are among the most important tools to counteract FHB. Biological control is an additional tool that can be used as part of an integrated management of FHB. Bacillus velezensisRC 218, Brevibacillus sp. RC 263 and Streptomyces sp. RC 87B were selected by their potential to control FHB and deoxynivalenol production. The aim of this work was to test the tolerance of these biocontrol agents to triazole-based fungicides such as prothioconazole, tebuconazole and metconazole. Bacterial growth was evaluated in Petri dishes using the spread plating technique containing the different fungicides. Bacillus velezensisRC 218 and Streptomyces sp. RC 87B showed better tolerance to fungicides than Brevibacillus sp. RC 263. Complete growth inhibition was observed at concentrations of 20 µg ml-1 for metconazole, 40 µg ml-1 for tebuconazole and 80 µg ml-1 for prothioconazole. The results obtained indicate the possibility of using these biocontrol agents in combination with fungicides as part of an integrated management to control FHB of wheat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study evaluates the possibility to use biocontrol agents (Bacillus velezensisRC 218, Brevibacillus sp. RC 263 and Streptomyces sp. RC 87B) in combination with triazole-based fungicides to control Fusarium head blight in wheat. The evaluation of biocontrol agents' growth under in vitro conditions was carried out in Petri dishes containing either prothioconazole, tebuconazole or metconazole. Viability studies demonstrated that B. velezensisRC 218 and Streptomyces sp. RC 87B were more tolerant to the fungicides evaluated. Results obtained reflect the possibility to use fungicides at low doses combined with biocontrol agents.

Screening of a beta-cypermethrin-degrading bacterial strain Brevibacillus parabrevis BCP-09 and its biochemical degradation pathway.

A novel beta-cypermethrin (Beta-CP)-degrading strain isolated from activated sludge was identified as Brevibacillus parabrevis BCP-09 based on its morphological and physio-biochemical characteristics, and 16S rRNA gene analysis. Strain BCP-09 could effectively degrade Beta-CP at pH 5.0-9.0, 20-40 °C, and 10-500 mg L-1 Beta-CP. Under optimal conditions (pH 7.41, 38.9 °C, 30.9 mg L-1 Beta-CP), 75.87% Beta-CP was degraded within 3 days. Beta-CP degradation (half-life, 33.45 h) and strain BCP-09 growth were respectively described using first-order-kinetic and logistic-kinetic models. Seven metabolites were detected by high-performance liquid chromatography and gas chromatography-mass spectrometry- methyl salicylate, catechol, phthalic acid, salicylic acid, 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 3-phenoxybenzaldehyde, and 3-phenoxybenzoic acid (3-PBA). The major Beta-CP metabolite, 3-PBA was further degraded into phenol, benzoic acid, and 4-methylhexanoic acid. BCP-09 also degraded aromatic compounds such as phenol, catechol, and protocatechuic acid. Beta-CP appears to be mainly degraded into 3-PBA, which is continuously degraded into smaller benzene or chain compounds. Thus, strain BCP-09 could form a complete degradation system for Beta-CP and might be considered a promising strain for application in the bioremediation of environments and agricultural products polluted by Beta-CP.

Biodegradation of malathion by Brevibacillus sp. strain KB2 and Bacillus cereus strain PU.

We report here the degradation of a pesticide, malathion, by Brevibacillus sp. strain KB2 and Bacillus cereus strain PU, isolated from soil samples collected from malathion contaminated field and an army firing range respectively. Both the strains were cultured in the presence of malathion under aerobic and energy-limiting conditions. Both strains grew well in the medium having malathion concentration up to 0.15%. Reverse phase HPLC-UV analysis indicated that Strain KB2 was able to degrade 72.20% of malaoxon (an analogue of malathion) and 36.22% of malathion, while strain PU degraded 87.40% of malaoxon and 49.31% of malathion, after 7 days of incubation. The metabolites mal-monocarboxylic acid and mal-dicarboxylic acid were identified by Gas chromatography/mass spectrometry. The factors affecting biodegradation efficiency were investigated and effect of malathion concentration on degradation rate was also determined. The strain was analyzed for carboxylesterase activity and maximum activity 210 ± 2.5 U ml(-1) and 270 U ± 2.7 ml(-1) was observed for strains KB2 and PU, respectively. Cloning and sequencing of putative malathion degrading carboxylesterase gene was done using primers based PCR approach.

