Environmental Engineering Reference
In-Depth Information
there is a fourth enzyme, cellodextrinase, which “attacks the chain ends of the cellulose polymers,
liberating glucose” (Mousdale 2008). For the best productivity (converting all of the sugars at high
rates), microorganisms that bring about fermentation should be able to withstand ethanol and inhibi-
tory compounds as well as be resistant to contamination. The following microbes were improved to
impart these properties: yeast (
Saccharomyces cerevisiae
),
Zymomonas mobilis
, and
Escherichia
coli
(Lu and Mosier 2008; Kim et al. 2010a,b; Sun and Cheng 2002).
Yeast can tolerate ethanol and exists at a low pH, reducing the risk of contamination of other
microorganisms. Various strains of yeast can ferment an array of sugars such as monosaccharides
(glucose, mannose) and also disaccharides such as sucrose and maltose to ethanol.
Z. mobilis
is a
Gram-negative bacterium that uses the Entner-Doudoroff pathway anaerobically to produce 5-10%
more ethanol than yeast. This strain also cannot ferment pentoses effectively.
E. coli
can generate
ethanol in small quantities because it can ferment sugars into lactic acid, formic acid, and acetic acid
(Lu and Mosier 2008). However, most strains of yeast are unable to ferment pentose sugars such as
xylose and arabinose that represent up to 40% of total biomass carbohydrates.
S. cerevisiae
,
E. coli
,
and
Z. mobilis
have been genetically modified to enable them to ferment pentose sugars (Lu and
Mosier 2008; Kim et al. 2010a, 2010b; Weber et al. 2010). Current trends indicate that corn grain
ethanol will be displaced by cellulosic ethanol, ethanol production from sugarcane will include
production from sugar and cellulosic biomass, considerable quantities of biodiesel will be produced
from nonedible oil and cellulosic feedstock, and ethanol may be replaced by higher energy (more
reduced) compounds, as biofuel discovery and practical applications of synthetic catalysts become
game-changers in the biofuel production scenario.
The future of genomics-based biotechnology research for bioenergy crops has been detailed in
a recent review by Yuan et al. (2008). Genetic improvement of biofuel crops through biotechnology
will play an important role in improving biofuel production and making biofuels more sustainable
(Gressel 2008; Vega-Sánchez and Ronald 2010; Harfouche et al. 2011). Incorporating new genes
into plants uses various techniques for delivery. These genes are then made part of the plant's
chromosomal DNA through recombination. Particle bombardment (gene gun) forces the genes into
the cell through pressure. To gain specificity in the plant cell requires the use of
Agrobacterium
tumefaciens
, which allows the genes to enter the nucleus and combine with the host DNA. Although
A. tumefaciens
is only found naturally among dicot species, certain strains are able to infect monocot
plants, such as corn, sorghum, and switchgrass (Sticklen 2008). The gene of interest is placed under
the control of a promoter, allowing for tissue-specific inducible expression. Adding a selectable or
screenable marker to the vector simplifies “the identification of transformed plants and increases the
efficiency of recovery of transgenic plants” (Skinner et al. 2004). The selectable markers typically
used are
gus
and
gfp
or those that confer antibiotic resistance (screenable): kanamycin, hygromycin,
glyphosate, etc. Being able to observe the level of gene expression of a species is very useful in
determining phenotypes that are desirable, leading to a better understanding of the specific genes
that affect important features (Heaton et al. 2008). For the confirmation of a successful transfor-
mation, PCR, Southern blotting, and progeny analysis can be used (Skinner et al. 2004). Using
the techniques of genomics allows for faster selection of desirable characteristics than traditional
cross-breeding. Biotechnology using tissue culture techniques is also an important area of research.
One method useful to researchers is somaclonal variation (spontaneous changes in plants), in which
the opportunity arises to “develop new germplasm, better adapted to end-user demands” (Schroder
et al. 2008). These variations could lead to better adaptations of plants to unfavorable conditions.
In addition to the use of genetics to improve bioenergy crops, it is equally important to use
genetically improved organisms that will lower the operating costs and allow for faster and more
efficient ethanol production. A recombinant strain of
S. cerevisiae
allows for the co-fermentation of
glucose and xylose. The transgenic
Z. mobilis
strain (
Z. mobilis
CP4), producing up to 95% ethanol,
can grow on a mixture of glucose and xylose to produce 95% ethanol. In
E. coli
, overexpressing
pyruvate decarboxylase (
PDC
) and alcohol dehydrogenase (
ADH
) genes can produce a high per-
centage of alcohol (Lu and Mosier 2008). The ATCC11303 (
E. coli
) strain B seems to be the best
