Bacillus subtilis SPB1 lipopeptide biosurfactant: antibacterial efficiency against the phytopathogenic bacteria Agrobacterium tumefaciens and compared production in submerged and solid state fermentation systems.

Bacillus subtilis SPB1 derived biosurfactants (BioS) proved its bio-control activity against Agrobacterium tumefaciens using tomato plant. Almost 83% of disease symptoms triggered by Agrobacterium tumefaciens were reduced. Aiming potential application, we studied lipopeptide cost-effective production in both fermentations systems, namely the submerged fermentation (SmF) and the solid-state fermentation (SSF) as well as the use of Aleppo pine waste and confectionery effluent as cheap substrates. Optimization studies using Box-Behnken (BB) design followed by the analysis with response surface methodology were applied. When using an effluent/sea water ratio of 1, Aleppo pine waste of 14.08 g/L and an inoculum size of 0.2, a best production yield of 17.16 ± 0.91 mg/g was obtained for the SmF. While for the SSF, the best production yield of 27.59 ± 1.63 mg/g was achieved when the value of Aleppo pine waste, moisture, and inoculum size were, respectively, equal to 25 g, 75%, and 0.2. Hence, this work demonstrated the superiority of SSF over SmF.

Optimization of textile effluent bacterial treatment and improvement of the process efficiency through SPB1 biosurfactant addition.

The current work aims to optimize biological textile effluent treatment through the use of newly selected bacterial consortia composed of two strains: Citrobacter sedlakii RI11 and Aeromonas veronii GRI. We assessed the effect of SPB1 biosurfactant addition on color removal (CR). The process was optimized by a Box-Bhenken by examining the effect of pH, consortia density and biosurfactant value on treatment efficiency. Firstly, physicochemical analyses of the studied effluent revealed an alkaline pH along with a high content of suspended materials and large amounts of organic matter. Optimal CR and a chemical oxygen demand abatement of about 94 and 86% were obtained when treating the textile effluent at pH 5 with a total optical density of 0.4 and by incorporating 0.01% SPB1 biosurfactant. Additionally, an abolishment of phytotoxicity was registered after treatment optimization. The evaluations of the action mode of both selected bacteria during textile effluent treatment suggested the occurrence of biodegradation phenomena of dyes through enzymatic activities.

Enhanced biosurfactant production by Bacillus subtilis SPB1 using developed fed-batch fermentation: effects of glucose levels and feeding systems.

Biosurfactants stand for highly useful and promising compounds. They basically serve for a variety of applications in multiple industries and aspects of human life. Therefore, it is highly required to improve their production yield especially through the development of new and more efficient fermentation processes. In this aim, batch and fed-batch were studied and compared in terms of their effective biosurfactant production by Bacillus subtilis SPB1. Experiments of fed-batch fermentations were carried out through three different glucose feeding strategies, namely the pulsed, the constant Donespeed and the exponential feeding. The comparison between different fermentation processes revealed that fed-batch process proved to be a more efficient cultivation strategy than the batch process in terms of cell biomass, biosurfactant production and productivity. Among the three different feeding strategies, the exponential feeding process achieved the highest fermentation results of final biosurfactant concentration. The latter increased more than twofolds compared to batch fermentation.

Surface properties and heavy metals chelation of lipopeptides biosurfactants produced from date flour by Bacillus subtilis ZNI5: optimized production for application in bioremediation.

The present study summarizes the valorization of date flour by the production of lipopeptide biosurfactant (BioS) by Bacillus subtilis ZNI5 (MW091416). A Taguchi design permitted the formulation of a medium composed only of 6% date flour and 0.5% yeast extract within 2 days of incubation at 150 rpm with a maximal surface tension (ST) reduction of about 27.8 mN/m. The characterization of the lipopeptide shows a CMC value of about 400 mg/L with a minimal ST of 30 mN/m and an ability to disperse oil to about 80 mm at 800 mg/L. Having reduced phytotoxicity, the ZNI5 BioS and ZNI5 strain were assayed for Copper and Cobalt chelation and biosorption. The improvement of the germination index of radish seeds irrigated by the treated contaminated water showed the great potential application of ZNI5 lipopeptide in the bioremediation of heavy metals.

