A simple scaled down system to mimic the industrial production of first generation fuel ethanol in Brazil

Abstract

Although first-generation fuel ethanol is produced in Brazil from sugarcane-based raw materials with high efficiency, there is still little knowledge about the microbiology, the biochemistry and the molecular mechanisms prevalent in the non-aseptic fermentation environment. Learning-by-doing has hitherto been the strategy to improve the process so far, with further improvements requiring breakthrough technologies. Performing experiments at an industrial scale are often expensive, complicated to set up and difficult to reproduce. Thus, developing an appropriate scaled down system for this process has become a necessity. In this paper, we present the design and demonstration of a simple and effective laboratory-scale system mimicking the industrial process used for first generation (1G) fuel ethanol production in the Brazilian sugarcane mills. We benchmarked this system via the superior phenotype of the Saccharomyces cerevisiae PE-2 strain, compared to other strains from the same species: S288c, baker's yeast, and CEN.PK113-7D. We trust that such a system can be easily implemented in different laboratories worldwide, and will allow a better understanding of the S. cerevisiae strains that can persist and dominate in this industrial, non-aseptic and peculiar environment.

A biorefinery concept for the production of fuel ethanol, probiotic yeast and whey protein from a by-product of the cheese industry

“…The next step consisted of cultivating the selected strains (individually) in a system previously validated as an adequate proxy for the industrial production of fuel ethanol from sugarcane molasses with cell recycling and sulfuric acid treatment, which is usually employed for fighting contaminating microorganisms in big scale non-aseptic fermentations(13). This system recapitulates the performance of a given yeast strain during consecutive fermentation cycles, a feature which cannot be captured using regular cultivation systems, such as flasks or tubes.…”

“…Since the original protocol published by Raghavendran et al (2017) (13) involved fed-batch fermentations (using 3 installments), we also evaluated the effects of using simple (or batch) fermentation instead. As may be observed from the results of K. marxianus CIDCA 9121 in Figure 5, cell viability remained unaffected and a slight improvement of the overall ethanol yield ( p < 0,001) was detected, indicating the process can also be carried out using the much simpler batch mode.…”

“…From this second screening round, four strains were selected for their evaluation in a miniaturized system that resembles the industrial production of fuel ethanol in Brazil from sugarcane, in terms of ethanol yield on lactose and cell viability. This system was originally developed and validated for the fermentation of diluted sugarcane molasses with the yeast Saccharomyces cerevisiae , with cell recycling and sulfuric acid treatment of the yeast biomass after each fermentation cycle, in order to reduce the contamination load, similarly to industrial practice (13). As a result, it was demonstrated that only after some fermentation cycles (typically five), the superior phenotype of some selected industrial strains becomes evident.…”

“…Raghavendran et al (2017) (13) approximated the industrial fed-batch mode of operation by adding the medium in 3 installments along each fermentation round, which is mainly to avoid high sugar concentrations and the undesired effects of glucose repression that are known to affect S. cerevisiae . Since in our study lactose is the sugar present in the medium, meaning that no glucose should build up in the extracellular environment (lactose metabolism in Kluyveromyces yeasts takes place via its transport into the cells and subsequent intracellular hydrolysis into glucose and galactose (19), we decided to verify whether a simple batch process could be used in our experiments.…”

“…This experiment was performed as described by Raghavendran et al . (2017) (13). Cells from one colony of the desired yeast strain were aseptically inoculated in 500 mL baffled flasks containing 100 mL of YPD medium (with 4% glucose) and incubated at 30 °C and 200 rpm.…”