Antibacterial Properties of Melanoidins Produced from Various Combinations of Maillard Reaction against Pathogenic Bacteria.

Novel melanoidins are produced by the Maillard reaction. Here, melanoidins with high antibacterial activity were tested by examining various combinations of reducing sugars and amino acids as reaction substrates. Twenty-two types of melanoidins were examined by combining two reducing sugars (glucose and xylose) and eleven l-isomers of amino acids (alanine, arginine, glutamine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and valine) to confirm the effects of these melanoidins on the growth of Listeria monocytogenes at 25°C. The melanoidins produced from the combination of d-xylose with either l-phenylalanine (Xyl-Phe) or l-proline (Xyl-Pro), for which absorbance at 420 nm was 3.5 ± 0.2, completely inhibited the growth of L. monocytogenes at 25°C for 48 h. Both of the melanoidins exhibited growth inhibition of L. monocytogenes which was equivalent to the effect of nisin (350 IU/mL). The antimicrobial spectrum of both melanoidins was also investigated for 10 different species of bacteria, including both Gram-positive and Gram-negative species. While Xyl-Phe-based melanoidin successfully inhibited the growth of Bacillus cereus and Brevibacillus brevis, Xyl-Pro-based melanoidin inhibited the growth of Salmonella enterica Typhimurium. However, no clear trend in the antimicrobial spectrum of the melanoidins against different bacterial species was observed. The findings in the present study suggest that melanoidins generated from xylose with phenylalanine and/or proline could be used as potential novel alternative food preservatives derived from food ingredients to control pathogenic bacteria. IMPORTANCE Although the antimicrobial effect of melanoidins has been reported in some foods, there have been few comprehensive investigations on the antimicrobial activity of combinations of reaction substrates of the Maillard reaction. The present study comprehensively investigated the potential of various combinations of reducing sugars and amino acids. Because the melanoidins examined in this study were produced simply by heating in an autoclave at 121°C for 60 min, the targeted melanoidins can be easily produced. The melanoidins produced from combinations of xylose with either phenylalanine or proline exhibited a wide spectrum of antibiotic effects against various pathogens, including Listeria monocytogenes, Bacillus cereus, and Salmonella enterica Typhimurium. Since the antibacterial effect of the melanoidins on L. monocytogenes was equivalent to that of a nisin solution (350 IU/mL), we might expect a practical application of melanoidins as novel food preservatives.

Following tyrothricin peptide production by Brevibacillus parabrevis with electrospray mass spectrometry.

The tyrocidines and analogues are cationic cyclodecapeptides [cyclo (D-Phe1-L-Pro2-L-(Phe3/Trp3)-D-(Phe4/Trp4)-L-Asn5-L-Gln6-L-(Tyr7/Phe7/Trp7)-L-Val8-L-(Orn9/Lys9)-L-Leu10], produced together with the neutral linear pentadecapeptide gramicidins, in the antibiotic tyrothricin complex by Brevibacillus parabrevis. Despite discovery 80 years ago, it was still uncertain whether these peptides are secreted or sequestered intracellularly. We resolved this by utilising high resolution electrospray mass spectrometry to confirm the predominantly intracellular sequestration of the peptides in the tyrothricin complex. A "peptidomics" approach allowed us to map the intracellular production of 16 cyclodecapeptides and 6 gramicidins over 16 days of culturing. Gramicidin production remained relatively constant, with Val-gramicidin A the predominant analogue produced throughout the 16 day fermentation period. The tyrothricin cyclodecapeptides have four variable positions and there was a culturing time related shift from the Phe-rich A analogues, containing a L-Phe3-D-Phe4 aromatic dipeptide unit, to the Trp-rich C analogues with L-Trp3-D-Trp4. For the other variable aromatic residue position, Tyr7 was preferentially incorporated above Trp7, with a minor incorporation of Phe7 over the whole culturing period. For the variable basic amino acid residue, there was time-sensitive shift from Orn9 to Lys9 incorporation. Modulation of the cyclodecapeptide profile over time does not correlate with the reported non-ribosomal peptide synthetase affinity, specifically for Trp in the variable aromatic residue positions, indicating additional supply-demand control in the cyclodecapeptides production by B. parabrevis. These novel observations are not only of importance for production and purification of selected peptide analogues from the tyrothricin complex, but also for insight into microbial control of non-ribosomal peptide production that extends beyond the peptide synthetase machinery.

