Pilot-scale production of Bacillus subtilis MSCL 897 spore biomass and antifungal secondary metabolites in a low-cost medium.

OBJECTIVES: Bacillus subtilis is a plant growth promoting bacterium (PGPB) that acts as a microbial fertilizer and biocontrol agent, providing benefits such as boosting crop productivity and improving nutrient content. It is able to produce secondary metabolites and endospores simultaneously, enhancing its ability to survive in unfavorable conditions and eliminate competing microorganisms. Optimizing cultivation methods to produce B. subtilis MSCL 897 spores on an industrial scale, requires a suitable medium, typically made from food industry by-products, and optimal temperature and pH levels to achieve high vegetative cell and spore densities with maximum productivity. RESULTS: This research demonstrates successful pilot-scale (100 L bioreactor) production of a biocontrol agent B. subtilis with good spore yields (1.5 × 109 spores mL-1) and a high degree of sporulation (>80%) using a low-cost cultivation medium. Culture samples showed excellent antifungal activity (1.6-2.3 cm) against several phytopathogenic fungi. An improved methodology for inoculum preparation was investigated to ensure an optimal seed culture state prior to inoculation, promoting process batch-to-batch repeatability. Increasing the molasses concentration in the medium and operating the process in fed-batch mode with additional molasses feed, did not improve the overall spore yield, hence, process operation in batch mode with 10 g molasses L-1 is preferred. Results also showed that the product quality was not significantly impacted for up to 12 months of storage at room temperature. CONCLUSION: An economically-feasible process for B. subtilis-based biocontrol agent production was successfully developed at the pilot scale.

Pichia pastoris growth-coupled heme biosynthesis analysis using metabolic modelling.

Soy leghemoglobin is one of the most important and key ingredients in plant-based meat substitutes that can imitate the colour and flavour of the meat. To improve the high-yield production of leghemoglobin protein and its main component-heme in the yeast Pichia pastoris, glycerol and methanol cultivation conditions were studied. Additionally, in-silico metabolic modelling analysis of growth-coupled enzyme quantity, suggests metabolic gene up/down-regulation strategies for heme production. First, cultivations and metabolic modelling analysis of P. pastoris were performed on glycerol and methanol in different growth media. Glycerol cultivation uptake and production rates can be increased by 50% according to metabolic modelling results, but methanol cultivation-is near the theoretical maximum. Growth-coupled metabolic optimisation results revealed the best feasible upregulation (33 reactions) (1.47% of total reactions) and 66 downregulation/deletion (2.98% of total) reaction suggestions. Finally, we describe reaction regulation suggestions with the highest potential to increase heme production yields.

High-level production of recombinant HBcAg virus-like particles in a mathematically modelled P. pastoris GS115 Mut+ bioreactor process under controlled residual methanol concentration.

Recombinant hepatitis B core antigen (HBcAg) molecules, produced in heterologous expression systems, self-assemble into highly homogenous and non-infectious virus-like particles (VLPs) that are under extensive research for biomedical applications. HBcAg production in the methylotrophic yeast P. pastoris has been well documented; however, productivity screening under various residual methanol levels has not been reported for bioreactor processes. HBcAg production under various excess methanol levels of 0.1, 1.0 and 2.0 g L-1 was investigated in this research. Results indicate that, under these particular conditions, the total process and specific protein yields of 876-1308 mg L-1 and 7.9-11.2 mg gDCW-1, respectively, were achieved after 67-75 h of cultivation. Produced HBcAg molecules were efficiently purified and the presence of highly immunogenic, correctly formed and homogenous HBcAg-VLPs with an estimated purity of 90% was confirmed by electron microscopy. The highest reported HBcAg yield of 1308 mg L-1 and 11.2 mg gDCW-1 was achieved under limiting residual methanol concentration, which is about 2.5 times higher than the next highest reported result. A PI-algorithm-based residual methanol concentration feed rate controller was employed to maintain a set residual methanol concentration. Finally, mathematical process models to characterise the vegetative, dead and total cell biomass (Xv, Xd and X), substrate (Glycerol and Methanol) concentration, reactor volume (V), and product (HBcAg) dynamics during cultivation, were identified. A rare attempt to model the residual methanol concentration during induction is also presented.

Application of In-Situ and Soft-Sensors for Estimation of Recombinant P. pastoris GS115 Biomass Concentration: A Case Analysis of HBcAg (Mut+) and HBsAg (MutS) Production Processes under Varying Conditions.

Microbial biomass concentration is a key bioprocess parameter, estimated using various labor, operator and process cross-sensitive techniques, analyzed in a broad context and therefore the subject of correct interpretation. In this paper, the authors present the results of P. pastoris cell density estimation based on off-line (optical density, wet/dry cell weight concentration), in-situ (turbidity, permittivity), and soft-sensor (off-gas O2/CO2, alkali consumption) techniques. Cultivations were performed in a 5 L oxygen-enriched stirred tank bioreactor. The experimental plan determined varying aeration rates/levels, glycerol or methanol substrates, residual methanol levels, and temperature. In total, results from 13 up to 150 g (dry cell weight)/L cultivation runs were analyzed. Linear and exponential correlation models were identified for the turbidity sensor signal and dry cell weight concentration (DCW). Evaluated linear correlation between permittivity and DCW in the glycerol consumption phase (<60 g/L) and medium (for Mut+ strain) to significant (for MutS strain) linearity decline for methanol consumption phase. DCW and permittivity-based biomass estimates used for soft-sensor parameters identification. Dataset consisting from 4 Mut+ strain cultivation experiments used for estimation quality (expressed in NRMSE) comparison for turbidity-based (8%), permittivity-based (11%), O2 uptake-based (10%), CO2 production-based (13%), and alkali consumption-based (8%) biomass estimates. Additionally, the authors present a novel solution (algorithm) for uncommon in-situ turbidity and permittivity sensor signal shift (caused by the intensive stirrer rate change and antifoam agent addition) on-line identification and minimization. The sensor signal filtering method leads to about 5-fold and 2-fold minimized biomass estimate drifts for turbidity- and permittivity-based biomass estimates, respectively.