3.3.3 Esterification conversion of organic acids and rectification of highly pure PDO from fermentation broth
The esterification between the organic acids and methanol in the desalinated fermentation broth was performed under the optimal conditions selected above: reaction temperature of 70oC, 10% catalyst loading and 2.5 h reaction time. As shown in Table 3 , 4.52% MA and 1.72% MB were detected in the esterified fermentation broth. This indicates that 97.3% acetic acid and 92.9% butyric acid were successfully converted to their corresponding methyl esters. The slightly lower conversion of acids compared to that in the synthetic broth could be due to the presence of a small amount of water (1.52%) in the desalinated broth before the esterification reaction. Since the conversion of both acetic and butyric acids can be maintained over 93% with an initial water content lower than 2.5% (Fig5D ), considering the process efficiency, water absorption treatment by molecular sieves or silica gel was not applied before the esterification. Followed by a simple vacuum distillation operated at 60oC and 200 mbar, methanol, MA and MB were completely evaporated from the fermentation broth, resulting in a colorless methanol solution containing 5.87% MA and 2.27% MB (Content 5 in Fig. 7 ). The total yield of the recovered acids in their methyl ester form was therefore more than 88%. These results demonstrate that the organic acids can be separated from PDO and extracted from the fermentation broth simply and efficiently by integration of the esterification conversion process. Furthermore, the content of PDO and glycerol in the fermentation broth was basically unchanged before and after the esterification and methanol recycling process (Table3 ), indicating that very few PDO or glycerol ester impurities were formed. On the contrary, more than 8% PDO was reported to be esterified and lost during the direct separation of PDO and free organic acids by vacuum distillation in our previous study (Zhang et al., 2021 ). Although the lost PDO could be recovered by a subsequent alkaline hydrolysis treatment, the elimination of a large amount of PDO ester impurities required more consumption of NaOH (50g NaOH /kg PDO) and resulted in a sodium organic acids content of more than 4% in the residual from the final PDO distillation step. This makes the residual glycerol impossible to be reused in the fermentation process due to the growth inhibition of organic acids to C. pasteurianum(Sabra et al., 2016 ). Therefore, because of the significantly reduced formation of PDO and glycerol ester impurities, the methyl esterification strategy also improved the process economy and efficiency for the final rectification of highly pure PDO. After neutralization of the excessive HCl by adding 50% NaOH solution, and active carbon treatment to polish odor and color, the residual was subjected to a rotary evaporator for the vacuum distillation of PDO. As a result, a colorless and odorless distillate product containing 96% PDO and 3% glycerol (Content 8 in Fig.7 ) was successfully obtained, with a total PDO recovery yield of 77% for the whole DSP. Organic acids were hardly detectable in the final product (Table3 ), indicating that the purified PDO has a high quality and could be directly used as an effective moisturizer in cosmetic manufacturing (Becker and Wittmann, 2015 ). The residual glycerol from the PDO distillation process still contained 10% PDO and a small amount of organic acids (0.31% acetic acid and 0.28% butyric acid). It is highly possible that these acids were released from the hydrolysis of the trace amount of PDO or glycerol esters in the neutralization step. Since the content of organic acids was very low, the residual glycerol from the new DSP is more promising to be directly reused in fermentation. In fact, if the water from the concentration step (4% PDO lost), and the residual glycerol from the final distillation step (5.5% PDO lost) can be both recycled in the fermentation, the total PDO recovery yield from the DSP would increase to 83%, which is economically more competitive.
Based on the successful lab-scale experimental results, a process flow diagram for the industrial-scale co-production of PDO and organic acid esters from the crude glycerol fermentation broth is presented in Fig.7 . Compared to the laboratory process, the most significant changes in the industrial process are listed as follow: 1. Two sets of triple effect evaporators are used for the removal of water and recycling of methanol to minimize the energy consumption; 2. The desalinated fermentation broth in methanol solution is fed to a fix-bed reactor packed with Amberlyst-15 for the continuous esterification conversion of organic acids, which is expected to reduce the reaction time and facilitate the reuse of catalyst (Salvi et al., 2018 ). Considering possible need of regeneration or replacement of the catalyst, the feed can be switched to a second fixed-bed reactor in series, and the regeneration of catalyst in the first fixed-bed reactor can be carried out simultaneously; 3. The recycled methanol is fed to a molecular sieves column for water absorption, and subsequently reused for the crystallization and esterification process. It should be noted that the content of methyl esters in the recycled methanol was less than 8% (Table 3 ). Accumulation of methyl esters by multiple reuse of the methanol solution in the esterification reaction with organic acids could be beneficial for the separation of methyl esters from methanol. Since the water content in the recycled methanol will be increased to over 4% due to the esterification reaction (data not shown), in order to ensure high acid conversion and avoid excessive use of energy, the absorption of water from the recycled methanol involving molecular sieves is therefore proposed; 4. The PDO containing stream from the polishing purification can be fed to different PDO rectification process according to product applications. For application in cosmetics, where glycerol and PDO are both used as moisturizers in the formula, the stream can be fed to a short path evaporator for the continuous and efficient distillation of PDO. In order to produce fiber grade PDO for PTT synthesis, the stream can be fed to a distillation column for a complete separation of PDO and glycerol. Apart from the above-mentioned designs for the industrial-scale production, it is also worth mentioning that other alcohols such as ethanol and 1-propanol can also be used in our integrated process for producing a variety of high value-added organic acid esters for different applications. Generally, the novel and complete industrial process presented here is able to exploit a variety of metabolic products from an efficient crude glycerol fermentation for the co-production of PDO and various organic acid esters. The process itself is flexible and environmental friendly without producing any waste water, giving it great potential for improving the economy and efficiency of industrial PDO bio-production.