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Bioetanol a partir de hidrolisados lenhocelulósicos simulados. Avaliação preliminar do papel dos sistemas de transporte membranar de Saccharomyces cerevisiae
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Resumo(s)
The environmental problems, rooted in our society, strongly contribute to an emerging
demand to find alternative energy sources. In this context, bioethanol, the leading renewable
biofuel, is noted for its strong contribution in reducing the negative environmental impacts
generated by the use of fossil fuels.
Despite being mainly produced nowadays from starch (first-generation), bioethanol can be
produced from lignocellulosic material making it an even more "green" fuel (biofuel of second
generation). However, stress conditions and / or presence of inhibitors may limit the whole
fermentation process and hence the production of bioethanol. The main agents that inhibit yeast
growth and fermentation itself are: substrate composition, ethanol concentration, temperature,
pH and osmotic pressure. The mechanisms involved in tolerance and adaptation of
Saccharomyces cerevisiae to inhibitors present in lignocellulosic hydrolysates are not clear yet.
However, it is proven that during the pre-treatment and hydrolysis phases several substances
are produced that inhibit the growth and ethanol production by S. cerevisiae, including furfural,
acetic acid and p-hydroxybenzoic acid. The sustainability of bioethanol production is in the
acquisition of microorganisms resistant to such adverse conditions during the fermentation
process.
In this work conditions similar to those found in lignocellulosic hydrolysates were created in
the laboratory environment. The strain of Saccharomyces cerevisiae BY4741 was initially
submitted to different media that differed in the concentration of the main constituents found in
such substrates. The time required to reach the early stationary phase was almost equal in all
cases, ranging between three and five days. However, it was noted that the presence of toxics
in the medium accelerated the fermentation process of the BY474 strain. On the other hand,
there was a difficulty in reaching the depletion of glucose in a medium with toxic deficiency.
From the growth curve of BY4741 strain it was observed that the period of fermentation
was relatively long (ranging from 8 to 12 days) and the values of D.O.640nm were low (never
exceeding 4,0). For this reason we studied the influence of the auxotrophic markers and
nitrogen concentrations added to the medium. It was concluded that the markers concentration
was essential for a reduction in fermentation time, and there was significant reduction in
fermentation time when the triple of supplements was added to the medium.
All studies of ethanol production, especially those related with the tolerance to different
stresses, are based on effects on growth. In ethanol production, most of the fermentation
process occurs when cells have their growth finished. For this reason, the present study sought
to assess the behavior of seventeen deletion mutants during stationary phase, comparing its
behavior to the exhaustion stage, and in analogy with the parental BY4741 strain behavior.
Throughout this study, the fermentation took place in 50 mL of medium, in Erlenmeyer flasks of 100 mL, and its monitoring was performed by frequent measurements of the values of optical
density (D.O.640nm) and glucose level. Stabilization of D.O.640nm indicated the beginning of the
stationary phase and the depletion of glucose, which was detected qualitatively by commercial
strips (DIABUR-TEST ® 5000), setting the end of fermentation.
The first achievement of this work was the development of a methodology to survey the
behavior of different strains in their ability to accomplish the production of bioethanol from
lignocellulosic hydrolysates. It was utilized to monitor the effect caused in the fermentative
process by the deletion of the genes involved in membrane transport.
There were distinct behaviors among the seventeen mutants: in some cases the
fermentative capacity was considerably better than in the BY4741 strain (as in GAL6 and TRK1
OPI3), in others the fermentative capacity was similar (as in GUP1, FPS1 and HXT3), and
sometimes it was much worse (as in STL1, PDR5, PDR12 and ERG2).
Four mutants that showed very different fermentation profiles compared with BY4741 strain
were chosen for further studies. TRK1 and OPI3 were selected being the ones with the most
rapid glucose exhaustion. PDR5 and SLT2 mutants were also chosen as representatives of the
most negatively affected. In this set of mutants we determined the levels of glucose, ethanol
and glycerol, and assessed their behaviors on the influx and efflux of protons accross the
plasma membrane. All results were compared with those obtained of the parental strain.
In the first place, it has been verified that the simple use of glucose test strips do work
as an indicator of glucose concentration during the fermentation, having the results been
confirmed through the determination of the sugar concentration with base in a more accurate
method (method UV (nzytech D-glucose Kit)).
The rates of influx and efflux of H+ protons trough the plasma membrane, supported the
existence of differences in the physiologic state of the cells, in both sampling points.
With this work, we realize that there are essential genes that facilitate the natural
fermentation process, making these strains suitable for future research in this area. By finding
strains with high fermentative capacity, high tolerance to inhibitors and stress conditions,
especially high concentrations of ethanol and toxic compounds present in lignocellulosic
hydrolysates. Those unique strains become more attractive to be properly investigated and
eventually used as biological tools suitable for the construction of strains with new functional
fermentative capabilities, which can be suitable for industrial processes.
Descrição
Mestrado em Engenharia de Sistemas Bioenergéticos - Instituto Superior de Agronomia
Palavras-chave
bioethanol lignocellulosic hydrolysate Saccharomyces cerevisiae alcoholic fermentation membrane transport bioetanol hidrolisado lenhocelulósico inibidores lenhocelulósicos fermentação alcoolica transporte membranar