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Saccharomyces cerevisiae transcriptional reprograming due to bacterial contamination during industrial scale bioethanol production.

Carvalho-Netto OV, Carazzolle MF, Mofatto LS, Teixeira PJ, Noronha MF, Calderón LA, Mieczkowski PA, Argueso JL, Pereira GA - Microb. Cell Fact. (2015)

Bottom Line: The bioethanol production system used in Brazil is based on the fermentation of sucrose from sugarcane feedstock by highly adapted strains of the yeast Saccharomyces cerevisiae.The formation of such particles is undesirable because it slows the fermentation kinetics and reduces the overall bioethanol yield.In this study, we investigated the molecular physiology of one of the main S. cerevisiae strains used in Brazilian bioethanol production, PE-2, under two contrasting conditions: typical fermentation, when most yeast cells are in suspension, and co-aggregated fermentation.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética, Evolução e Bioagentes, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brazil. osmar@lge.ibi.unicamp.br.

ABSTRACT

Background: The bioethanol production system used in Brazil is based on the fermentation of sucrose from sugarcane feedstock by highly adapted strains of the yeast Saccharomyces cerevisiae. Bacterial contaminants present in the distillery environment often produce yeast-bacteria cellular co-aggregation particles that resemble yeast-yeast cell adhesion (flocculation). The formation of such particles is undesirable because it slows the fermentation kinetics and reduces the overall bioethanol yield.

Results: In this study, we investigated the molecular physiology of one of the main S. cerevisiae strains used in Brazilian bioethanol production, PE-2, under two contrasting conditions: typical fermentation, when most yeast cells are in suspension, and co-aggregated fermentation. The transcriptional profile of PE-2 was assessed by RNA-seq during industrial scale fed-batch fermentation. Comparative analysis between the two conditions revealed transcriptional profiles that were differentiated primarily by a deep gene repression in the co-aggregated samples. The data also indicated that Lactobacillus fermentum was likely the main bacterial species responsible for cellular co-aggregation and for the high levels of organic acids detected in the samples.

Conclusions: Here, we report the high-resolution gene expression profiling of strain PE-2 during industrial-scale fermentations and the transcriptional reprograming observed under co-aggregation conditions. This dataset constitutes an important resource that can provide support for further development of this key yeast biocatalyst.

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Related in: MedlinePlus

Bacterial diversity during industrial fermentation. A- Bacterial community represented by the family taxon level. The number of individuals from each family was obtained from the average number of reads identified for the time points of both conditions examined (TF – typical fermentation; FL – flocculated fermentation). B- The percentage of L. fermentum among the total Lactobacillus that were identified in the microbial community. The Lactobacillaceae family reads were subtracted from reads previously classified as bacteria in A. C- Picture taken at the time of sample collection in the plant. Flocs are under suspension due to high level of CO2 formed during fermentation. D- Illustration of flocculation assay at laboratory scale. E- Scanning electron micrograph showing co-aggregation between PE-2 yeast cells and L. fermentum at 5,000 times magnification. The image was captured after 30 hours of yeast and bacterium co-culture under laboratory conditions in D. Both microorganisms were isolated from the FL biological samples.
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Fig1: Bacterial diversity during industrial fermentation. A- Bacterial community represented by the family taxon level. The number of individuals from each family was obtained from the average number of reads identified for the time points of both conditions examined (TF – typical fermentation; FL – flocculated fermentation). B- The percentage of L. fermentum among the total Lactobacillus that were identified in the microbial community. The Lactobacillaceae family reads were subtracted from reads previously classified as bacteria in A. C- Picture taken at the time of sample collection in the plant. Flocs are under suspension due to high level of CO2 formed during fermentation. D- Illustration of flocculation assay at laboratory scale. E- Scanning electron micrograph showing co-aggregation between PE-2 yeast cells and L. fermentum at 5,000 times magnification. The image was captured after 30 hours of yeast and bacterium co-culture under laboratory conditions in D. Both microorganisms were isolated from the FL biological samples.

Mentions: The material used to prepare the sequencing libraries also included some non-mRNA molecules, which were also sequenced and generated reads. We took advantage of this feature of the data and mined it for sequences derived from the bacterial cells present in the fermentations. We performed rRNA identification through alignment of the RNA-Seq reads to the SILVA rRNA database [23]. An average of approximately 5% of the total reads were classified as ribosomal sequences, with 0.26% being assigned to a bacterial origin (Additional file 1). The bacterial read counts per taxon were calculated for the different taxonomic levels using the SILVA rRNA database. The family level distribution of the bacterial sequences detected in the two fermentation conditions sampled are shown in Figure 1A. Interestingly, TF and FL had a similar overall distribution of bacterial families. However, within the Lactobacillaceae family, most of the reads derived from the flocculated condition were assigned to a single species, Lactobacillus fermentum (~93%) (Figure 1B). In contrast, only 41% of the Lactobacillaceae reads belonged to this species in the typical fermentations. This observation was significant since L. fermentum has been reported to induce sedimentation in S. cerevisiae [9,10]. To evaluate in principle the ability of L. fermentum to induce co-agregation with PE-2, we isolated bacterial colonies from this species from our FL samples and confirmed their identity by 16S rDNA PCR and Sanger sequencing. These isolates were co-cultured with PE-2 under laboratory conditions and a comparable behavior to that observed at the distillery was observed (Figure 1C and D). The PE-2 yeast cells became co-aggregated and sedimented when co-cultured with greater than 1 × 105 L. fermentum cells/mL. A representative scanning electron micrograph of PE-2 yeast and L. fermentum bacterial cells from these co-cultures under laboratory conditions is shown in Figure 1E.Figure 1


Saccharomyces cerevisiae transcriptional reprograming due to bacterial contamination during industrial scale bioethanol production.