Multi-functional magnetic bacteria as efficient and economical Pickering emulsifiers for encapsulation and removal of oil from water.

HYPOTHESIS: Effective removal of oil pollutants from the surface of water is important in oil-polluted environments. Since living microorganisms can be used as particle-stabilizers for oil emulsification, magnet-responsive oil-degrading bacteria (M-Bacteria) are expected to integrate three intriguing properties, such as Pickering emulsification, magnet-responsiveness and bioactivity. Hence, by acting as an efficient Pickering emulsifier to encapsulate oil pollutants, it should be possible to eliminate oil from water under the application of an external magnetic field. EXPERIMENTS: Oil-degrading bacteria, Brevibacillus parabrev, were successfully coated with a shell of magnetic Fe3O4 nanoparticles using polycations. The morphology and physicochemical characteristics of M-Bacteria were characterized by various techniques. A systematic study on Pickering emulsification of M-Bacteria and the removal of five types of oils were performed. Specific adsorption of M-Bacteria at the oil droplet surface was observed through optical, fluorescence, and scanning electronic microscopy images. The biodegradation process of oil was monitored using gas chromatography. FINDINGS: Eco-friendly M-Bacteria not only acts as an effective particle emulsifier to realize encapsulation and magnetic separation of oil contaminants but also shows a strong ability for further conversion of oil. This is the first report of oil removal via Pickering emulsification of living bacterial cells, which shows the potential of bacterial cells as functional colloidal materials in treating oily wastewater.

Manganese(II) oxidation by the multi-copper oxidase CopA from Brevibacillus panacihumi MK-8.

Manganese contamination of groundwater exists worldwide. Manganese removal is primarily performed through catalytic oxidation by manganese-oxidizing bacteria. In this study, we identified a new manganese(II) oxidase (CopA) from Brevibacillus panacihumi MK-8. The copA gene was cloned and expressed in Escherichia coli strain BL21(DE3), and the recombinant strain BL21-pET-copA was able to remove 85.87% of Mn(II) from LB medium containing 1 mM Mn(II) after seven days. The optimum Mn(II) oxidase CopA activity was obtained at 37 °C in 10 mM HEPES buffer (pH 8.0) containing 0.4 mM CuCl2. Purified CopA removed 51.98% of manganese(II) under the optimal conditions. The copA gene-deleted strain (MK-8-ΔcopA) barely oxidized manganese, further demonstrating that the copA gene is the manganese oxidase gene. Biogenic Mn oxides were analyzed by scanning electron microscopy and X-ray diffraction. Thus, we suggest that the recombinant BL21-pET-copA strain and oxidase CopA have the potential to be used in biological manganese removal technology.

Kinetic analysis and degradation pathway for m-dichlorobenzene removal by Brevibacillus agri DH-1 and its performance in a biotrickling filter.

A strain, Brevibacillus agri DH-1, isolated from dry lands was used to remove m-dichlorobenzene. After 48h culturing, the concentrations of m-dichlorobenzene decreased from 26-130 to 7.87-28.87mg/L and dry cell weight for bacterial growth reached 52.43-75.05mg/L. The growth and degradation kinetics were analyzed by the fitting of Haldane-Andrews model and pseudo first-order model. A degradation pathway was proposed according to major intermediates (phenol), chloride ion variation, ring-opening enzyme activity, and high mineralization (0.47gCl-/gm-dichlorobenzene, 0.65 gco2/gm-dichlorobenzene, 0.15 gDCW/gm-dichlorobenzene). In addition, the performance in a biotrickling filter (BTF) was evaluated through removal efficiency and pressure drop values with increasing inlet loading rate from 4.10 to 122.57g/m3/h at three empty bed residence time points (30s, 60s, and 90s). The results demonstrated that strain DH-1 possessed high removal efficiency and stable operation in a BTF.

Characteristics and proteomic analysis of pyrene degradation by Brevibacillus brevis in liquid medium.

