Biology subjects

Gombert, A. K.

Publications and source records attributed to Gombert, A. K..

8 recordsLinked to original sources

bioRxiv2026

DYNAMICS OF CONTAMINANT MICROBES IN BIOETHANOL PRODUCTION FROM SUGARCANE

The dynamics and impact of microbial contaminants in industrial sugarcane bioethanol production in Brazil were investigated through a two-year metagenomic study across two biorefineries. Shotgun metagenomic sequencing revealed that temporal shifts in the contaminant microbiome dynamics within production seasons were more pronounced than inter-annual or inter-mill variations. While Saccharomyces spp. dominated, bacterial communities, primarily within the Firmicutes phylum and dominated by the genera Lactobacillus, Limosilactobacillus, and Bacillus, exhibited dynamic changes. Correlation analyses with industrial process parameters revealed a complex interplay: lower Lactobacillus levels in one mill were associated with increased ethanol yield, whereas higher levels in another mill correlated with reduced yeast viability and increased flocculation. The presence of Limosilactobacillus was linked to decreased yeast viability, whereas Bacillus showed potential for inhibiting both Lactobacillus and Limosilactobacillus. These findings highlight the nuanced and species-specific impacts of bacterial contaminants on bioethanol production, underscoring the need for strain-level functional studies and targeted interventions to optimize fermentation efficiency and stability in industrial settings.

microbiology↗

bioRxiv2024

Simultaneous enumeration of yeast and bacterial cells in the context of industrial bioprocesses

In scenarios where yeast and bacterial cells coexist, e.g. the food and bioethanol industries, it would be of interest to simultaneously quantify the concentrations of both cell types, since traditional methods used to determine these concentrations individually take more time and resources. Here, we compared different methods for quantifying the fuel ethanol Saccharomyces cerevisiae PE-2 yeast strain and cells from the probiotic Lactiplantibacillus plantarum strain in microbial suspensions. Individual suspensions were prepared ([~]107, yeast cells/mL or [~]109, bacterial cells/mL) and mixed in 1:1 or 100:1 yeast-to-bacteria ratios, covering the range typically encountered in sugarcane biorefineries. The following methods were used: bright field microscopy, manual and automatic Spread-plate and Drop-plate counting, flow cytometry (at 1:1 and 100:1 ratios), and a Coulter counter (at 1:1 and 100:1 ratios). By subjecting the same mixed cell suspension to each technique, we observed that for yeast cell counts in the mixture (1:1 and 100:1 ratios), flow cytometry, the Coulter counter, and both Spread-plate options (manual and automatic CFU counting) yielded statistically similar results, while the Drop-plate and microscopy-based methods gave statistically different results. For bacterial cell quantification, the microscopy-based method, Drop-plate, and both Spread-plate plating options and flow cytometry (1:1 ratio) produced no significantly different results (P>0.05). In contrast, the Coulter counter (1:1 ratio) and flow cytometry (100:1 ratio) presented results statistically different (P<0.05). Additionally, quantifying bacterial cells in a mixed suspension at a 100:1 ratio wasnt possible due to an observed overlap between yeast cell debris and bacterial cells. The results from this work indicate that each method has limitations, advantages, and disadvantages, meaning that the best option will always depend on the application. We present a comparison of the techniques, in terms of time-to-results, cost of analysis and equipment, range of detectable cell/particle diameters, adequacy for simultaneous enumeration, and general pros and cons. Graphical AbstractThis study compares methods for simultaneously quantifying yeast and bacterial cells in a mixed sample, highlighting that in different cell proportions, some methods cannot quantify both cell types and present distinct advantages and limitations regarding time, cost, and precision. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/600471v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@875599org.highwire.dtl.DTLVardef@104b849org.highwire.dtl.DTLVardef@ded39borg.highwire.dtl.DTLVardef@11692b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

