Differentiated pellicle organization and lipopeptide production in standing culture of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains

Pellicle formation and lipopeptide production was analysed in standing cultures of different Bacillus subtilis strains producing two or three families of lipopeptides. Despite its ability to produce surfactin, B. Subtilis ATCC 6633 was unable to form stable pellicle at air–water interface. For the ATTC 21332 and ATCC 9943 strains, it was shown for the first time that the lipopeptides were also produced in standing cultures at productivities similar or lower than those obtained when the culture medium is agitated. A differentiated behaviour was observed between these strains in repetitive batch cultures. B. subtilis 9943 formed a wrinkled, thinner and more resistant pellicle than B. subtilis 21332. The structure of the pellicle determined by electron microscopy observations showed that cells of B. subtilis 9943 formed microcolonies whereas those of B. subtilis 21332 rapidly died. Under these conditions, surfactin production by strain 21332 decreased after 2 days whereas it remained stable for B. subtilis 9943 during the 6 days of the cultures. These data indicate that cells of B. subtilis strains growing in pellicle can produce lipopeptides differently depending on their cellular organisation. M. Chollet-Imbert and F. Gancel have contributed equally to the scientific work.     

Production of surfactin and fengycin by Bacillus subtilis in a bubbleless membrane bioreactor

Surfactin and fengycin are lipopeptide biosurfactants produced by Bacillus subtilis. This work describes for the first time the use of bubbleless bioreactors for the production of these lipopeptides by B. subtilis ATCC 21332 with aeration by a hollow fiber membrane air–liquid contactor to prevent foam formation. Three different configurations were tested: external aeration module made from either polyethersulfone (reactor BB1) or polypropylene (reactor BB2) and a submerged module in polypropylene (reactor BB3). Bacterial growth, glucose consumption, lipopeptide production, and oxygen uptake rate were monitored during the culture in the bioreactors. For all the tested membranes, the bioreactors were of satisfactory bacterial growth and lipopeptide production. In the three configurations, surfactin production related to the culture volume was in the same range: 242, 230, and 188 mg l⁻¹ for BB1, BB2, and BB3, respectively. Interestingly, high differences were observed for fengycin production: 47 mg l⁻¹ for BB1, 207 mg l⁻¹ for BB2, and 393 mg l⁻¹ for BB3. A significant proportion of surfactin was adsorbed on the membranes and reduced the volumetric oxygen mass transfer coefficient. The degree of adsorption depended on both the material and the structure of the membrane and was higher with the submerged polypropylene membrane.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.     

Optimization of inactivation of endospores of Bacillus cereus by antimicrobial lipopeptides from Bacillus subtilis fmbj strains using a response surface method

Bacillus subtilis fmbj can produce a lipopeptide antimicrobial substance, the main components of which are surfactin and fengycin. In this paper, the sensitivity of Bacillus cereus to antimicrobial lipopeptides from B. subtilis fmbj was observed, and the effect of the microstructure of antimicrobial lipopeptide on spores of B. cereus was investigated. At the same time, the optimization of the inactivation of antimicrobial lipopeptides to spores of B. cereus by a response surface methodology was studied. Results showed that B. cereus had high sensitivity to it, whose minimal inhibitory concentration was 156.25 μg/ml. It could result in the death of spores by destroying the structure of resting spores and sprouting spores, as was observed by transmission electron microscopy. The optimization result indicated that spores of B. cereus could be inactivated by 2 orders of magnitude when the temperature was 29.6°C, the action time was 7.6 h, and the concentration was 3.46 mg·ml⁻¹.     

Biological control of root rot fungus <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> and growth enhancement of <Emphasis Type="Italic">Pinus roxburghii</Emphasis> (Sarg.) by rhizosphere competent <Emphasis Type="Italic">Bacillus subtilis</Emphasis> BN1

