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.

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

Iturins, which belong to antibiotic cyclic lipopeptides mainly produced by Bacillus sp., have the potential for application in biomedicine and biocontrol because of their hemolytic and antifungal properties. Bacillus amyloliquefaciens LL3, isolated previously by our lab, possesses a complete iturin A biosynthetic pathway as shown by genomic analysis. Nevertheless, iturin A could not be synthesized by strain LL3, possibly resulting from low transcription level of the itu operon. In this work, enhanced transcription of the iturin A biosynthetic genes was implemented by inserting a strong constitutive promoter C2up into upstream of the itu operon, leading to the production of iturin A with a titer of 37.35 mg l−1. Liquid chromatography-mass spectrometry analyses demonstrated that the strain produced four iturin A homologs with molecular ion peaks at m/z 1044, 1058, 1072 and 1086 corresponding to [C14 + 2H]2+, [C15 + 2H]2+, [C16 + 2H]2+ and [C17 + 2H]2+. The iturin A extract exhibited strong inhibitory activity against several common plant pathogens. The yield of iturin A was improved to 99.73 mg l−1 by the optimization of the fermentation conditions using a response surface methodology. Furthermore, the yield of iturin A was increased to 113.1 mg l−1 by overexpression of a pleiotropic regulator DegQ. To our knowledge, this is the first report on simultaneous production of four iturin A homologs (C14–C17) by a Bacillus strain. In addition, this study suggests that metabolic engineering in combination with culture conditions optimization may be a feasible method for enhanced production of bacterial secondary metabolites.

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Enhanced production of antifungal lipopeptide iturin A by Bacillus amyloliquefaciens LL3 through metabolic engineering and culture conditions optimization

Biosurfactants are amphiphilic molecules produced by a variety of microorganisms, including bacteria, yeast and filamentous fungi. Unlike chemically synthesized surfactants, biosurfactants present advantages, such as biodegradability, low toxicity, high selectivity and activity under extreme temperature, pH and salinity conditions, as well as a low critical micelle concentration. Moreover, they can be produced from agro-industrial waste and renewable sources. Their structural diversity and functional properties mean that they have potential applications in various industrial processes as wetting agents, dispersants, emulsifiers, foaming agents, food additives and detergents, as well as in the field of environmental biotechnology. However, opportunities for their commercialization have been limited due to the low yields obtained in the fermentation processes involved in their production as well as the use of refined raw materials, which means higher cost in production. In an attempt to solve these limitations on the commercialization of biosurfactants, various research groups have focused on testing the use of inexpensive alternative sources, such as agro-industrial waste, as substrates for the production of different biosurfactants. In addition to enabling the economical production of biosurfactants, the use of such waste aims to reduce the accumulation of compounds that cause environmental damage. This review shows advances in biosurfactant production carried out using different waste materials or by-products from agro-industrial activities.

Advances on research in the use of agro-industrial waste in biosurfactant production

Statistical optimization of antifungal iturin A production from Bacillus amyloliquefaciens RHNK22 using agro-industrial wastes.

Rapeseed meal and waste molasses are two important agro-industrial by-products which are produced in large quantities. In this study, solid state fermentation and fungal autolysis were performed to produce rapeseed meal hydrolysate (RMH) using fungal strains of Aspergillus oryzae, Penicillium oxalicum and Neurospora crassa. The hydrolysate was used as fermentation feedstock for heterotrophic growth of microalga Crypthecodinium cohnii that produce docosahexaenoic acid (DHA). The addition of waste molasses as a supplementary carbon source greatly increased the biomass and DHA yield. In the batch fermentations using media composed of diluted RMH (7%) and 1-9% waste molasses, the highest biomass concentration and DHA yield reached 3.43 g/L and 8.72 mg/L, respectively. The algal biomass produced from RMH and molasses medium also had a high percentage of DHA (22-34%) in total fatty acids similar to that of commercial algal biomass. RMH was shown to be rich in nitrogen supply comparable to the commercial nitrogen feedstock like yeast extract. Using RMH as sole nitrogen source, waste molasses excelled other carbon sources and produced the highest concentration of biomass. This study suggests that DHA production of the marine dinoflagellate C. cohnii could be greatly improved by concomitantly using the cheap by-products rapeseed meal hydrolysate and molasses as alternative feedstock.