Valorization of date juice by the production of lipopeptide biosurfactants by a Bacillus mojavensis BI2 strain: bioprocess optimization by response surface methodology and study of surface activities.

Lipopeptides biosurfactants (BioS) are natural surface-active compounds produced by a variety of microorganisms. They have great interest in environmental, biomedical and agro-industrial fields. However their large-scale application and production is limited by the cost of culture media and the low yield of production. Therefore, the improvement of the production yields and the development of efficient and cost-effective bioprocess became of a great interest. In this aim, we applied the response surface method to optimize an economic BioS production by a newly isolated strain Bacillus mojavensis BI2 on date Juice called "Luegmi" as unique carbon and nitrogen source. Using a Box-Bhenken design, we studied the effect of three independent variables on lipopeptide production; Leugmi concentration, Na2HPO4 and incubation time. The results of this study showed that Leugmi concentration at 25%, Na2HPO4 at 0.1% and incubation time of 24 h were optimal conditions for BioS  production, with a maximum Surface Tension (ST) decreasing capacity of 55% corresponding to 27 mN/m and an Oil Dispersing Activity (ODA) of 30 cm2 corresponding to a diameter of 6 cm. Preliminary characterization of the BioS produced on Luegmi by UV-Spectra and Thin Layer Chromatography showed its lipopeptide nature. Physic-chemical characterization of the produced lipopeptide on Leugmi showed its great surface activities and stabilities at different pH, temperature and salts concentration. The results of this study suggested that Leugmi, an agricultural byproducts can be used as a low-cost substrate to enhance the yield of lipopeptide BioS with great surface activities for potential environmental application.

Enhancement of Aeribacillus pallidus strain VP3 lipase catalytic activity through optimization of medium composition using Box-Behnken design and its application in detergent formulations.

Lipases are hydrolytic enzymes owing much importance in industrial applications. These enzyme-based detergents are ecofriendly and produce a wastewater with low level of COD (chemical oxygen demand). In the present work, a novel halophilous, thermoalkaline, and detergent-tolerant lipase produced by a newly isolated Aeribacillus pallidus strain VP3 was studied. Considerable interest has been given to this lipase by the improvement of its catalytic activity through the optimization of the pH, the (C/N) ratio, and the inoculum size, using the response surface methodology based on the Box-Behnken design of experiments. A total of 16 experiments were conducted, and the optimized pH, (C/N) ratio, and inoculum size were 10, 1, and 0.3, respectively. The results of the analysis of variance (ANOVA) test indicated that the established model was significant (p value < 0.05). The optimization of the production conditions leads to 2.83-fold of increase in the catalytic activity calculated as the ratio of the activity obtained after optimization (68 U) and the initial activity before optimization (24 U). All in all, the lipase of Aeribacillus pallidus could be considered as a potential candidate to be incorporated in detergent formulations since it shows a good stability towards detergents and wash performance.

Glycolipid biosurfactants: main properties and potential applications in agriculture and food industry.

Glycolipids, consisting of a carbohydrate moiety linked to fatty acids, are microbial surface active compounds produced by various microorganisms. They are characterized by high structural diversity and have the ability to decrease the surface and interfacial tension at the surface and interface, respectively. Rhamnolipids, trehalolipids, mannosylerythritol lipids and cellobiose lipids are among the most popular glycolipids. They have received much practical attention as biopesticides for controlling plant diseases and protecting stored products. As a result of their antifungal activity towards phytopathogenic fungi and larvicidal and mosquitocidal potencies, glycolipid biosurfactants permit the preservation of plants and plant crops from pest invasion. Also, as a result of their emulsifying and antibacterial activities, glycolipids have great potential as food additives and food preservatives. Furthermore, the valorization of food byproducts via the production of glycolipid biosurfactant has received much attention because it permits the bioconversion of byproducts on valuable compounds and decreases the cost of production. Generally, the use of glycolipids in many fields requires their retention from fermentation media. Accordingly, different strategies have been developed to extract and purify glycolipids. © 2016 Society of Chemical Industry.

Improvement of methyl orange dye biotreatment by a novel isolated strain, Aeromonas veronii GRI, by SPB1 biosurfactant addition.