Bacterial isolates having antifungal and good plant growth-promoting attributes were isolated from chir-pine (Pinus roxburghii) rhizosphere. An isolate, Bacillus subtilis BN1 exhibited strong antagonistic activity against Macrophomina phaseolina, and other phytopathogens including Fusarium oxysporum and Rhizoctonia solani. It was characterized and selected for the present studies. BN1 resulted in vacuolation, hyphal squeezing, swelling, abnormal branching and lysis of mycelia. The cell-free culture filtrate of BN1 inhibited the growth of M. phaseolina. Pot trial study resulted in statistically significant increase in seedling biomass besides reduction in root rot symptoms in chir-pine seedlings. BN1 treatment resulted in 43.6% and 93.54% increases in root and shoot dry weights respectively, as compared to control. Also, 80–85% seed viability was recorded in treatments receiving BN1 either alone or in the presence of M. phaseolina, compared to 54.5% with M. phaseolina. Bioinoculant formulation study suggested that maximum viability of bacteria was in a sawdust-based carrier. B. subtilis BN1 produced lytic enzymes, chitinase and β-1,3-glucanase, which are known to cause hyphal degradation and digestion of the cell wall component of M. phaseolina. In the presence of M. phaseolina, population of B1 was 1.5 × 10⁴ c.f.u. g⁻¹ root after one month, which increased to 4.5 × 10⁴ c.f.u. g⁻¹ root in three months. Positive root colonization capability of B. subtilis BN1 proved it as a potent biocontrol agent.     

Antiviral Activity of Antimicrobial Lipopeptide from Bacillus subtilis fmbj Against Pseudorabies Virus, Porcine Parvovirus, Newcastle Disease Virus and Infectious Bursal Disease Virus in Vitro

Bacillus subtilis fmbj can produce lipopeptide antimicrobial substance, whose main components were surfactin and fengycin. In the study, the antiviral activity of antimicrobial lipopeptides (AMLs) from B. subtilis fmbj (CGMCC No. 0934) against Pseudorabies Virus (PRV), Porcine Parvovirus (PPV), Newcastle Disease Virus (NDV) and Infectious Bursal Disease Virus (IBDV) was evaluated in vitro. The AMLs represented a direct inactivation effect on cell-free virus stocks of PRV, PPV, NDV and IBDV, and it could effectively inhibit infection and replication of the NDV and IBDV, but failed to affect PRV and PPV. The AMLs were represented higher toxicity for the Porcine Kidney (PK-15) cells (50% cytotoxic concentration (CC₅₀) value was 32.87 μM) and lower for the Chicken Embryo Fibroblasts (CEF) cells (CC₅₀ value was 89.16 μM). The Selectivity index of AMLs on PRV, PPV, NDV and IBDV was 1.44, 2.23, 8.40 and 12.19, respectively.     

Surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity

The synthesis of extracellular molecules such as biosurfactants should have major consequences on bacterial adhesion. These molecules may be adsorbed on surfaces and modify their hydrophobicities. Certain strains of Bacillus subtilis synthesize the lipopeptides, which exhibit antibiotic and surface active properties. In this study the high-performance liquid chromatography (HPLC) analysis of the culture supernatants of the seven B. subtilis strains, showed that the lipopeptide profile varied greatly according to the strain. Among the three lipopeptide types, only iturin A was produced by all B. subtilis strains. Bacterial hydrophobicity, evaluated by the water contact angle measurements and the hydrophobic interaction chromatography, varied according to the strain. Two strains (ATCC 15476 and ATCC 15811) showing extreme behaviors in term of hydrophobicity were selected to study surfactin and iturin A effects on bacterial hydrophobicity. The two lipopeptides modified the B. subtilis surface hydrophobicity. Their effects varied according to the bacterial surface hydrophobic character, the lipopeptide type and the concentration. Lipopeptide adsorption increased the hydrophobicity of the hydrophilic strain but decreased that of the hydrophobic. Comparison of lipopeptide effects on B. subtilis surface hydrophobicity showed that surfactin was more effective than iturin A for the two strains tested.     

Mineralization of diuron [3-(3,4-dichlorophenyl)-1, 1-dimethylurea] by co-immobilized <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. and <Emphasis Type="Italic">Delftia acidovorans</Emphasis>

Mineralization of diuron has not been previously demonstrated despite the availability of some bacteria to degrade diuron into 3,4-dichloroaniline (3,4-DCA) and others that can mineralize 3,4-DCA. A bacterial co-culture of Arthrobacter sp. N4 and Delftia acidovorans W34, which respectively degraded diuron (20 mg l⁻¹) to 3,4-DCA and mineralized 3,4-DCA, were able to mineralize diuron. Total diuron mineralization (20 mg l⁻¹) was achieved with free cells in co-culture. When the bacteria were immobilized (either one bacteria or both), the degradation rate was higher. Best results were obtained with free Arthrobacter sp. N4 cells co-cultivated with immobilized cells of D. acidovorans W34 (mineralization of diuron in 96 h, i.e., 0.21 mg l⁻¹ h⁻¹ vs. 0.06 mg l⁻¹ h⁻¹ with free cells in co-culture).     

