Antimicrobial lipopeptide production by Bacillus spp. for post-harvest biocontrol

[摘要] ENGLISH ABSTRACT: As overpopulation threatens the world's ability to feed itself, food has become an invaluable resource. Unfortunately, almost a third of the food produced for human consumption is lost annually. Pests including insects, phytopathogens and weeds are responsible for more than a third of the annual major crop losses suffered around the world.The majority of current post-harvest control strategies employ synthetic agents. These compounds, however, have been found to be detrimental to the environment as well as human health, which has led researchers to investigate alternative strategies. Biocontrol agents are environmentally compatible, have a lower toxicity and are biodegradable, making them an attractive alternative to the synthetic control agents. The lipopeptides produced by Bacillus spp. in particular, have shown great potential as biocontrol agents against various post-harvest phytopathogens.Most biocontrol strategies apply the biocontrol organism, for example Bacillus, directly, whereas this study focused on the use of the lipopeptide itself as an antifungal agent. This is advantageous as the lipopeptides are less sensitive to their surroundings, such as temperature and pH, compared to living organisms, allowing for the production of a standardized product.This study investigated the production of the Bacillus lipopeptides surfactin, fengycin and iturin under controlled batch conditions. Parameters increasing lipopeptide production were quantified, focussing on antifungal lipopeptides (iturin and fengycin), and lipopeptide production was optimized. Experiments were performed in a fully instrumented 1.3 L bench-top bioreactor and lipopeptide analyses were performed via high pressure liquid chromatography (HPLC) and liquid chromatography-mass spectroscopy (LC-MS).After screening four Bacillus spp., Bacillus amyloliquefaciens DSM 23117 was found to be the best antifungal candidate. This was based on it outperforming other candidates in terms of maximum antifungals produced, Yp/x,antifungals (yield per cells), and antifungal productivity.Nitrate, in the form of NH4NO3, was critical for lipopeptide production and an optimum concentration was observed above which the CDW (cell dry weight) no longer increased significantly and both μmax (maximum specific growth rate, h-1) and lipopeptide production decreased. For μmax, the optimum NH3NO4 concentration was 10 g/L and for lipopeptides it was 8 g/L. At these respective NH4NO3 concentrations μmax = 0.58 (h-1), the maximum antifungals (fengycin and iturin) were 285.7 mAU*min and the maximum surfactin concentration was 302 mg/L.The lipopeptides produced by B. amyloliquefaciens, the antifungals (fengycin and iturin) and surfactin, are secondary metabolites, regardless of the optimization treatment, i.e. increased NH4NO3 concentrations.Using 30% enriched air extended the nitrate utilization period, suggesting that when increasing supply concentration, more oxygen was available to act as electron acceptors, allowing nitrate to be used for lipopeptide production.The number of iturin and fengycin homologues generally increased with an increase in nitrate concentration. This suggested that process conditions, such as nitrate concentration, can be used to manipulate homologue ratios, allowing for the possibility to tailor-make biocontrol-agent upstream, during the production process, and possibly increase the efficacy of the biocontrol strategy.The lipopeptides produced by B. amyloliquefaciens showed complete inhibition against Botryotinia fuckeliana and diminished the growth capabilities of Botrytis cinerea. No inhibition was observed against Penicillium digitatum. These results indicate potential of the biocontrol strategy, although scale-up and fed-batch studies are recommended, especially when considering commercial implementation. Studies regarding the lipopeptide application method, i.e. a single application or multiple applications, should also be investigated as this will influence the efficacy of the lipopeptides against the target organisms.

[发布机构] Stellenbosch University