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L.P. Thapa et al. / Process Biochemistry xxx (2015) xxx–xxx
strain [32,33]. The increased microbial cell growth of the mutant
could be largely attributed to the reduced production of lactic acid,
which lowers the acidification rate of the media [34]. Eventually,
increase in cell growth of E. aerogenes SUMI014 favors an increase in
the ethanol concentration become maximum in culture broth the
cell growth gets decreased. Deletion of ldhA gene increased the con-
centration of NADH and a redox balance be achieved. The greater
availability of NADH led to a shift towards production of bioethanol,
the major fermentation product [35]. Converti and Perego [36] also
explained that amongst the intracellular metabolic pathways in E.
aerogenes, lactate production was the most NADH-consuming path-
way. Thus, the deletion of ldhA gene alters the metabolic flux and
increases the production of the reduced metabolite, bioethanol.
pH affected growth and bioethanol production indicate that E. aero-
genes SUMI014 is sensitive to changes in pH.
3.4. Effect of phosphate salts
Excessive changes in the hydrogen ion concentration (pH) of
the medium were prevented by the addition of a phosphate salt
(which acts a buffer). Therefore, the engineered strain, E. aero-
genes SUMI014, was fermented with various concentrations of
both KH2PO4 and K2HPO4 at a ratio (1:1) in order to deter-
concentrations of both phosphate salts and reached a maximum
(28.99 g/L) at a concentration of 6.5 g/L at 34 ◦C for 48 h and pH 7.5.
productivity (Fig. 5). The effect of phosphate salts on ethanol pro-
duction was investigated because the addition of phosphate salts
improves the efficiency of the fermentation process by increasing
the formation of adenosine triphosphate (ATP), which is the main
source of energy in cells [41]. Starvation of E. aerogenes SUMI014
with respect to potassium, phosphate, or magnesium ions leads to a
reversible increase in the rate of protein degradation and inhibition
of ribonucleic acid (RNA) synthesis [42]. Therefore, the addition of
phosphate salts in the production medium is required to increase
the generation of ATP molecules by preventing protein degrada-
tion as well as the inhibition of RNA synthesis during bioethanol
production.
3.3. Effect of temperature, time profile and initial medium pH
Physical parameters such as temperature and time profile are
sensitive primary factors for the fermentation of microorganisms.
Therefore, E. aerogenes SUMI014 was incubated in 100 mL produc-
tion medium at 32 ◦C, 34 ◦C, and 37 ◦C for various time intervals by
adjusting the medium to pH 7 at 200 rpm. E. aerogenes SUMI014
was grown very well at 34 ◦C and produced 22.96 g/L of bioethanol
as compared with 21.73 g/L at 32 ◦C and 21.77 g/L at 37 ◦C after 48 h
of cultivation (Fig. 4A).
Due to its influence on bacterial metabolism, pH level is one of
the most important factors affecting cell growth and production
of target products in bio-industry [37]. Due to the activity of liv-
ing cells, various organic acids are produced and accumulated in
culture medium. The resulting decrease in pH causes a significant
decrease in bioethanol production. Here, the effect of the initial pH
of the medium was investigated in order to determine the most
suitable pH for the growth of E. aerogenes SUMI014 and bioethanol
production. E. aerogenes SUMI014 was fermented at 34 ◦C for a
period of 48 h in aerobic conditions in media adjusted to varying pH
(value range 4–8.5) by the addition of hydrochloric acid and NaOH,
accordingly. Fig. 4B shows that the concentration of bioethanol
gradually increased with increasing pH and reached a maximum
(28.65 g/L) at pH 7.5 for 48 h incubation. Production declined at
dient between the intracellular and extracellular compartments. As
the pH gradient increases, the proton-motive force decreases, and
hence, more ATP is required for the extrusion of the same number
of protons. Therefore, less ATP remains for biosynthesis and growth
under aerobic conditions [38]. This means that glycerol utilization
was limited by the pH gradient between the inside and the outside
of the cells. The pH gradient prevented cells from transporting pro-
tons (H+) and utilizing substrate. There was an inhibitory effect of
pH on glycerol utilization and ethanol production by E. aerogenes
in the pH range 6–7.5. In this pH range, E. aerogenes SUMI014 pro-
duced 17.70 ∼ 24.50 g/L bioethanol by utilizing 80 ∼ 95% glycerol of
80 g/L. This result can be explained by the ratio of reduced to oxi-
dized nicotinamide adenine dinucleotide (the NADH/NAD+ ratio),
which affects the distribution of carbon flux through the metabolite
routes under aerobic conditions. Nakashimada et al. [39], demon-
strated the relationship between culture pH and NADH/NAD+ ratios
in various pH ranges. In the pH range 6–6.7, NADH/NAD+ ratios
were higher than at other pH values and this affected hydrogen pro-
duction by E. aerogenes. From a reaction engineering perspective, a
higher NADH/NAD+ ratio benefits reactions in which NADH is used
as a cofactor. Enterobacter aerogenes is known to utilize NADH as
a cofactor for the production of ethanol and 2,3-butanediol. Zhang
et al. [40] reported that ethanol production was enhanced by the
addition of NADH. The findings that a change in growth medium
3.5. Overexpression of adhE gene
The adhE gene of E. aerogenes KCTC 2190, which is 86 % identi-
cal to the alcohol dehydrogenase (AdhY) from Citrobacter youngae
ATCC 29220 and 85 % identical to that of E. coli str. K-12 substr.
MG1655 at the amino acid level, was cloned into plasmid pGEX-
4T-3 under the control of the Tac promoter. ADH, a key enzyme
SUMI014. Expression of the gene can be induced by adding 0.1 mM
IPTG solution after 2 h of incubation. Then the proper expression
of adhE gene was checked by measuring the ADH activity after
42 h of incubation in production media (Table 2). The enzyme data
showed that the adhE overexpression recombinant mutant strain
E. aerogenes SUMI2008 has highest enzyme activity as compared
with other strains. On the other hand, mutant strain has higher
ADH activity as compared with that of wild type strain because
the deletion of ldhA gene increased the ADH activity [34]. The
adhE-overexpressing recombinant mutant E. aerogenes SUMI2008,
mutant E. aerogenes SUMI014, wild E. aerogenes KCTC 29007 and
control strain containing only plasmid pGEX-4T-3 were incubated
in 100 mL production media containing 80 g/L glycerol as a carbon
source at 34 ◦C for 78 h. The adhE-overexpressed strain produced
38.32 g/L bioethanol, greater by an average of about 3.78 g/L than
that of parental mutant strain of 34.54 g/L, and by 25.23 g/L than
Table 3
The titer, yield and productivity of bioethanol produced by E. aerogenes ATCC 29007,
E. aerogenes SUMI014, E. aerogenes SUMI2008, and the control E. aerogenes SUMI014
strain with empty plasmid at optimized conditions.
Strains
Titer
(g/L)
Yield
(g/g)
Productivity
(g/L h)
E. aerogenes ATCC
29007
13.09
0.1561
0.1614
E. aerogenes SUMI014
E. aerogenes SUMI2008
E. aerogenes SUMI014
with empty plasmid
34.22
38.32
33.34
0.4278
0.4792
0.4168
0.4387
0.4912
0.4274
Please cite this article in press as: L.P. Thapa, et al., Improved bioethanol production from metabolic engineering of Enterobacter aerogenes