A Fully Defined Synthetic Medium Mimicking Sugar Cane Molasses

BackgroundYeast research in the context of food/beverage production and industrial biotechnology faces a dilemma: to use real industrial media or to use fully defined laboratory media? While the former option might lead to experiments closer to industrial conditions, the latter has the advantage of allowing for reproducibility and comparability of results among different laboratories, as well as being suitable for the investigation of how different individual components affect microbial or process performance. It is undoubtable that the development of a synthetic must a few decades ago led to important advances in wine yeast research. ResultsWe developed a fully defined medium that mimics sugarcane molasses, a frequently used medium in different industrial processes where yeast is cultivated. The medium, named 2SMol, builds upon a previously published semi-defined formulation and is conveniently prepared from some stock solutions: C-source, organic N, inorganic N, organic acids, trace elements, vitamins, Mg+K, and Ca. We validated the 2SMol recipe in a scaled-down sugarcane biorefinery model, comparing the performance of different yeast strains in different real molasses-based media. We also showcase the flexibility of the medium by investigating the effect of nitrogen availability on the ethanol yield during fermentation. ConclusionsHere we present in detail the development of a fully defined synthetic molasses medium, and we hope the 2SMol formulation will be valuable to researchers both in academia and industry to obtain new insights and developments in industrial yeast biotechnology.

bioengineering↗

bioRxiv2022

Comparative proteome analysis of different Saccharomyces cerevisiae strains during growth on sucrose and glucose

Both the identity and the amount of a carbon source present in laboratory or industrial cultivation media have major impacts on the growth and physiology of a microbial species. In the case of the yeast Saccharomyces cerevisiae, sucrose is arguably the most important sugar used in industrial biotechnology, whereas glucose is the most common carbon and energy source used in research, with many well-known and described regulatory effects, e.g. glucose repression. Here we compared the label-free proteomes of exponentially growing S. cerevisiae cells in a defined medium containing either sucrose or glucose as the sole carbon source. For this purpose, bioreactor cultivations were employed, and three different strains were investigated, namely: CEN.PK113-7D (a common laboratory strain), UFMG-CM-Y259 (a wild isolate), and JP1 (an industrial bioethanol strain). These strains present different physiologies during growth on sucrose; some of them reach higher specific growth rates on this carbon source, when compared to growth on glucose, whereas others display the opposite behavior. It was not possible to identify proteins that commonly presented either higher or lower levels during growth on sucrose, when compared to growth on glucose, considering the three strains investigated here, except for one protein, named Mnp1 - a mitochondrial ribosomal protein of the large subunit, which had higher levels on sucrose than on glucose, for all three strains. Interestingly, following a Gene Ontology overrepresentation and KEGG pathway enrichment analyses, an inverse pattern of enriched biological functions and pathways was observed for the strains CEN.PK113-7D and UFMG-CM-Y259, which is in line with the fact that whereas the CEN.PK113-7D strain grows faster on glucose than on sucrose, the opposite is observed for the UFMG-CM-Y259 strain.

systems biology↗

bioRxiv2022

Yeast population dynamics in Brazilian bioethanol production

The large scale and non-aseptic fermentation of sugarcane feedstocks into fuel ethanol in biorefineries represents a unique ecological niche, in which the yeast Saccharomyces cerevisiae is the predominant organism. Several factors, such as sugarcane variety, process design, and operating and weather conditions, make each of the [~]400 industrial units currently operating in Brazil a unique ecosystem. Here, we track yeast population dynamics in two different biorefineries through two production seasons (April to November of 2018 and 2019), using a novel statistical framework on a combination of metagenomic and clonal sequencing data. We find that variation from season to season in one biorefinery is small compared to the differences between the two units. In one biorefinery, all lineages present during the entire production period derive from one of the starter strains, while in the other, invading lineages took over the population and displaced the starter strain. However, despite the presence of invading lineages and the non-aseptic nature of the process, all yeast clones we isolated are phylogenetically related to other previously sequenced bioethanol yeast strains, indicating a common origin from this industrial niche. Despite the substantial changes observed in yeast populations through time in each biorefinery, key process indicators remained quite stable through both production seasons, suggesting that the process is robust to the details of these population dynamics.

evolutionary biology↗

bioRxiv2021

Aerobic growth physiology of Saccharomyces cerevisiae on sucrose is strain-dependent

