For more than a decade now, scientists at Stellenbosch University (SU) have been working on a biobased fungicide to control postharvest diseases on fresh fruit. Given food-safety and consumer-preference issues with traditional chemical treatments, the need for green chemistry to control fungal and bacterial postharvest diseases, and to replace commercial chemical compounds, has become more urgent in the agricultural sector.
In 2011 Prof Kim Clarke, who holds a PhD in Chemical Engineering from SU, put together a dynamic, multi-disciplinary team of specialists whose expertise ranged from life science to chemical engineering, to investigate and determine the best way to produce a new bioproduct to control fruit and plant disease. Increasingly, the integration of engineering and life science disciplines is recognised as the key that unlocks the potential of new bioproducts to progress from the initial research stage to successful production and implementation.
Prof Clarke’s search for the right biocandidate led her team to the bacteria Bacillus amyloliquefaciens for three reasons: its GRAS (generally regarded as safe) status; its demonstrated control against postharvest pathogens such as Penicillium and Botrytis spp; and it produces the lipopeptide (LP) bioproducts surfactin, iturin and fengycin that exhibit antibacterial and antifungal properties.
Numerous trials confirmed the efficacy of these LPs. However, the real-life application of this biotechnology continues to be limited by production processes. Large-scale fermentation and – more importantly – purification processes have not yet been developed for these compounds. As a result, production remains uneconomical.
“Over the years, the methodology to produce LPs in shake flasks in a laboratory was developed and perfected, but production has to be scaled for the product to be applied to an entire crop,” says Prof Robbie Pott, associate professor at the Department of Process Engineering at SU. “Our challenge is to make a controlled, consistent product whose concentration is known and matches the requirement – rather than spraying some bacteria on the fruit and hoping for the best. The goal is to separate the LP bioproducts from the bacteria that produce them, and then to collect, purify and concentrate them into a saleable product.”
And a particular challenge in the production of LPs is that the producing organism commonly consumes the product over time. This reduces final product titre, or concentration. In searching for a solution, Prof Pott focused on developing a new type of reactor configuration that builds on knowledge from a previous Hortgro project. In this project, aqueous two-phase separation proved successful in recovering LPs into a separate phase. “Using this knowledge, PhD candidate George Teke designed a reactor that uses in situ fermentation, in other words, removing the product from the fermentation phase so that it cannot be consumed,” explains Prof Pott. And MEng. candidate Kirsten van Niekerk focused on developing a suitable solvent phase that collects and concentrates the LPs from the fermentation broth.
The two-year project to develop a bioreactor specifically designed to produce LPs, was jointly funded by Hortgro and the Postharvest Innovation Programme (PHI).
In addition to building, modelling and testing the new reactor, the study also investigated other process options, specifically, adhered growth of Bacillus on a solid substrate (sugarcane bagasse, in this case).
Results and the way forward
The project met its primary objective, which was to design, build and test a new type of bioreactor that can successfully produce LPs, as well as other fermentation products. In so doing, the project team helped to bring a biobased antifungal agent closer to commercialisation.
To properly understand the multiphase nature of this novel bioreactor configuration, computational fluid dynamics (CFD) were used to create a model of the reactor. The model proved valuable in confirming that the reactor operates as it was designed to, and can furthermore be used to determine extraction flow rates and map expected reactor performance.
The experiments done with sugarcane bagasse have shown that adhered growth of Bacillus to produce LPs is not advised. As a result, planktonic culture is recommended for LP production.
Economic analyses were conducted on three potential LP fermentation processing routes – one batch, two continuous – while sensitivity analyses determined process bottlenecks. Though continuous fermentation is more economical, all three process options are severely constrained by high capital cost requirements, suggesting that flow sheets that use fewer unit operations, would be better.
“The take-home message for the industry is that we have built a reactor that makes in situ extractive fermentation possible,” says Prof Pott. “However, product yield and concentration need to be improved significantly before the economics of LP production will make bottom-line sense.”
While the economics of LPs cannot yet compete with synthetic fungicides, this work has illustrated which process options are likely to yield the best results, namely continuous operation, and a focus on improving product titre.
“If the industry is serious about using LPs as an alternative fungicide, the questions posed and answers offered in this project are a significant step towards achieving that,” concludes Prof Pott.