Identification of lipopeptide antibiotics of a <Emphasis Type="Italic">Bacillus subtilis</Emphasis> isolate and their control of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> diseases in maize and wheat

Substances produced by Bacillus subtilis D1/2, a bacterium isolated from cultivated soil, were found to inhibit Fusarium graminearum. The antifungal activity of the bacterium was attributable to major extracellular lipopeptides isolated and identified as fengycins. Their synthesis was enhanced by casamino acids added to the culture medium. The unpurified cell-free spent medium elicited hemolysis with increasing concentration. Its application to field-cultivated maize and chamber-grown wheat suppressed gibberella ear rot and Fusarium head blight, respectively, when the plants were inoculated with F. graminearum macroconidia. The treatment of maize ears consistently arrested ear-rot development, while the treatment of wheat spikes retarded the progress of Fusarium head blight. Although the deoxynivalenol and ergosterol contents of treated maize kernels were halved, they remained high because of the experimental requirement to inoculate with a high number (1.5 × 10⁴) of macroconidia. As a potential antifungal agent for controlling Fusarium diseases, B. subtilis D1/2 can be further developed as a useful component of integrated pest management. Handling Editor: Reijo Karjalainen.     

Root-promoting rhizobacteria in <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> cuttings

Cloned Eucalyptus spp. plantations are based in greenhouse production of plants generated by vegetative propagation. Diverse studies have demonstrated that rhizospheric bacteria can stimulate plant growth, and more recently that they can increase rooting in vegetative material. Considering this potential, the objective of this study was to verify the effect of bacterial strains on rooting Eucalyptus globulus. A total of 132 bacterial strains isolated from the rhizosphere of E. globulus and Eucalyptus nitens were studied. The bacterial inoculums in a concentration of 4 × 10⁸ cfu/ml were applied to the rooting substrate at the cutting installation and 45 days after by irrigation. Rooting was evaluated on days 60 and 75 after cutting installation, considering the number of roots as well as their fibrosity and roots biomass. Of the 132 strains evaluated, 26 significantly increased cutting rooting in a range of 191.4–69.4% with respect to the control. Additionally, some strains stimulated the development of fine roots and incremented the roots biomass. The strains identificated that produced a rooting effect were: Bacillus firmus, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis, B. subtilis/amyloliquefaciens, Bacillus circulans, Brevibacillus brevis, Paenibacillus lautus and Stenotrophomona maltophilia. These first trials suggest the potential of these bacteria to be used in clonal production programs for E. globulus.     

A modified electro-transformation method for Bacillus subtilis and its application in the production of antimicrobial lipopeptides

A modified electroporation method using trehalose is presented for the transformation of Bacillus subtilis. The new method improved the transformation efficiency of B. subtilis nearly 2,000-fold compared with the usual method, giving 4 × 10⁵ transformants/μg DNA. Using this method, B. subtilis was engineered to improve production of antimicrobial lipopeptides and produced 1.8-fold more surfactin and 2.9-fold more fengycin.     

Marine Bacterium Derived Lipopeptides: Characterization and Cytotoxic Activity Against Cancer Cell Lines

A marine Bacillus circulans DMS-2 was able to grow and produce biosurfactant on glucose mineral salts medium (GMSM) with a reduction in the surface tension up to 27 mN m⁻¹. The microorganism produced 1.64 ± 0.1 g l⁻¹ of crude biosurfactant. The lipopeptide nature of the produced biosurfactant was confirmed by primulin and ninhydrin assays using High Performance Thin Layer Chromatography (HPTLC). Preparative thin layer chromatography (TLC) was performed to purify the lipopeptides from the crude biosurfactant. The critical micelle concentrations (CMC) of the crude and purified products were found to be 90 and 40 mg l⁻¹ respectively. Fourier transform infrared spectrophotometer (FTIR) and matrix assisted laser desorption/ionization time of flight (MALDI-ToF) mass spectral analysis revealed the identity of the produced lipopeptides as surfactin (m/z 1,023 Da) and fengycin (m/z 1,495 Da) isoforms. The purified marine lipopeptides displayed a significant antiproliferative activity against the human colon cancer cell lines HCT-15 (IC₅₀ 80 μg ml⁻¹) and HT-29 (IC₅₀ 120 μg ml⁻¹).     