Carvalho-Netto OV, Carazzolle MF, Mofatto LS, Teixeira PJ, Noronha MF, Calderón LA, Mieczkowski PA, Argueso JL, Pereira GA - Microb. Cell Fact. (2015)

Bacterial diversity during industrial fermentation. A- Bacterial community represented by the family taxon level. The number of individuals from each family was obtained from the average number of reads identified for the time points of both conditions examined (TF – typical fermentation; FL – flocculated fermentation). B- The percentage of L. fermentum among the total Lactobacillus that were identified in the microbial community. The Lactobacillaceae family reads were subtracted from reads previously classified as bacteria in A. C- Picture taken at the time of sample collection in the plant. Flocs are under suspension due to high level of CO2 formed during fermentation. D- Illustration of flocculation assay at laboratory scale. E- Scanning electron micrograph showing co-aggregation between PE-2 yeast cells and L. fermentum at 5,000 times magnification. The image was captured after 30 hours of yeast and bacterium co-culture under laboratory conditions in D. Both microorganisms were isolated from the FL biological samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4318157&req=5

Fig1: Bacterial diversity during industrial fermentation. A- Bacterial community represented by the family taxon level. The number of individuals from each family was obtained from the average number of reads identified for the time points of both conditions examined (TF – typical fermentation; FL – flocculated fermentation). B- The percentage of L. fermentum among the total Lactobacillus that were identified in the microbial community. The Lactobacillaceae family reads were subtracted from reads previously classified as bacteria in A. C- Picture taken at the time of sample collection in the plant. Flocs are under suspension due to high level of CO2 formed during fermentation. D- Illustration of flocculation assay at laboratory scale. E- Scanning electron micrograph showing co-aggregation between PE-2 yeast cells and L. fermentum at 5,000 times magnification. The image was captured after 30 hours of yeast and bacterium co-culture under laboratory conditions in D. Both microorganisms were isolated from the FL biological samples.
Mentions: The material used to prepare the sequencing libraries also included some non-mRNA molecules, which were also sequenced and generated reads. We took advantage of this feature of the data and mined it for sequences derived from the bacterial cells present in the fermentations. We performed rRNA identification through alignment of the RNA-Seq reads to the SILVA rRNA database [23]. An average of approximately 5% of the total reads were classified as ribosomal sequences, with 0.26% being assigned to a bacterial origin (Additional file 1). The bacterial read counts per taxon were calculated for the different taxonomic levels using the SILVA rRNA database. The family level distribution of the bacterial sequences detected in the two fermentation conditions sampled are shown in Figure 1A. Interestingly, TF and FL had a similar overall distribution of bacterial families. However, within the Lactobacillaceae family, most of the reads derived from the flocculated condition were assigned to a single species, Lactobacillus fermentum (~93%) (Figure 1B). In contrast, only 41% of the Lactobacillaceae reads belonged to this species in the typical fermentations. This observation was significant since L. fermentum has been reported to induce sedimentation in S. cerevisiae [9,10]. To evaluate in principle the ability of L. fermentum to induce co-agregation with PE-2, we isolated bacterial colonies from this species from our FL samples and confirmed their identity by 16S rDNA PCR and Sanger sequencing. These isolates were co-cultured with PE-2 under laboratory conditions and a comparable behavior to that observed at the distillery was observed (Figure 1C and D). The PE-2 yeast cells became co-aggregated and sedimented when co-cultured with greater than 1 × 105 L. fermentum cells/mL. A representative scanning electron micrograph of PE-2 yeast and L. fermentum bacterial cells from these co-cultures under laboratory conditions is shown in Figure 1E.Figure 1

Bottom Line: The bioethanol production system used in Brazil is based on the fermentation of sucrose from sugarcane feedstock by highly adapted strains of the yeast Saccharomyces cerevisiae.The formation of such particles is undesirable because it slows the fermentation kinetics and reduces the overall bioethanol yield.In this study, we investigated the molecular physiology of one of the main S. cerevisiae strains used in Brazilian bioethanol production, PE-2, under two contrasting conditions: typical fermentation, when most yeast cells are in suspension, and co-aggregated fermentation.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética, Evolução e Bioagentes, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brazil. osmar@lge.ibi.unicamp.br.

ABSTRACT

Background: The bioethanol production system used in Brazil is based on the fermentation of sucrose from sugarcane feedstock by highly adapted strains of the yeast Saccharomyces cerevisiae. Bacterial contaminants present in the distillery environment often produce yeast-bacteria cellular co-aggregation particles that resemble yeast-yeast cell adhesion (flocculation). The formation of such particles is undesirable because it slows the fermentation kinetics and reduces the overall bioethanol yield.

Results: In this study, we investigated the molecular physiology of one of the main S. cerevisiae strains used in Brazilian bioethanol production, PE-2, under two contrasting conditions: typical fermentation, when most yeast cells are in suspension, and co-aggregated fermentation. The transcriptional profile of PE-2 was assessed by RNA-seq during industrial scale fed-batch fermentation. Comparative analysis between the two conditions revealed transcriptional profiles that were differentiated primarily by a deep gene repression in the co-aggregated samples. The data also indicated that Lactobacillus fermentum was likely the main bacterial species responsible for cellular co-aggregation and for the high levels of organic acids detected in the samples.

Conclusions: Here, we report the high-resolution gene expression profiling of strain PE-2 during industrial-scale fermentations and the transcriptional reprograming observed under co-aggregation conditions. This dataset constitutes an important resource that can provide support for further development of this key yeast biocatalyst.

Show MeSH
Related in: MedlinePlus