Polycyclic aromatic hydrocarbons (PAHs) are widely spread in various ecosystems and are of great concern due to their potential toxicity, mutagenicity and carcinogenicity. Bioremediation has been proposed as an effective approach to remove PAHs. In this study, the physiological responses and proteome of Brevibacillus brevis under exposure to pyrene, a four-ring compound from PAHs family, were investigated. The changes of cell viability of B. brevis were observed during the degradation of pyrene by means of flow cytometry. The results indicated that pyrene stimulated superoxide dismutase (SOD) activity from 93.9 to 100.6 U mg-1 prot, whereas inhibited catalase (CAT) activity from 29.1 to 20.3 U mg-1 prot. The main compositions of B. brevis changed during pyrene degradation, with the proportion of unsaturated fatty acids increased by 13.4%. In addition, we performed a proteomic approach (two-dimensional gel electrophoresis and MALDI-TOF/TOF-MS) in order to explore how B. brevis survived upon treatment with pyrene. It was showed that the expression of 13 proteins increased whereas 10 other decreased after pyrene-treatment. The differentially expressed proteins were identified and the results indicated that they were involved in multiple biological processes including energy metabolism, biosynthesis, transmembrane transport and oxidative stress. Overall, these findings offered a new insights into the cellular response strategy developed by B. brevis to overcome the pyrene stress.

Biodegradation of malodorous thiols by a Brevibacillus sp. strain isolated from a Tunisian phosphate factory.

Hydrogen sulfide (H2S) and thiols (RSH) generated by the phosphate industry cause harmful effects on human health and quality of life. The present study aims to investigate and evaluate a bacterial strain CAT37 isolated from gas-washing wastewaters in terms of its properties and ability to degrade malodorous thiols. Gas-washing wastewater samples were submitted to physicochemical analyses and used for the isolation of thiol-degrading bacteria. The results from gas chromatography-mass spectrometry (GC-MS) analysis revealed that the isolated strain CAT37 was able to oxidize ∼99% of each thiol, decanethiol and dodecanethiol used as sole carbon and energy sources after 30 days of incubation at 37°C. The strain CAT37 displayed a biodegradative potential on several thiols known by their toxicity and odors. The results from phylogenetic and phenotypic analysis revealed that the CAT37 isolate belonged to the genus Brevibacillus, showing the highest sequence similarity to Brevibacillus agri. Overall, the results indicated that the strain CAT37 exhibited a number of attractive biodegradation abilities against thiols and could be considered a promising candidate for industrial application in future thiol biodeodorization strategies.

Biodegradation of crude oil using an efficient microbial consortium in a simulated marine environment.

Ochrobactrum sp. N1, Brevibacillus parabrevis N2, B. parabrevis N3 and B. parabrevis N4 were selected when preparing a mixed bacterial consortium based on the efficiency of crude oil utilization. A crude oil degradation rate of the N-series microbial consortium reached upwards of 79% at a temperature of 25 °C in a 3.0% NaCl solution in the shake flask trial. In the mesocosm experiment, a specially designed device was used to simulate the marine environment. The internal tank size was 1.5 m (L)×0.8 m (W)×0.7 m (H). The microbial growth conditions, nutrient utilization and environmental factors were thoroughly investigated. Over 51.1% of the crude oil was effectively removed from the simulated water body. The escalation process (from flask trials to the mesocosm experiment), which sought to represent removal under conditions more similar to the field, proved the high efficiency of using N-series bacteria in crude oil degradation.

Determinative factors of competitive advantage between aerobic bacteria for niches at the air-liquid interface.

We focused on bacterial interspecies relationships at the air-liquid interface where the formation of pellicles by aerobes was observed. Although an obligate aerobe (Brevibacillus sp. M1-5) was initially dominant in the pellicle population, a facultative aerobe (Pseudoxanthomonas sp. M1-3) emerged and the viability of M1-5 rapidly decreased due to severe competition for oxygen. Supplementation of the medium with carbohydrates allowed the two species to coexist at the air-liquid interface. These results indicate that the population dynamics within pellicles are primarily governed by oxygen utilization which was affected by a combination of carbon sources.

[Analysis of microbial diversity of nitrifying bacteria by terminal restriction fragment length polymorphism].

We analyzed the microbial diversity and quantity of nitrifying bacteria in the enrichment reactor by Terminal Restriction Fragment Length Polymorphism (T_RFLP), a cultured-independent molecular technique. The result indicated that nitrobacteria enriched the best, and the diversity index decreased 62.80% compared with the initial data. Nitrobacteria were predominant in the reactor. Meanwhile, we studied the microbial diversity before and after adding Nitrobacteria into shrimp ponds, and analyzed several major bacterial species that existed stably in the pond. According to the analysis by T_RFLP program, species including Brevibacillus brevis, Microbacterium lactium, Azoarcus indigens and Bordetella holmesii were the dominant bacteria in the ponds.