Page 74 - Advances in bioenergy (2016)

the fermentative performance of this flocculating engineered S. cerevisiae strain acquired some
beneficial characteristics for ethanol production processes from agro-industrial substrates
under HG. Therefore, this differential induction of gene expression directed by TPS1 promoter
(under ethanol induction) added evidence that controlled gene expression can endow
beneficial yeast phenotypes for industrial applications.

High Gravity Ethanol Production Processes

The main advantage of biomass bioconversion under HG (initial dry matter concentrations
above 12% w/w) or very high gravity (VHG, above 30% w/w) conditions is the generation of
high final ethanol concentrations (above 5% v/v), decreasing the cost of the distillation step,
95
which is considered one of the main constraints in the bioethanol industry. However, serious
inconveniences come along with increase in initial dry matter: (1) The high sugar content may
cause osmotic stress which leads to a number of cellular responses which in turn affect the

ethanol yield and productivity. (2) Mixing of solids, enzymes, and yeast cells becomes very
difficult using conventional reactors due to low water content, which increases the viscosity of
the substrate material. (3) Ethanol has an inhibitory effect on cell growth and viability already
at relatively low concentrations, which becomes more severe as the ethanol concentration
increases. (4) The presence of toxic compounds, generated during the pretreatment step in high
concentrations, makes the bioconversion almost impossible in some cases.

The design of new type of reactors was a major concern at the beginning of the research in the
field. Free fall rotating systems were proven very efficient for HG processes. Jørgensen et
96
al. reported a successful free fall bioreactor design for the bioconversion of lignocellulose
4
into ethanol using initial dry matter up to 40%. Recently, Matano et al., in an attempt to
improve the mixing efficiency, used a similar rotary fermentation system, consisting of a

fermentation vessel positioned horizontally in a drum-type heat block which axially rotated
under controlled temperature. By using a recombinant cellulolytic S. cerevisiae strain and 10
−1
FPU g DM, 89% of the theoretical ethanol yield was achieved from 20% (w/v) rice straw.
Various process improvement approaches have been tested to cope with the stressful HG
conditions imposed to yeast cells. The detoxification of the initial material has in many cases
97
proved beneficial to the overall fermentative performance, whereas, in other cases, the
addition of nutrient supplementations during fermentation enhanced the ethanol yield and rate
of the process. 98

Evolutionary Engineering and Inverse Engineering

The majority of evolutionary engineering applications have focused on two areas: evolutionary

engineering of stress resistance and substrate utilization. S. cerevisiae is probably the most
robust microorganism used in biofuels applications. It has an innate tolerance to some of the
inhibitory compounds in lignocellulose hydrolysates, such as hydroxymethylfurfural (HMF),
furfural, and phenolics, due to its ability to convert them to less harmful molecules. However,
it can only tolerate certain levels of these inhibitory compounds, whereas its fermentative
performance can be completely inhibited in the presence of low concentrations of some other