Production of antimicrobial lipopeptides by Bacillus sp. for biological control of postharvest phytopathogens in the perishable fruit industry

Fungal phytopathogens cause considerable damage to postharvest fruit crops. Synthetic chemical fungicides used to control postharvest disease have been associated with adverse environmental impact and health issues. A novel environmentally benign bio-fungicide (lipopeptide molecules) has been produced from Bacillus sp. and shown to be effective against postharvest fungal phytopathogens associated with perishable fruit crops. The research has primarily focused on optimising the upstream production kinetics and downstream purification of the lipopeptide product on the laboratory scale.

Bacillus cultures were conducted in shaking incubators and in instrumented bioreactors. Microbial growth was quantified using a spectrophotometer (Varian) and cell dry weight (Millipore). Glucose concentration was determined using high pressure liquid chromatography or HPLC (Aminex HPX-87H) and by colorimetric analysis (dinitrosalicylic acid method). Nitrate concentration was measured using an ion chromatograph (Dionex) and ammonium concentration by colorimetric analysis (nitroprusside method). Lipopeptide concentrations were determined by HPLC (Phenomenex Luna C18).

Purification was effected by acid precipitation (four pH values), solvent extraction (11 solvents) and adsorption onto macroporous resin (Diaion HP 20). Lipopeptide analysis was conducted by HPLC and by Thin Layer Chromatography (TLC) using surfactin and fengycin standards. Protein and lipid impurities were assessed by TLC (ninhydrin and primuline analyses respectively).

Efficacy verification: Antifungal efficacy against phytopathogens prevalent on postharvest fruit was determined by means of radial diffusion assays.

Upstream processing: The effects of nitrogen concentration and nitrogen source on the antifungal lipopeptide production kinetics have been quantified.

Downstream processing: Percentage purities and recoveries for acid precipitation and solvent extraction purification procedures have been quantified.

Efficacy: Antifungal efficacy of a 10-fold concentrate was confirmed for Alternaria brassicicola, Botrytis cinerea, Rhizopus stolonifera and Penicillium expansum

Antifungal lipopeptides were produced by Bacillus amyloliquefaciens DSM 23117 and quantified by reverse phase high pressure liquid chromatography (RP-HPLC). Using discrete ratios of ammonium to nitrate, the nitrogen source was shown to exhibit a significant influence on the production kinetics. The antifungal concentration in the culture supernatant exhibited a general downward trend with decreasing amounts of nitrate with a 2.6-fold difference in the maximum and minimum lipopeptide concentrations in the cultures containing only nitrate or ammonium respectively. A similar trend was observed in cell concentration which exhibited a 3.3-fold reduction, and a corresponding 1.3-fold increase in specific antifungal concentration, over the same range. Purification of the culture supernatant was effected by acid precipitation to pH 1, 2, 3 and 4. Precipitates were recovered by centrifugation and drying and the lipopeptides quantified by RP-HPLC. High recoveries (70-80 %) were attained at pH 1-3, with optimal purity at pH 3. Thin layer chromatography confirmed the low purity at pH 4 was due to contaminating proteins. The solvents methanol and isopropanol showed the highest antifungal lipopeptide recovery while methanol yielded the highest purity. Antifungal efficacy of a 10-fold concentrate was confirmed for Alternaria brassicicola, Botrytis cinerea, Rhizopus stolonifera and Penicillium expansum in zone clearing assays, demonstrating the successful production of a concentrated and partially purified biofungicide effective against postharvest phytopathogens, specifically those affecting postharvest fruit.