3.3 Determination of influencing factors for succinic acid
production from xylan using microbial consortium containing strain M5
and 130Z
The influence of single factors, such as mixed time (inoculation time ofA. succinogenes to T. thermosaccharolyticum M5), initial
xylan concentration, initial yeast extract concentration, pH,
and MgCO3
concentration on succinic acid production was explored. During these
variables, mixed time, initial xylan concentration and
MgCO3 concentration were found to have greater impacts
on succinic acid production. As shown in Fig 4A, the optimal
inoculation time of strain A.
succinogenes 130Z to T. thermosaccharolyticum M5 was 72 h. The
possible reason could be that when the
inoculation time was too early,
xylanase and
β-xylosidase activities secreted by
strain M5 was relatively low (Fig 4B), which was not conducive to the
subsequent xylan degradation. However, if the inoculation time was too
late, strain M5 was in the decline period, and xylanase and β-xylosidase
secretion efficiency would be reduced. When strain 130Z was inoculated
at 72 h, strain M5 was in the exponential growth period, and a large
amount of xylanase and β-xylosidase was secreted. In this microbial
co-cultivation system, xylanase activity was relatively stable with 0.41
U/mL when temperature was shifted to 37℃, indicating that xylan could
still be continuously degraded.
Generally, higher xylan
concentration would lead to higher xylose production in the microbial
co-cultivation system. However, when xylan concentration was increased
to a certain extent, the growth of strain M5 would be inhibited, namely
the substrate inhibition occurred. Moreover, a product feedback
inhibition of xylanase and β-xylosidase caused by xylose also existed,
which has been
mentioned
above (Fig 4C). Hence, when xylan concentration was at 80 g/L, the
highest succinic acid concentration of 22.07 g/L was obtained (Fig 4D).
Further increase of xylan would reduce succinic acid production, as only
21.81 g/L of succinic aicd was produced from 100 g/L of xylan.
Organic acids produced in the fermentation process could significantly
reduce the pH value. MgCO3 was usually added as an acid
neutralizer to maintain the pH values in the fermentation medium.
During the reaction,
MgCO3 can also release CO2 to maintain
the CO2 environment needed for succinic acid
fermentation (Hyohak et al., 2010). In addition, MgCO3
can provide magnesium ions for phosphoenolpyruvate carboxykinase, which
is a key enzyme in succinic acid production (Zou, Zhu, Li, & Tang,
2011). As reflected in Fig 4E, when MgCO3 concentration
was 60 g/L, the highest succinic acid titer was up to 28.97 g/L. When
MgCO3 concentration was increased from 40 g/L to 60 g/L,
succinic acid concentration was also increased simultaneously. However,
further increase in MgCO3 concentration reduced succinic
acid production. For instance, when MgCO3 concentration
was increased to 80 g/L, succinic acid titer was just 17.30 g/L.
3.4 Optimization of fermentation conditions for enhancedsuccinic acid
production from xylan using microbial co-cultivation system containing
strain M5 and 130Z
Based on the one-factor-at-a-time (OFAT) experiments, the optimal
conditions for three important factors (mixed time, initial xylan
concentration, and MgCO3 concentration) were determined
using response surface methodology (RSM). For the response surface
analysis, 17 experiments with triplicates were conducted according to
RSM design as shown in Table 1, where X1 denotes initial
xylan concentration (g/L), X2 denotes mixed time,
X3 denotes MgCO3 concentration, and Y is
succinic acid production (g/L). According to multiple regression
analysis of experimental data, second-order polynomial equation of
empirical relationship between response and screening variables was
obtained as follows:
(2)
The
adequacy of this model was further verified using analysis of variance
(ANOVA), which was tested using Fisher’s statistical analysis (Table 2)
(Isar, Agarwal, Saran, Kaushik, & Saxena, 2007).
Value
of ”Prob > F” was less than 0.0500, indicating that
model terms were significant
(Bezerra, Santelli, Oliveira, Villar, & Escaleira, 2008). The lack of
fit F-value of 1.71 implied that the lack of fit was not significant.
The non-significant lack of fit suggested that obtained experimental
responses sufficiently fit with the model (Ganjali Dashti, Abdeshahian,
Wan Yusoff, Kalil, & Abdul Hamid,
2014).
The response surface is a three-dimensional spatial surface map, which
can visually reflect the interaction between various factors and
response values, and show the relationship between the observed response
value and test parameter level (Cui et al., 2009). The above multiple
regression equation was used to make the response surface curve. As
shown in Fig 5A-C, there was interaction between each pair of variables,
and significant interaction between the three selected variables. The
optimal succinic acid production value predicted from the response
surface model was 32.39 g/L under the optimal conditions, in which xylan
concentrations was 84.05 g/L, mixed time was 67 h and
MgCO3 concentration was 62.35 g/L, respectively. To
verify the accuracy of model prediction, three sets of parallel
experiments were carried out under the optimal conditions. As seen in
Fig 5D, succinic acid production could reach
32.50 g/L with yield of 0.41 g/g,
which was basically consistent with the model prediction value. In this
microbial co-cultivation system, strain M5 was able to constantly
hydrolyze xylan to xylose during first 67 h, and approximately 12.23 g/L
of xylose occurred in the fermentation broth before strain 130Z was
inoculated. After dosage of strain
130Z, xylose could be immediately consumed, and succinic acid was
accumulated. Owning to the constant hydrolysis of
xylanase and β-xylosidase, xylose
concentration in the fermentation broth was maintained at 6-7 g/L. In
addition, the activities of xylanase and β-xylosidase were always
maintained at 0.38-0.43 U/mL and 6.02-7.34 U/mL, respectively during the
co-cultivation process. Before 96 h, succinic acid production increased
exponentially, and the highest succinic acid titer of 32.50 g/L was
obtained at 240 h. Taken together, this microbial co-cultivation system
could not only reduce feedback inhibition of enzymes, but also make it
possible to produce succinic acid directly from xylan through CBP.