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Analysis of methods for quantifying yeast cell concentration in complex lignocellulosic fermentation processes.

ABSTRACT: Cell mass and viability are tightly linked to the productivity of fermentation processes. In 2^nd generation lignocellulose-based media quantitative measurement of cell concentration is challenging because of particles, auto-fluorescence, and intrinsic colour and turbidity of the media. We systematically evaluated several methods for quantifying total and viable yeast cell concentrations to validate their use in lignocellulosic media. Several automated cell counting systems and stain-based viability tests had very limited applicability in such samples. In contrast, manual cell enumeration in a hemocytometer, plating and enumeration of colony forming units, qPCR, and in situ dielectric spectroscopy were further investigated. Parameter optimization to measurements in synthetic lignocellulosic media, which mimicked typical lignocellulosic fermentation conditions, resulted in statistically significant calibration models with good predictive capacity for these four methods. Manual enumeration of cells in a hemocytometer and of CFU were further validated for quantitative assessment of cell numbers in simultaneous saccharification and fermentation experiments on steam-exploded wheat straw. Furthermore, quantitative correlations could be established between these variables and in situ permittivity. In contrast, qPCR quantification suffered from inconsistent DNA extraction from the lignocellulosic slurries. Development of reliable and validated cell quantification methods and understanding their strengths and limitations in lignocellulosic contexts, will enable further development, optimization, and control of lignocellulose-based fermentation processes.

SUBMITTER: Wang R

PROVIDER: S-EPMC8163860 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Analysis of methods for quantifying yeast cell concentration in complex lignocellulosic fermentation processes.

Wang Ruifei R Lorantfy Bettina B Fusco Salvatore S Olsson Lisbeth L Franzén Carl Johan CJ

Scientific reports 20210528 1

Cell mass and viability are tightly linked to the productivity of fermentation processes. In 2<sup>nd</sup> generation lignocellulose-based media quantitative measurement of cell concentration is challenging because of particles, auto-fluorescence, and intrinsic colour and turbidity of the media. We systematically evaluated several methods for quantifying total and viable yeast cell concentrations to validate their use in lignocellulosic media. Several automated cell counting systems and stain-b ...[more]

PMID: 34050249

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Project description:BackgroundThe eco-friendly transformation of agro-industrial wastes through microbial bioconversion could address sustainability challenges in line with the United Nations' Sustainable Development Goals. The bulk of agro-industrial waste consists of lignocellulosic materials with fermentable sugars, predominantly cellulose and hemicellulose. A number of pretreatment options have been employed for material saccharification toward successful fermentation into second-generation bioethanol. Biological and/or enzymatic pretreatment of lignocellulosic waste substrates offers eco-friendly and sustainable second-generation bioethanol production opportunities that may also contribute to waste management without affecting food security. In this study, we isolated a promising filamentous bacterium from the guts of cockroaches with commendable cellulolytic activity. The matrices of sequential statistics, from one-factor-at-a-time (OFAT) through significant variable screening by Placket-Burman design (PBD) to Box‒Behnken design of a surface methodology (BBD-RSM), were employed for major medium variable modeling and optimization by solid-state fermentation. The optimized solutions were used to saccharify lignocellulose in real time, and the kinetics of reducing sugar accumulation were subsequently evaluated to determine the maximum concentration of sugars extracted from the lignocellulose. The hydrolysate with the highest reducing sugar concentration was subjected to fermentation by Saccharomyces cerevisiae, Klyuveromyces marxianus and a mixture of both, after which the ethanol yield, concentration and fermentation efficiency were determined.ResultsSequential statistics revealed that rice husk powder, corn cob powder, peptone and inoculum volume were significant variables for the bioprocess at 59.8% (w/w) rice husk powder, 17.8% (w/w) corn cob powder, 38.8% (v/w; 109 cfu/mL) inoculum volume, and 5.3% (w/w) peptone. These conditions mediated maximum cellulolytic and xylanolytic activities of 219.93 ± 18.64 FPU/mL and 333.44 ± 22.74 U/mL, respectively. The kinetics of saccharification of the lignocellulosic waste under optimized conditions revealed two peaks of reducing sugar accumulation between 16 and 32 h and another between 56 and 64 h.ConclusionsAlthough K. marxianus had a significantly greater fermentation efficiency than S. cerevisiae, fermentation with a 50:50 (% v/v) mixture of both yeasts led to 88.32% fermentation efficiency with 55.56 ± 0.19 g/L crude bioethanol, suggesting that inexpensive, eco-friendly and sustainable bioethanol production could be obtained from renewable energy sources.

Project description:The isolation and selection of yeast strains to improve the quality of the cachaça-Brazilian Spirit-have been studied in our research group. Our strategy considers Saccharomyces cerevisiae as the predominant species involved in sugarcane juice fermentation and the presence of different stressors (osmolarity, temperature, ethanol content, and competition with other microorganisms). It also considers producing balanced concentrations of volatile compounds (higher alcohols and acetate and/or ethyl esters), flocculation capacity, and ethanol production. Since the genetic bases behind these traits of interest are not fully established, the whole genome sequencing of 11 different Saccharomyces cerevisiae strains isolated and selected from different places was analyzed to identify the presence of a specific genetic variation common to cachaça yeast strains. We have identified 20,128 single-nucleotide variants shared by all genomes. Of these shared variants, 37 were new variants (being six missenses), and 4,451 were identified as missenses. We performed a detailed functional annotation (using enrichment analysis, protein-protein interaction network analysis, and database and in-depth literature searches) of these new and missense variants. Many genes carrying these variations were involved in the phenotypes of flocculation, tolerance to fermentative stresses, and production of volatile compounds and ethanol. These results demonstrate the existence of a genetic profile shared by the 11 strains under study that could be associated with the applied selective strategy. Thus, this study points out genes and variants that may be used as molecular markers for selecting strains well suited to the fermentation process, including genetic improvement by genome editing, ultimately producing high-quality beverages and adding value.IMPORTANCEThis work demonstrates the existence of new genetic markers related to different phenotypes used to select yeast strains and mutations in genes directly involved in producing flavoring compounds and ethanol, and others related to flocculation and stress resistance.

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Analysis of methods for quantifying yeast cell concentration in complex lignocellulosic fermentation processes.

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Analysis of methods for quantifying yeast cell concentration in complex lignocellulosic fermentation processes.

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