Bioreactor and process development for the production of antimicrobial lipopeptides produced by Bacillus spp. for biological control of postharvest phytopathogens in the perishable fruit industry

Lipopeptides have proven to be excellent biologically-based control agents for the prevention of phytopathogens. However, the technology has been significantly limited by production processes – large scale fermentations, and more importantly, purification processes have not been developed for these compounds. This leaves production uneconomical or unfeasible currently.

Building on a novel separation method in a previous Hortgro project, based on Aqueous Two Phase Separation, this project aims to develop a reactor specifically designed for production of LPs. The reactor integrates LP extraction from the broth, and their recovery as a salable product.

The bioreactor was designed by fabricating prototypes, testing fluid mechanics, and iterating. A final prototype was integrated into an existing fermenter, and tested. CFD analyses of the modified reactor were conducted in silico, using Fluent software.

Growth experiments of Bacillus cultivated in a suspension of cellulosic solids (sugarcane bagasse) were conducted, using standard medium formulation using methodologies developed in previous theses (Mazibuko, Johannes, Dlamini et al). Lipopeptide concentrations were monitored over time in the liquid phase, using HPLC.

Process economics were simulated in silico using data generated in this project, as well as literature data, for three process options: i) batch fermentation of LPs, ii) continuous production of LPs using the semi-partition reactor developed in this project, and iii) continuous production of LPs using a foam fractionation reactor. Sensitivity analyses on the key process parameters were conducted.

A thorough literature review was conducted, which supported the project’s initial assertion that technologies for in situ fermentation were limited.

A semi-partition bioreactor was designed to facilitate in situ fermentation. This reactor was fabricated, and tested with hydrodynamic and mixing studies. The reactor successfully allows continuous collection of an extractant phase. A CFD model of the reactor was developed, in order to better understand the mixing and operability. This work successfully demonstrates the range of operability of this reactor, and recommends its use for in situ production of LPs.

A growth and LP production study of Bacillus grown on a solid cellulosic substrate (sugarcane bagasse) was conducted. Results gave no LP production, not recommending the use of solid substrates in LP fermentation.

Economic analyses based on heuristics and available data were conducted for three potential LP production flowsheets. All analyses confirm that product yield and titre effect process economics significantly. Continuous processes are favoured over a batch process (although there are processing risks inherent in continuous processing). Capital expenses are limiting for potential processes, and so proposed processes with simplified unit operations are economically favoured – the SPB performs well in this analysis

A semi-partition bioreactor has been designed, fabricated, and tested. This reactor will have applicability in the production of LPs, as well as other fermentation products. A CFD model of this reactor has been developed, with single-phase and multi-phase flows. The analyses indicate that the reactor operates as designed, and the CFD model can be used to determine extraction flow rates, and expected reactor performance.

On the basis of experiments performed using sugarcane bagasse, adhered growth of Bacillus for the production of LPs is not advised. On this basis, planktonic culture is recommended for LP production.

Economic analyses were conducted on three potential LP fermentation processing routes – one batch, two continuous. Sensitivity analyses determined process bottlenecks. The key process parameter which needs improvement is product yield and titre. Additionally, continuous fermentations are more economical, and all process options are severely constrained by high capital cost requirements, suggesting the choice of flowsheets which use fewer unit operations.

A new reactor, specifically designed for in situ extractive fermentation has been developed and demonstrated. However, while LPs show promise, their production economics are still hampered by low product titres.