3.3.1 Process design and preparation for the esterification of
organic acids in crude glycerol fermentation broth
Fermentation in 1 m3 bioreactor clearly shown that,
along with 80 g/L PDO, almost 30 g/L organic acids (acetate and
butyrate) were co-produced by C. pasteurianum G8, which make up
more than 20% of the carbon atoms of glycerol consumed. Therefore, the
process economy can be significantly improved if the organic acids are
recovered as co-products along with PDO from the fermentation broth. In
this scenario, the total product titer, overall productivity and yield
(PDO + organic acids) from the 1m3 crude glycerol
fermentation reached 110 g/L, 5.78 g/L/h and 0.67 g/g, respectively.
However, a direct separation of free organic acids from PDO usually
requires the usage of a distillation column with a high reflux ratio,
which increases the investment and reduces the production efficiency
(Kaeding et al., 2015 ;Rousseaux et al., 2013 ). More
seriously, PDO and the organic acids can form esters during the
distillation process, and the high boiling point of the PDO ester
impurities significantly reduces the recovery yield of organic acids and
the final purity of PDO (Zhang et al.,
2021 ). To overcome this problem, a DSP that can convert the organic
acids to alcohol esters with low boiling point before the acid
separation is of special interest. In fact, a conversion of acetic and
butyric acids to their corresponding methyl esters is beneficial to the
acid recovery via evaporation owing to the significantly reduced boiling
point for the esters (acetic acid 117.9oC vs. MA
56.8oC; butyric acid 164.35 oC vs.
MB 102.8 oC, at atmosphere pressure). The occupation
of the carboxylic group in the methyl esters also prevents the formation
of PDO-derived esters during the thermal separation process. This can
significantly increase the efficiency and reduce the costs of PDO
recovery. Furthermore, both MA and MB have a higher market price than
those of the free acids (acetic acid 0.6 USD/kg vs. MA 1.1 USD/kg;
butyric acid 1.01 USD/kg vs. MB 4.04 USD/kg, according to market survey
made on 16 Aug. 2021), which represent a great gain or “compensation”
for the production cost of PDO.
Based on the advantages mentioned above, the development of a novel DSP
for the co-production of PDO, MA and MB was investigated using the
fermentation broth from the 1 m3 bioreactor. It is
well known that the presence of water will greatly inhibit the
esterification process (Dange et al., 2015 ). In order to achieve
high conversions of acetic and butyric acids, the removal of water from
the fermentation broth is necessary. In the study by Orjuela et
al ., a similar process was developed to recover fermentation-derived
succinic acid by first converting the succinic acid to diethyl succinate
through esterification with ethanol (Orjuela et al., 2013 ). By
partially concentrating the fermentation broth, sodium succinate was
precipitated and completely separated from water. The dried salt was
then mixed with ethanol and sulfuric acid for the esterification
reaction. In our case, however, acetate and butyrate salts cannot be
crystallized and precipitated from a concentrated broth due to their
solubility in PDO and glycerol. Moreover, acetic and butyric acids are
more easily evaporated along with water compared to succinic acid with a
high boiling point (236.15 oC), resulting in a high
acid loss in the water removal process (Kaeding et al., 2015 ;Zhang et al., 2021 ). To ensure a high
recovery yield of the acids and a complete water removal at the same
time, a NaOH solution of high concentration (50% w/w) was used to
adjust the pH of the ultra-filtrated fermentation broth to pH9 for
preventing the evaporation of acids by forming sodium salts of acetate
and butyrate. In addition, the glycerol content in the fermentation
broth was increased to 4% by adding crude glycerol to prevent
precipitation of the inorganic salts during the water evaporation
process. As shown in Fig. 7 (Content 3)
and Table 3 , owing to the dissolution
of adequate PDO and glycerol, no obvious salt crystallization and
precipitation were observed after a nearly complete evaporation of water
(rest water content <1%). Meanwhile, the recovery yield of
PDO, acetic acid and butyric acid in the residue were 96, 92 and 96%,
respectively. Surprisingly, only 3 g/L PDO but no acids were detected in
the distillated water (data not shown), indicating a complete retention
of sodium acetate and sodium butyrate in the residue. The loss of acids
might be only due to the dead volume and sampling from the rotary
evaporator. The distillated water with a small amount of PDO can be
directly reused in the fermentation process (Fig.7 ), leading to a theoretical yield of
100% for PDO, acetic and butyric acid in the water removal process.
To further convert the acetic acid and butyric acid in the dewatered
broth to their corresponding methyl esters, fresh anhydrous methanol was
added, and 37% concentrated HCl solution was used to acidify the
mixture for liberating the free organic acids from the sodium salts. As
a result, the medium salts and the NaCl formed by the acidification of
the sodium acid salts were precipitated due to the reduced solubility in
the methanol solution and subsequently removed by centrifugation
(Content 4 in Fig. 7 ), and the
remaining mixture containing methanol, PDO, glycerol and free acetic and
butyric acids was ready for the esterification reaction. Gao et
al . previously evaluated the salt crystallization effect by adding
ethanol to the concentrated fermentation broth of PDO
(Gao et al., 2007 ). It was reported that
the volume ratio of ethanol to the condensed broth should be over 2: 1,
and the pH of the mixture should be reduced to 2 for a complete
crystallization of the salts. Similarly, the mass ratio of methanol to
the dewatered broth in this study was set at 2.5: 1 and the pH was
reduced to 2 to ensure a complete removal of inorganic salts. In this
way, the molar ratios of methanol, PDO and glycerol to the total organic
acids (acetic acid + butyric acid) were 27: 1, 2.3: 1, and 1.1: 1,
respectively (Table 3 ). Considering the
fact that the organic acids can be also esterified with the
poly-hydroxyl PDO and glycerol, a high molar ratio of methanol to the
organic acids was used to greatly increase the esterification
selectivity to the targeted methyl esters.