Aeromonas veronii GRI (KF964486), isolated from acclimated textile effluent after selective enrichment on azo dye, was assessed for methyl orange biodegradation potency. Results suggested the potential of this bacterium for use in effective treatment of azo-dye-contaminated wastewaters under static conditions at neutral and alkaline pH value, characteristic of typical textile effluents. The strain could tolerate higher doses of dyes as it was able to decolorize up to 1000 mg/l. When used as microbial surfactant to enhance methyl orange biodecolorization, Bacillus subtilis SPB1-derived lipopeptide accelerated the decolorization rate and maximized slightly the decolorization efficiency at an optimal concentration of about 0.025%. In order to enhance the process efficiency, a Taguchi design was conducted. Phytotoxicity bioassay using sesame and radish seeds were carried out to assess the biotreatment effectiveness. The bacterium was able to effectively decolorize the azo dye when inoculated with an initial optical density of about 0.5 with 0.25% sucrose, 0.125% yeast extract, 0.01% SPB1 biosurfactant, and when conducting an agitation phase of about 24 h after static incubation. Germination potency showed an increase toward the nonoptimized conditions indicating an improvement of the biotreatment. When comparing with synthetic surfactants, a drastic decrease and an inhibition of orange methyl decolorization were observed in the presence of CTAB and SDS. The nonionic surfactant Tween 80 had a positive effect on methyl orange biodecolorization. Also, studies ensured that methyl orange removal by this strain could be due to endocellular enzymatic activities. To conclude, the addition of SPB1 bioemulsifier reduced energy costs by reducing effective decolorization period, biosurfactant stimulated bacterial decolorization method may provide highly efficient, inexpensive, and time-saving procedure in treatment of textile effluents.

Purification and identification of Bacillus subtilis SPB1 lipopeptide biosurfactant exhibiting antifungal activity against Rhizoctonia bataticola and Rhizoctonia solani.

This study reports the potential of a soil bacterium, Bacillus subtilis strain SPB1, to produce lipopeptide biosurfactants. Firstly, the crude lipopeptide mixture was tested for its inhibitory activity against phytopathogenic fungi. A minimal inhibitory concentration (MIC), an inhibitory concentration at 50% (IC50%), and an inhibitory concentration at 90% (IC90%) values were determined to be 0.04, 0.012, and 0.02 mg/ml, respectively, for Rhizoctonia bataticola with a fungistatic mode of action. For Rhizoctonia solani, a MIC, an IC50%, and IC90% values were determined to be 4, 0.25, and 3.3 mg/ml, respectively, with a fungicidal mode of action. For both of the fungi, a loss of sclerotial integrity, granulation and fragmentation of hyphal mycelia, followed by hyphal shriveling and cell lysis were observed with the treatment with SPB1 biosurfactant fraction. After extraction, separation, and purification, different lipopeptide compounds were identified in the culture filtrate of strain SPB1. Mass spectroscopic analysis confirmed the presence of different lipopeptide compounds consisting of surfactin isoforms with molecular weights of 1007, 1021, and 1035 Da; iturin isoforms with molecular weights of 1028, 1042, and 1056 Da; and fengycin isoforms with molecular weights of 1432 and 1446 Da. Two new clusters of lipopeptide isoforms with molecular weights of 1410 and 1424 Da and 973 and 987 Da, respectively, were also detected. This study reported the ability of a B. subtilis strain to co-produce lipopeptide isoforms with potential use as antifungal compounds.

Malachite green bioremoval by a newly isolated strain Citrobacter sedlakii RI11; enhancement of the treatment by biosurfactant addition.

Citrobacter sedlackii RI11, isolated from acclimated textile effluent after selective enrichment on synthetic dyes, was assessed for malachite green (MG) biotreatment potency. Results indicate that this bacterium has potential for use in effective treatment of MG contaminated wastewaters under shaking conditions at neutral and alkaline pH value, characteristic of typical textile effluents. Also, the newly isolated strain can tolerate higher doses of dye and decolorize up to 1,000 mg/l of dye. When used as microbial surfactant to enhance MG biodecolorization, Bacillus subtilis SPB1-derived lipopeptide accelerated the decolorization rate and maximized the decolorization efficiency at an optimal concentration of biosurfactant of about 0.075%. Studies ensured that MG removal by this strain could be due to biodegradation and/or adsorption. Results on germination potencies of different seeds using the treated dyes under different conditions favor the use of SPB1 biosurfactant for the treatment of MG.