Cyclic lipopeptide profile of three <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains; antagonists of <Emphasis Type="Italic">Fusarium</Emphasis> head blight

The objective of the study was to identify the lipopetides associated with three Bacillus subtilis strains. The strains are antagonists of Gibberella zeae, and have been shown to be effective in reducing Fusarium head blight in wheat. The lipopeptide profile of three B. subtilis strains (AS43.3, AS43.4, and OH131.1) was determined using mass spectroscopy. Strains AS43.3 and AS43.4 produced the anti-fungal lipopeptides from the iturin and fengycin family during the stationary growth phase. All three strains produced the lipopeptide surfactin at different growth times. Strain OH131.1 only produced surfactin under these conditions. The antifungal activity of the culture supernatant and individual lipopeptides was determined by the inhibition of G. zeae. Cell-free supernatant from strains AS43.3 and AS43.4 demonstrated strong antibiosis of G. zeae, while strain OH131.1 had no antibiosis activity. These results suggest a different mechanism of antagonism for strain OH131.1, relative to AS43.3 and AS43.4.     

Comparative genomic study of <Emphasis Type="Italic">spo0E</Emphasis> family genes and elucidation of the role of Spo0E in <Emphasis Type="Italic">Bacillus anthracis</Emphasis>

The propensity of bacterium to sporulate or retain the vegetative form depends on the amount of phosphorylated Spo0A (Spo0A^-P), regulated by Spo0E multigene family of phosphatases (Spo0E, YisI and YnzD). Phylogenetic analysis revealed that Spo0E multigene family of phosphatases (SMFP) descends in two distinct clades of aerobic (Bacillus cluster) and anaerobic (Clostridia cluster) sporulating bacteria. High sequence conservation within species gives a notion that these members could have evolved through lineage and species-specific duplication event. Of the five genes in Bacillus cereus group, three are pathogen specific, and their synteny suggests that these paralogs could be involved in the regulation of amino acid metabolism and its transport. Overexpression of B. subtilis Spo0E, an ortholog of SMFP members in B. anthracis (BAS1251), resulted in sporulation deficient phenotype in B. anthracis. B. anthracis Spo0A^-P binds to a consensus DNA sequence 5′-TGNCGAA-3′ (‘0A-like box’) and loses its DNA binding ability following treatment with B. subtilis Spo0E. Thus, B. subtilis Spo0E acts on B. anthracis Spo0A^-P and, therefore could complement the function of BAS1251. Further, since ‘0A-like box’ are present in the promoter region of abrB gene, a known regulator of anthrax toxin gene expression, cross talk among SMFP members and Spo0A^-P–AbrB could regulate the expression of anthrax toxin genes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Relevance of bacterioplankton abundance and production in the oligotrophic equatorial Indian Ocean

Bacterioplankton abundance and production, chlorophyll a (Chl a) concentrations and primary production (PP) were measured from the equatorial Indian Ocean (EIO) during northeast (NEM), southwest (SWM) and spring intermonsoon (SpIM) seasons from 1°N to 5°S along 83°E. The average bacterial abundance was 0.52 ± 0.29, 0.62 ± 0.33 and 0.46 ± 0.19 (× 10⁸ cells l⁻¹), respectively during NEM, SWM and SpIM in the top 100 m. In the deep waters (200 m and below), the bacterial counts averaged ∼0.35 ± 0.14 × 10⁸ cells l⁻¹ in SWM and 0.39 ± 0.16 × 10⁸ cells l⁻¹ in SpIM. The 0–120 m column integrated bacterial production (BP) ranged from 19 to 115 and from 10 to 51 mg C m⁻² d⁻¹ during NEM and SWM, respectively. Compared with many open ocean locations, bacterial abundance and production in this region are lower. The bacterial carbon production, however, is notably higher than that of phytoplankton PP (BP:PP ratio 102% in SWM and 188% in NEM). With perpetually low PP (NEM: 20, SWM: 18 and SpIM: 12 mg C m⁻² d⁻¹) and Chl a concentration (NEM: 16.5, SWM: 15.0 and SpIM: 20.9 mg m⁻²), the observed bacterial abundance and production are pivotal in the trophodynamics of the EIO. Efficient assimilation and mineralization of available organics by bacteria in the euphotic zone might serve a dual role in the ultra-oligotrophic regions including EIO. Thus, bacteria probably sustain microheterotrophs (micro- and meso-zooplankton) through microbial loop. Further, rapid mineralization by bacteria will make essential nutrients available to autotrophs.     

