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.