Biosorption of Congo Red from aqueous solution by Bacillus weihenstephanensis RI12; effect of SPB1 biosurfactant addition on biodecolorization potency.

Bacillus weihenstephanensis RI12, isolated from hydrocarbon contaminated soil, was assessed for Congo Red bio-treatment potency. Results suggested the potential of this bacterium for use in effective treatment of Congo Red contaminated wastewaters under shaking conditions at acidic and neutral pH value. The strain could tolerate higher doses of dyes as it could decolorize up to 1,000 mg/l of Congo Red. When used as microbial surfactant to enhance Congo Red biodecolorization, Bacillus subtilis SPB1-derived lipopeptide accelerated the decolorization rate and maximized the decolorization efficiency at an optimal concentration of biosurfactant of about 0.075%. Studies ensured that Congo Red removal by this strain could be due to an adsorption phenomena. Germination potencies of tomato seeds using the treated dyes under different conditions showed the efficient biotreatment of the azo dye Congo Red especially with the addition of SPB1 biosurfactant. To conclude, the addition of SPB1 bioemulsifier reduced energy costs by reducing the effective decolorization period; the biosurfactant stimulated bacterial decolorization method may provide a highly efficient, inexpensive and time-saving procedure in the treatment of textile effluents.

Antifungal efficiency of a lipopeptide biosurfactant derived from Bacillus subtilis SPB1 versus the phytopathogenic fungus, Fusarium solani.

Bacillus subtilis SPB1 lipopeptides were evaluated as a natural antifungal agent against Fusarium solani infestation. In vitro antifungal assay showed a minimal inhibitory concentration of about 3 mg/ml with a fungicidal mode of action. In fact, treatment of F. solani by SPB1 lipopeptides generated excessive lyses of the mycelium and caused polynucleation and destruction of the related spores together with a total inhibition of spore production. Furthermore, an inhibition of germination potency accompanied with a high spore blowing was observed. Moreover, in order to be applied in agricultural field, in vivo antifungal activity was proved against the dry rot potato tubers caused by F. solani. Preventive treatment appeared as the most promising as after 20 days of fungi inoculation, rot invasion was reduced by almost 78%, in comparison to that of non-treated one. When treating infected tomato plants, disease symptoms were reduced by almost 100% when applying the curative method. Results of this study are very promising as it enables the use of the crude lipopeptide preparation of B. subtilis SPB1 as a potent natural fungicide that could effectively control the infection of F. solani in tomato and potato tubers at a concentration similar to the commercial fungicide hymexazol and therefore prevent the damage of olive tree.

Microbial derived surface active compounds: properties and screening concept.

Biosurfactants are surface-active biomolecules that are produced by a variety of microorganisms. They have gained biotechnologist interest for high diversity and their efficient action in comparison to synthetic emulsifiers. So, we discussed a wide array of screening method based on direct and indirect surface and interfacial tension measurements. Also, this review describes biosurfactant physicochemical properties and natural role in the environment. Also, it presents their tolerance to extreme conditions of temperature, pH and ionic strength, low toxicity and biodegradability. Functional properties like emulsification, foaming, solubilizing and membrane permeabilizing activities were also discussed along with their related application.

Biodegradation of diesel oil by a novel microbial consortium: comparison between co-inoculation with biosurfactant-producing strain and exogenously added biosurfactants.