Isolation of antifouling compounds from the marine bacterium, <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> SCH0402

Two compounds, 2-hydroxymyristic acid (HMA) and cis-9-oleic acid (COA), were isolated from a chloroform extract of the marine bacterium, Shewanella oneidensis SCH0402. In a spectrophotometer-based chemotaxis assay, HMA completely eliminated the optical density (OD) of Alteromonas marina SCH0401 and Bacillus atrophaeus SCH0408, motile, fouling bacteria, at 100 and 1000 μg ml⁻¹, respectively. COA similarly decreased the OD of A. marina and B. atrophaeus by 100% at 1000 μg ml⁻¹. The commercially available, highly toxic anti-fouling compound, tributyltin oxide (TBTO) never reduced the OD of the target bacteria by 100% even at higher concentration. Instead, all the test bacterial cells were killed at higher than 1000 μg ml⁻¹ of concentration. Both HMA and COA inhibited germination of Ulva pertusa spores completely at 10 and 100 μg ml⁻¹, respectively, while TBTO inhibited germination at 0.01 μg ml⁻¹. However, in field assays, both HMA and COA showed anti-fouling activities as potent as TBTO against a wide range of fouling organisms, including micro- and macro-algae, barnacles, and mussels. The average fouling coverage on the surface of the control panel was 93 ± 6% after 1.5 years but no fouling was observed on the surface of the test panel onto which each compound was applied separately. Thus, bacterial repellent compounds can be used as substitutes for potent toxic anti-fouling compounds, resulting in higher standards of environmental safety without loss of anti-fouling performance.     

Synergistic effect of green tea extract and probiotics on the pathogenic bacteria, <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis>

The aim of this study was to assess the effect of a commercial green tea extract (TEAVIGO™) on the microbial growth of three probiotic strains (Lactobacillus and Bifidobacterium), as well as three pathogenic bacteria. MIC and co-culture studies were performed. The MICs of the green tea extract against Staphylococcus aureus and Streptococcus pyogenes (100 μg ml⁻¹) were considerably lower than those against the probiotic strains tested (>800 μg ml⁻¹) and Escherichia coli (800 μg ml⁻¹). In co-culture studies, a synergistic effect of the probiotic strains and the green tea extract was observed against both Staph. aureus and Strep. pyogenes. Green tea extract in combination with probiotics significantly reduced the viable count of both pathogens at 4 h and by 24 h had completely abolished the recovery of viable Staph. aureus and Strep. pyogenes. These reductions were more significant than the reductions induced by probiotics or green tea extracts used separately. These results demonstrate the potential for combined therapy using the green tea extract plus probiotics on microbial infections caused by Staph. aureus and Strep. pyogenes. As probiotics and the green tea extract are derived from natural products, treatment with these agents may represent important adjuncts to, or alternatives to, conventional antibiotic therapy.     

Factors controlling the colonial structure of <Emphasis Type="Italic">Pediastrum tetras</Emphasis> (Chlorophyceae)

Accounting for morphological plasticity in phytoplankton populations is relevant for taxonomy, systematic/evolutionary, and ecological studies. In this work, the green alga Pediastrum tetras (Ehrenberg) Ralfs was used to describe the variation in population size structure over its growth cycle and to analyze responses to changes in biotic and abiotic factors. Pediastrum cultures reached a final stable concentration in approximately 10 days. This density (8 × 10⁵ cells ml⁻¹) remained stable for at least another 13 days and the intrinsic growth rate was 0.24 ± 0.01 day⁻¹. In the exponential phase, the relative number of single cells and the proportion of large cells (with vesicles inside) within colonies increased. When density peaked, a relative increase of single cells as well as small cells in new colonies took place. Finally, during the stationary phase, the trend reversed: fewer single cells and a larger cell size (without vesicles) were observed. Results indicated that nutrient supply could affect population structure, diminishing the proportion of eight-cell colonies. Daphnia magna Straus significantly reduced the Pediastrum population density due to predation, and this led to a significant decrease in the density of the largest colonies. In addition, info-chemicals induced a slight increase in the density of the largest colonies compared to the control treatment. Our study suggests a possible trade-off in P. tetras colonial size in natural environments: during the stationary growth period in a lake, Pediastrum populations tend to increase in size for efficient use of nutrients, while they decrease in size in the presence of herbivores. Handling editor: J. Padisak