Present knowledge on the quantitative aerobic physiology of the yeast Saccharomyces cerevisiae during growth on sucrose as sole carbon and energy source is limited to either adapted cells or to the model laboratory strain CEN.PK113-7D. To broaden our understanding of this matter and open novel opportunities for sucrose-based biotechnological processes, we characterized three strains, with distinct backgrounds, during aerobic batch bioreactor cultivations. Our results reveal that sucrose metabolism in S. cerevisiae is a strain-specific trait. Each strain displayed a distinct extracellular hexose concentration and invertase activity profiles. Especially, the inferior maximum specific growth rate (0.21 h-1) of the CEN.PK113-7D strain, with respect to that of strains UFMG-CM-Y259 (0.37 h-1) and JP1 (0.32 h-1), could be associated to its low invertase activity (0.04 to 0.09 U mgDM-1). Moreover, comparative experiments with glucose or fructose alone, or in combination, suggest mixed mechanisms of sucrose utilization by the industrial strain JP1, and points out the remarkable ability of the wild isolate UFMG-CM-259 to grow faster on sucrose than on glucose in a well-controlled cultivation system. This work hints to a series of metabolic traits that can be exploited to increase sucrose catabolic rates and bioprocess efficiency. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/432870v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@132c7a3org.highwire.dtl.DTLVardef@d0b790org.highwire.dtl.DTLVardef@c38ef4org.highwire.dtl.DTLVardef@de6bee_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2020

How to characterize a strain? The neglected influence of clonal heterogeneity on the phenotypes of industrial Saccharomyces

Populations of microbes are constantly evolving heterogeneity that selection acts upon, yet heterogeneity is non-trivial to assess methodologically. The practice of isolating single cell colonies for establishing, transferring, and using a strain results in single-cell bottlenecks with a generally neglected effect on the characteristics of the strain. We used six industrial yeasts to assess the level of heterogeneity in clonal populations, especially in terms of stress tolerance. First, we uncovered the existence of genome structure variants in available sequenced genomes of clonal lineages of thes strains. Subsequent phenotyping of strains and their newly isolated subclones showed that single-cell bottlenecks during isolation can considerably influence the observable phenotype. Next, we decoupled fitness distributions on the level of individual cells from clonal interference by plating single cell colonies. We used the obtained data on colony area for statistical modeling of the heterogeneity in phenotypes. One strain was further used to show how individual subclonal lineages are remarkably different not just in phenotype, but also in the level of heterogeneity. Thereby we call attention to the fact that choosing an initial clonal lineage from an industrial yeast strain may vastly influence downstream performances and observations on geno- and phenotype, and also on heterogeneity.

microbiology↗

bioRxiv2018

Physiology and lipid accumulation capacity of different Yarrowia lipolytica and Rhodosporidium toruloides strains on glycerol

ObjectiveTo compare physiological and process parameters, as well as lipid accumulation capacity, of six strains of Yarrowia lipolytica and two strains of Rhodosporidium toruloides in media containing glycerol as the main carbon and energy source.\n\nResultsThe strains Y. lipolytica IMUFRJ 50678, Po1g, W29 and CCT 5443 displayed very similar physiological parameters, with {micro}max, [Formula] and [Formula] average values of 0.31 h-1, 0.53 (g DW/g S), 0.60 (g S/g DW.h) and 0.46 (g DW/g S), respectively. The two strains of R. toruloides presented physiological and process parameters with no significant difference, with average values of 0.084 h-1, 0.53 (g DW/g S), 0.17 (g S/g DW.h) and 0.44 (g DW/g S). Among all Y. lipolytica strains, Y. lipolytica CCT 5443 strain presented the highest YLip/S, 0.054 (g Lip/g S), and PLipof 0.040 (g Lip/l.h). Among all investigated strains (Y. lipolytica and R. toruloides), the yeast R. toruloides CCT 7815 displayed the highest lipid accumulation capacity, with YLip/Sequal to 0.11 (g Lip/g S) and PLipequal to 0.10 (g Lip/l.h).\n\nConclusionAmong all strains investigated in our study, the yeast strain R. toruloides CCT 7815 presents the most promising characteristics for industrial single cell oil production.\n\nList of abbreviations

microbiology↗