Bioremediation, involving the use of microorganisms to detoxify or remove pollutants, is the most interesting strategy for hydrocarbon remediation. In this aim, four hydrocarbon-degrading bacteria were isolated from oil-contaminated soil in Tunisia. They were identified by the 16S rDNA sequence analysis, as Lysinibacillus bronitolerans RI18 (KF964487), Bacillus thuringiensis RI16 (KM111604), Bacillus weihenstephanensis RI12 (KM094930), and Acinetobacter radioresistens RI7 (KJ829530). Moreover, a lipopeptide biosurfactant produced by Bacillus subtilis SPB1, confirmed to increase diesel solubility, was tested to increase diesel biodegradation along with co-inoculation with two biosurfactant-producing strains. Culture studies revealed the enhancement of diesel biodegradation by the selected consortium with the addition of SPB1 lipopeptide and in the cases of co-inoculation by biosurfactant-producing strain. In fact, an improvement of about 38.42 and 49.65 % of diesel degradation was registered in the presence of 0.1 % lipopeptide biosurfactant and when culturing B. subtilis SPB1 strain with the isolated consortium, respectively. Furthermore, the best improvement, evaluated to about 55.4 %, was recorded when using the consortium cultured with B. subtilis SPB1 and A. radioresistens RI7 strains. Gas chromatography analyses were correlated with the gravimetric evaluation of the residual hydrocarbons. Results suggested the potential applicability of the selected consortium along with the ex situ- and in situ-added biosurfactant for the effective bioremediation of diesel-contaminated water and soil.

Review lipopeptides biosurfactants: Mean classes and new insights for industrial, biomedical, and environmental applications.

Lipopeptides are microbial surface active compounds produced by a wide variety of bacteria, fungi, and yeast. They are characterized by high structural diversity and have the ability to decrease the surface and interfacial tension at the surface and interface, respectively. Surfactin, iturin, and fengycin of Bacillus subtilis are among the most popular lipopeptides. Lipopepetides can be applied in diverse domains as food and cosmetic industries for their emulsification/de-emulsification capacity, dispersing, foaming, moisturizing, and dispersing properties. Also, they are qualified as viscosity reducers, hydrocarbon solubilizing and mobilizing agents, and metal sequestering candidates for application in environment and bioremediation. Moreover, their ability to form pores and destabilize biological membrane permits their use as antimicrobial, hemolytic, antiviral, antitumor, and insecticide agents. Furthermore, lipopeptides can act at the surface and can modulate enzymes activity permitting the enhancement of the activity of certain enzymes ameliorating microbial process or the inhibition of certain other enzymes permitting their use as antifungal agents. This article will present a detailed classification of lipopeptides biosurfactant along with their producing strain and biological activities and will discuss their functional properties and related applications.

High molecular weight bioemulsifiers, main properties and potential environmental and biomedical applications.

High molecular weight bioemulsifiers are amphipathic polysaccharides, proteins, lipopolysaccharides, lipoproteins, or complex mixtures of these biopolymers, produced by a wide variety of microorganisms. They are characterized by highly structural diversity and have the ability to decrease the surface and interfacial tension at the surface and interface respectively and/or emulsify hydrophobic compounds. Emulsan, fatty acids, phospholipids, neutral lipids, exopolysaccharides, vesicles and fimbriae are among the most popular high molecular weight bioemulsifiers. They have great physic-chemical properties like tolerance to extreme conditions of pH, temperature and salinity, low toxicity and biodegradability. Owing their emulsion forming and breaking capacities, solubilization, mobilization and dispersion activities and their viscosity reduction activity; they possess great environmental application as enhancer of hydrocarbon biodegradation and for microbial enhanced oil recovery. Besides, they are applied in biomedical fields for their antimicrobial and anti-adhesive activities and involvement in immune responses.

Direct injection method for HPLC/MS/MS analysis of acrylamide in aqueous solutions: application to adsorption experiments.

Polyacrylamides are polymers used in many fields and represent the main source of release of the highly toxic acrylamide in the environment. In this work, a simple, rapid, and sensitive analytical method was developed with HPLC/MS/MS and direct injection for acrylamide analysis in water and adsorption samples. AFNOR standards NF T90-210 and NF T90-220 were used for the analytical method validation and uncertainty estimation. Limit of quantification (LOQ) for acrylamide was 1 µg/L, and accuracy was checked at three acrylamide levels (1, 6, and 10 µg/L). Uncertainties were estimated at 34.2, 22, and 12.4 % for acrylamide concentrations at LOQ, 6 µg/L, and 10 µg/L, respectively. Acrylamide adsorption on clays (kaolinite, illite) and sludge was then studied as a function of pH, time, and acrylamide concentrations. Acrylamide adsorption on kaolinite, illite, and sludge was found to be very weak since adsorption percentages were inferior to 10 %, whatever the pH value and the initial acrylamide concentration. The low affinity of acrylamide for clays and sludge is likely due to its hydrophilic property, small size, and charge neutrality.

