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NewsLife Sciences


RAPID AND AUTOMATED MICROBIAL QUANTIFICATION SUPPORTS RESEARCH INTO RENEWABLE
BIOFUELS

SelectScience

Find out how advanced cell counting technology is helping scientists improve the
efficiency of bioethanol production

6 Dec 2023
Georgina Wynne Hughes
Editorial Assistant
Dr. Mariane Raposo, postdoctoral researcher at the ‘Luiz de Queiroz’ College of
Agriculture, University of São Paulo

Increasing concerns about energy security, sustainability, and global climate
change have inspired a surge of research into biofuel production as an
alternative to fossil fuel-based energy. Bioethanol, the current major biofuel,
is produced industrially via alcoholic fermentation using the yeast
Saccharomyces cerevisiae. However, contamination by bacteria and wild yeasts is
frequently observed during the fermentation process, which can reduce production
efficiency and yield.

In this article, SelectScience® speaks with Dr. Mariane Raposo, a postdoctoral
researcher in the laboratory of Dr. Carlos A. Labate at the ‘Luiz de Queiroz’
College of Agriculture, University of São Paulo, to learn more about her work
developing strategies for identifying contaminating microorganisms during
bioethanol production. Raposo shares the challenges of conventional microscopy
methods for microbial quantification and describes how the QUANTOM Tx™ from
Logos Biosystems has expedited her research by enabling near-instant
characterization of bacteria and yeast with high sensitivity and accuracy.

A streamlined microbial quantification workflow

To evaluate methods for identifying microbial contamination, Raposo is both
analyzing industrial samples and conducting experiments in the lab using
bioreactors to simulate industrial alcoholic fermentation under controlled
conditions. “My routine lab work includes isolating bacteria and yeasts from
complex samples, preparing selective media to obtain the microorganisms of
interest, and performing morphological and physiological analyses of
microorganisms for their characterization,” she says.

A critical part of this workflow, she adds, is determining the concentration and
cell viability of yeasts and bacteria present in both industrial and laboratory
samples. To this end, her lab utilizes the QUANTOM Tx™ Microbial Cell Counter,
which they have found to significantly speed up and streamline the process
compared to conventional methods. “Previously, we determined concentration and
cell viability using optical microscopy analysis for yeast and colony counting
on solid medium for bacteria,” explains Raposo. “These techniques were extremely
time-consuming and required multiple personnel to prepare materials and analyze
samples, and to obtain results for bacteria, we had to wait for at least 24
hours of incubation.”

“In contrast, using the QUANTOM Tx, we’re able to obtain results for both yeasts
and bacteria instantly and without the need for culturing,” Raposo enthuses,
noting that the system also avoids the inherent variability of conventional
colony counting.

The QUANTOM Tx™ Microbial Cell Counter from Logos Biosystems

The QUANTOM Tx uses automated fluorescence imaging to identify and count
individual microbial cells in mere minutes. Prior to analysis, samples are
stained and mixed with loading buffer, loaded into QUANTOM™ M50 Cell Counting
Slides, and spun in a QUANTOM™ Centrifuge to evenly distribute the cells
throughout the counting chamber. Once a slide is inserted, the QUANTOM Tx
autofocuses and scans up to 10 fields of view to count fluorescent-stained
microbial cells. Results can then be verified instantly on the touch-screen
display, viewed graphically, and saved to an SSD. “Preparing samples takes a few
minutes and the whole workflow requires one person from sample prep to
analysis,” says Raposo. “Its rapid data acquisition, the eliminated need to
prepare materials like culture media beforehand, and the effortless execution of
the analysis are its biggest advantages for us.”

Adjustable protocols for diverse microbes

Raposo’s research focuses on multiple types of bacteria, including Gram-positive
lactic bacteria and Gram-negative acetic bacteria, as well as different yeast
strains both within and outside the Saccharomyces genus. “Lactic bacteria (LAB)
belonging to the genus Lactobacillus are the most frequent contaminants and the
most responsible for the drop in the yield of fuel ethanol production,” she
says. “Among yeasts, we mostly look at strains of the species Saccharomyces
cerevisiae, including strains responsible for carrying out alcoholic
fermentation as well as contaminating strains.”

According to Raposo, one of the greatest challenges of this work is dealing with
complex samples and determining the concentration of bacteria and yeast present
in the same sample. This, she shares, is another area where the QUANTOM Tx is
particularly advantageous. The instrument employs a sophisticated cell detection
and declustering algorithm to distinguish individual cells regardless of cell
shape, size, or arrangement. While the software is optimized for bacterial
cells, the ability to create custom protocols with parameters such as size,
detection sensitivity, declustering level, and roundness have enabled Raposo and
her team to accurately distinguish and count both bacteria and yeast strains.

“Using the QUANTOM Tx we’re able to determine the microbiological parameters of
our samples much faster and easier than using conventional techniques,” says
Raposo. “We have in our favor the fact that the difference in size and
morphology between these two microorganisms is considerable, which allows us to
direct the software to count bacteria and yeasts separately.”

Future outlooks

Research into bacteria and yeasts that can effectively produce fossil-fuel
alternatives is expanding rapidly, and Raposo believes this trend is set to
continue. “There’s a lot of work being done in this area,” she says. “This
includes studies obtaining genetically modified yeasts that present high
efficiency in the conversion of hexoses and pentoses to ethanol, as well as
those identifying bacteria and archaea capable of producing biogas obtained from
the metabolization of organic matter.”

“Microorganisms are great allies for the production of alternative ‘clean’
fuels, and I’m excited to see their potential continue to be explored in the
future,” she concludes.


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TAGS

MicrobiologyFlow Cytometry / Cell CountingCell / Tissue CultureCell
AnalysisBioethanolMicrobial DetectionBacteria


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