Page 56 - Refining Biomass Residues for Sustainable Energy and Bioproducts

Bacterial production of fatty acid and biodiesel: opportunity and challenges 33

Significant development has been observed during the last few decades relating to
the genetic modification of E. coli as well as cyanobacteria to enhance the production
and secretion of FFAs (Table 2.3). The natural secretion of fatty acid components in
the growth medium would eliminate the intermediate steps such as harvesting, drying,
and chemical extraction of fatty acid from bacterial cells hence makes the production
process cost effective. Naturally E. coli is not capable to accumulate fatty acid,
but after inducing genetic modification, E. coli becomes a striking producer owing
to its extensively studied lipid metabolism pathway among all the prokaryotes
and various tools well known for its genetic manipulation. Therefore a significant
production of fatty acid can be achieved by overexpression or restricted expression of
genes involved in fatty acid biosynthesis or degradation pathway, respectively.
The overexpression of wild or recombinant thioesterases results in the release of
acyl moiety from ACP, which triggers the FFA accumulation (Lennen and Pfleger,
2012). Applying recombinant E. coli in fed-batch fermentation using glycerol
or glucose along with woody biomass hydrolysate as carbon source produced
up to 4.8 and 3.8 g/L FFA respectively (Table 2.3). The cyanobacterium strain,
Synechocystis sp. SD277, was comprehensively improved for the generation and
release of FFA in the production media by removing and masking several genes
responsible for the biosynthesis of PHB or cyanophycin leading to weakening of cell
wall integrity. This engineered strain was able to produce up to 200 mg FFA/L by
overexpressing a number of thioesterase genes (Liu et al., 2011), Table 2.3. Even
though the yield obtained via this process is quite low, the utilization of CO 2 as a
carbon source and simultaneous production of FFA make this process much more
sustainable.
R. opacus is able to store a good amount of TAG in comparison to other
bacterial strains as discussed previously. Various improved genetic engineering
methods have been directed to increase its substrate range for the production of
more cost-effective and sustainable lipids from cheap carbon sources such as
glycerol, lignocellulosic-derived sugars, or plant hydrolysate (Table 2.3). By
adopting targeted manipulation and an adaptive evolution mechanism, production
of more than 50% of CDW or 16 g/L TAG was succeeded, while using corn stover
hydrolysate as the sole carbon source (Kurosawa et al., 2014). The achieved yields
are considerably more than the yields obtained by previously reported wild-type
R. opacus strain using various hydrolyzed plant biomass. Table 2.1 highlights the
significance of strain optimization for the production of bacterial lipids from waste
feedstock. There are several research focusing on TAG storage in E. coli; for
example, an overexpression of vital WS/DGAT genes is targeted for a combined
production of fatty acid and diacylglycerol together (Ro ¨ttig et al., 2015).
Nevertheless, the attainable yields are still not comparable with natural lipid produ-
cers, so this process requires significant improvement.

2.3.4 Biosynthesis of fatty acid ethyl ester by engineered
bacteria
At present, fatty acid methyl esters (FAMEs) are mainly produced by methanol
derived from fossil resources, ethanol has also been used as an alternative to