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Improvement of Omega-3 Docosahexaenoic Acid Production by Marine Dinoflagellate Crypthecodinium cohnii Using Rapeseed Meal Hydrolysate and Waste Molasses as Feedstock

The feasibility of using untreated rapeseed meal as a nitrogen source for iturin A production by Bacillus subtilis 3–10 in submerged fermentation was first evaluated by comparison with two different commercial nitrogen sources of peptone and ammonium nitrate. A significant promoting effect of rapeseed meal on iturin A production was observed and the maximum iturin A concentration of 0.60 g/L was reached at 70 h, which was 20% and 8.0 fold higher than that produced from peptone and ammonium nitrate media, respectively. It was shown that rapeseed meal had a positive induction effect on protease secretion, contributing to the release of soluble protein from low water solubility solid rapeseed meal for an effective supply of available nitrogen during fermentation. Moreover, compared to raw rapeseed meal, the remaining residue following fermentation could be used as a more suitable supplementary protein source for animal feed because of the great decrease of major anti-nutritional components including sinapine, glucosinolate and its degradation products of isothiocyanate and oxazolidine thione. The results obtained from this study demonstrate the potential of direct utilization of low cost rapeseed meal as a nitrogen source for commercial production of iturin A and other secondary metabolites by Bacillus subtilis.

https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0111171&type=printable

Direct bio-utilization of untreated rapeseed meal for effective iturin A production by Bacillus subtilis in submerged fermentation.

The invention belongs to the field of biotechnology. A method for producing iturin through liquid state fermentation with rapeseed meal as a raw material is characterized by including the following steps of firstly, activating strains to obtain activated bacillus subtilis; secondly, conducting shake-flask culture; thirdly, conducting culture in a fermentation cylinder, wherein the bacillus subtilis where shake-flask culture is conducted is fermented in a fermentation medium, and the fermentation conditions include that the inoculum size ranges from 2%(v/v) to 10%(v/v), the pH is 7.0, the fermentation to ventilation ratio is 1-2:1 (vvm), the stirring rotation speed ranges from 300 rpm to 800 rpm, and the fermentation cycle is 72 hours; fourthly, collecting fermentation supernatant and rapeseed meal residues. While high-concentration iturin is obtained through liquid state fermentation with the rapeseed meal as a nitrogen source, the rapeseed meal residues which are appropriately dried can be used as forage protein rich in probiotics and can also be used as bio-organic fertilizers with the bacteriostasis function, and therefore high-valued full utilization of rapeseed meal resources is achieved.

Method for producing iturin through liquid state fermentation with rapeseed meal as raw material

The invention belongs to the technical field of iturin yield increasing. A method for promoting bacillus subtilis to continuously express iturin through a two-stage feeding strategy is characterized by comprising the following steps of (1) strain activation, (2) shake-flask culture, (3) fermentation tank culture and (4) feeding culture, wherein in the step (1), slant culture is conducted on the bacillus subtilis3-10 for 24 hours in an incubator at the temperature of 28 DEG C, an LB agar culture medium is used and activated bacillus subtilis is obtained; feeding is started when the concentration of reducing sugar is reduced to 3-4 g/L in the fermentation process, a feeding culture medium is formed by 500 g of glucose and 1 L of water, the two-stage control strategy is adopted in the feeding culture process, the feeding speed of the glucose at the earlier stage is controlled to be 0.56 g/L/h, the feeding speed is reduced to 0.28 g/L/h after 80 h until 120-hour fermentation ends, and supernate is obtained. By means of the method, continuous and efficient expression of the iturin can be realized and no glucose accumulation exists in the whole fermentation process.

Niu Yanxing

Papers

Direct bio-utilization of untreated rapeseed meal for effective iturin A production by Bacillus subtilis in submerged fermentation.

Method for producing iturin through liquid state fermentation with rapeseed meal as raw material

Method for promoting bacillus subtilis to continuously express iturin through two-stage feeding strategy