Treatment of diesel- and kerosene-contaminated water by B. subtilis SPB1 biosurfactant-producing strain.

This study investigated the efficiency of hydrocarbon utilization by B. subtilis SPB1, a biosurfactant-producing strain. Microbial growth, biosurfactant production, and hydrocarbon biodegradation were studied in a liquid mineral medium, supplemented with 2% hydrocarbons in both the absence and in the presence of 0.1% yeast extract. Preliminary studies showed that maximum growth was registered with a 2% hydrocarbon solution. Results showed that the addition of yeast extract greatly stimulated microbial growth and thus induced biosurfactant production. Furthermore, biodegradation efficiencies were higher in the presence of yeast extract. Kerosene fuel was more recalcitrant to biodegradation than diesel oil. This study's findings suggest that the addition of an organic nitrogen source stimulates tension-active agents' production, which emulsifies hydrophobic compounds and enhances their biodegradation and microbial growth.

Evaluation of B. subtilis SPB1 biosurfactants' potency for diesel-contaminated soil washing: optimization of oil desorption using Taguchi design.

Low solubility of certain hydrophobic soil contaminants limits remediation process. Surface-active compounds can improve the solubility and removal of hydrophobic compounds from contaminated soils and, consequently, their biodegradation. Hence, this paper aims to study desorption efficiency of oil from soil of SPB1 lipopeptide biosurfactant. The effect of different physicochemical parameters on desorption potency was assessed. Taguchi experimental design method was applied in order to enhance the desorption capacity and establish the best washing parameters. Mobilization potency was compared to those of chemical surfactants under the newly defined conditions. Better desorption capacity was obtained using 0.1% biosurfacatnt solution and the mobilization potency shows great tolerance to acidic and alkaline pH values and salinity. Results show an optimum value of oil removal from diesel-contaminated soil of about 87%. The optimum washing conditions for surfactant solution volume, biosurfactant concentration, agitation speed, temperature, and time were found to be 12 ml/g of soil, 0.1% biosurfactant, 200 rpm, 30 °C, and 24 h, respectively. The obtained results were compared to those of SDS and Tween 80 at the optimal conditions described above, and the study reveals an effectiveness of SPB1 biosurfactant comparable to the reported chemical emulsifiers. (1) The obtained findings suggest (a) the competence of Bacillus subtilis biosurfactant in promoting diesel desorption from soil towards chemical surfactants and (b) the applicability of this method in decontaminating crude oil-contaminated soil and, therefore, improving bioavailability of hydrophobic compounds. (2) The obtained findings also suggest the adequacy of Taguchi design in promoting process efficiency. Our findings suggest that preoptimized desorption process using microbial-derived emulsifier can contribute significantly to enhancement of hydrophobic pollutants' bioavailability. This study can be complemented with the investigation of potential role in improving the biodegradation of the diesel adsorbed to the soil.

Improvement of bread dough quality by Bacillus subtilis SPB1 biosurfactant addition: optimized extraction using response surface methodology.

BACKGROUND: Statistically based experimental designs were applied to Bacillus subtilis SPB1 biosurfactant extraction. The extracted biosurfactant was tested as an additive in dough formulation. RESULTS: The Plackett-Burman screening method showed that methanol volume, agitation speed and operating temperature affect biosurfactant extraction. The effect was studied and adjusted using response surface methodology. The optimal values were identified as 5 mL methanol, 180 rpm and 25 °C, yielding predicted responses of 2.1 ± 0.06 for the purification factor and 87.47% ± 1.58 for the retention yield. Study of the incorporation of purified lipopeptide powder into the dough preparation in comparison with a commercial surfactant - soya lecithin - reveal that SPB1 biosurfactant significantly improves the textural properties of dough (hardness, springiness, cohesion and adhesion) especially at 0.5 g kg⁻¹. At the same concentration (0.5 g kg⁻¹), the effect of SPB1 biosurfactant was more pronounced than that of soya lecithin. Also, this biosurfactant considerably enhanced the gas retention capacity in the course of fermentation. CONCLUSION: These results show that SPB1 biosurfactant could be of great interest in the bread-making industry. A method for preparative extraction of lipopeptide biosurfactant with methanol as the extraction solvent has been effectively established.