3.3.2 Optimization of the esterification process using synthetic
broth
Before performing the esterification of organic acids in the real
fermentation broth, a synthetic broth containing similar compositions of
the desalinated fermentation broth (Table3 ) was prepared and used for
optimization of the esterification conditions. To achieve better
separation and recovery of catalyst from the reaction mixture, a
heterogeneous catalyst Amberlyst-15 resin, which is reported to have an
excellent catalytic activity in many esterification reactions
(Dange et al., 2014 ), was used for the formation of methyl esters
instead of using homogeneous catalysts such as sulfuric acid or
para-toluene-sulfonic acid. As shown in Fig.5A and5B , the conversion of both acetic and
butyric acid increased markedly as the reaction temperature was
increased from 25 to 70oC. An equilibrium maximum
conversion of 99.54 ± 0.14% in 90 min reaction time for acetic acid
(XeA), and 98.27 ± 0.03% in 150 min for butyric acid
(XeB), were reached at the boiling temperature of
70oC, respectively. In addition, it can be also seen
that the XeB decreased to 62.02 ± 0.80% when the
temperature was reduced to 25oC, while a relatively
high XeA of 90.88 ± 4.4% was maintained at the same
reaction temperature, indicating that the activation energy needed for
the formation of MB is higher than that for MA. The reaction temperature
was fixed at 70oC with a reaction time of 2.5 h for
the rest of the esterification experiments in order to ensure a high
conversion of both acetic and butyric acid and thus a high recovery
yield of the organic acids from the fermentation broth.
Catalyst loading was also observed to have positive influence on the
conversion of acetic and butyric acids (Fig5C ). Regardless of whether the catalyst
loading was 10 or 20%, the conversion of both acids were over 98%. A
reduction to 5% loading led to a similar acetic acid conversion,
whereas the butyric acid conversion slightly decreased to 96%. By
contrast, in control samples without catalyst less than 10% acid
conversion was reached, indicating an excellent catalytic activity of
Amberlyst-15 for simultaneous synthesis of MA and MB. We also examined
whether the high catalyst loading of 20% can accelerate the reaction
rate for reaching the same maximum acid conversion yield, but no
significant reduction of the time was observed (data not shown). These
findings illustrate that a 10% loading of Amberlyst-15 is enough for
the efficient conversion of acetic and butyric acids in the synthetic
broth.
In contrast to catalyst loading, the initial water content presented in
the synthetic broth negatively affected the acid conversion (Fig.5D ). The XA and
XB under the optimal reaction conditions decreased to
94.08 ± 0.69% and 87.21 ± 1.11% with 10% water, which was 5.5% and
11.3% lower than those achieved in reactions without water,
respectively. Thus the initial water content in the fermentation broth
should be carefully controlled before starting the esterification
reaction. In our designed DSP, water from the fermentation medium can be
completely removed by vacuum distillation. However, some water will be
inevitably introduced to the fermentation broth during the acidification
step using HCl solution. To remove the trace amount of water and improve
the equilibrium conversion in the real fermentation broth, addition of
molecular sieves or silica gel that has good water absorption can be
considered in the DSP.
In principle, alcohols other than methanol, such as ethanol and
1-propanol can also be esterified with the free organic acids in the
fermentation broth, forming corresponding ethyl ester or propyl esters
of acetic and butyric acid, which are also high value-added products
widely used in manufacturing perfumeries and flavoring food (Dange
et al., 2015 ; Xue et al., 2020 ;Nemati et al., 2021 ). In fact, we
found that ethanol and 1-propanol were also effective for salt
crystallization from the fermentation broth. Therefore, the reactivity
of ethanol and 1-propanol with acetic and butyric acids in the synthetic
broth was also investigated. As shown in Fig.6A , both XA and
XB slightly decreased to around 96% when the alcohol
was changed to same molar of ethanol or 1-propanol. The total
consumption of acids by the esterification reaction maintained at a
similar level regardless of the carbon chain length of the used alcohol.
However, there are large differences in the esterification selectivity
of the organic acids to the different alcohols including PDO and
glycerol under the same reaction conditions (Fig.6B ). The content of PDO and glycerol
decreased markedly after the esterification reaction as the number of
carbons in the linear alkyl chain of alcohol increased, indicating more
acids were esterified with the polyols when ethanol or 1-propanol was
used. In particular, the reduction of PDO in all three different alcohol
based synthetic broths was almost 2 times higher than the corresponding
reduction of glycerol. This may be attributed to the increased steric
hindrance and thus reduced reactivity between the acids and the
ortho-hydroxylated glycerol compared to the meta-hydroxylated PDO
(Cui et al., 2017 ). Although
esterification between the acids and the polyols is inevitable, both
acetic and butyric acids exhibited the highest esterification
selectivity to methanol in the synthetic mixture. The reacted polyols
(PDO + glycerol) in the methanol based reaction was 1.6 and 5.4 times
lower than those in the ethanol and 1-propanol based reactions,
respectively (Fig 6B ). More than 95%
of the reacted acetic and butyric acids were successfully converted to
the corresponding methyl esters (data not shown). As discussed above,
less PDO derived ester impurities facilitate the separation of acids and
the rectification of highly pure PDO. Therefore, considering the ester
recovery and the PDO purification efficiency, methanol is a good choice
for the esterification conversion of organic acids in the fermentation
broth.