Preliminary experiment: the influence of different nutrient supplementation on D-lactic acid production
The effects of supplementation with different nutrient sources (YE, meat extract (ME), peptone (PEP) and corn steep liquor (CSL)) on D-lactic acid fermentation by L. delbrueckii were investigated in model fermentation broth. The amount of each nutrient source added was equivalent to a nitrogen dose of 10 g/l YE (corresponding to 0.1% nitrogen). The D-lactic acid produced after 18 h fermentation was used for comparison.
The highest D-lactic acid concentration was yielded by YE, confirming that YE was the most appropriate supplement for D-lactate production using L. delbrueckii (data not shown). To determine the optimum YE dose for fermentation, 1, 3, 5, 10 and 20 g/l YE was individually added to the model medium (abbreviated YE1, YE3, YE5, YE10 and YE20), and the influence of YE amount on cell mass, D-lactic acid productivity and yield and byproduct formation was depicted in Fig. 1. A linear relationship between byproduct and YE dose was observed. The byproduct increased from 0.21 to 3.75 g/l as the initial YE dose varied from 1 to 20 g/l. However, the productivity, yield and cell mass changed logarithmically, the maximum D-lactate yield of 0.95 g/g did not increase at dose higher than YE10, and more YE yielded no obvious enhancement in cell mass and productivity. The observations indicated that high level of YE addition induced higher titer byproduct, which led to inhibition of cell growth and fermentative ability. On the other hand, excess byproduct might cause the additional cost of product recovery and downstream processing. Therefore, the optimal dose of YE for D-lactic acid fermentation by L. delbrueckii was 10 g/l.
Screening essential B vitamin for D-lactic acid production
B vitamins are integral nutrient for LAB growth and lactate fermentation. To understand the influence of individual B vitamins on D-lactic acid production by L. delbrueckii, screening experiments were conducted by means of excluding single B vitamins from the synthetic fermentation medium containing YE3. Nine B vitamins presented in YE were selected, and the individual supplementation amount was equivalent to that of YE10. Moreover, fermentation in medium comprising YE3, YE10 and YE3 coupled with full B vitamins was performed as a control. As Fig. 2 defined, the maximum and minimum cell mass and D-lactic acid titer was obtained from the control medium of YE10 and YE3, respectively. By supplementing full B vitamins in YE3 medium, the cell mass and D-lactate titer were significantly improved to levels close to those of YE10, implying the combined supplementation of YE3 and B vitamins could fulfill the nutritional requirement of L. delbrueckii, consequently overcoming the inhibition of cell growth and fermentation capacities caused by YE reduction, and maintaining the cell mass and D-lactic acid production at a high level. The elimination of vitamins B6, B7, B8, B9 and B12 gave no obvious influence on cell growth and fermentation abilities. The initial glucose of 50.6 g/l was almost consumed after 18 h fermentation with the maximum cell mass of 5.23, 5.17, 5.14 and 5.03 g/l and D-lactic acid titer of 45.5, 45.1, 43.9 and 42.8 g/l, close to that of control medium supplemented with YE3 and full B vitamins (5.27 and 46.3 g/l). In contrast in the case of vitamins B1, B2, B3 and B5 absence, the cell growth was similar to that of control medium with YE3, presenting a significant inhibitory profile. Accordingly, glucose consumption was severely delayed, over 20 g/l glucose left after 18 h fermentation, leading to a considerable reduction in D-lactic acid titers (28.1, 25.3, 26.0 and 23.1 g/l). The observations revealed that VB1, VB2, VB3 and VB5 were essential for L. delbrueckii growth and fermentation, which coincided with the published report showing that the four B vitamins played important roles in the regulatory mechanisms governing LAB growth and fermentation abilities. Klotz et al.  reported that VB5 was a component of coenzyme A (CoA) and acyl-carrier-protein (CAP) involved in protein and fatty acid synthesis of LAB. VB2 and VB3 were the precursors of cofactors such as FAD and NAD. NADH directly impacts lactic acid production as a cofactor of lactate dehydrogenase (LDH) [37,38]. In addition, some studies figured out that B vitamin supplementation enhanced the activities of key enzymes of LA biosynthetic pathway in terms of phosphofructokinase (PFK) and L(+)-LDH. VB1 promoted glycolysis rate while VB2 accelerated electron transfer, thus increasing the metabolic flux of EMP pathway [38, 39].
Effect of trace elements on L. delbrueckii D-lactic acid production
It is already known that trace elements have positive effect on lactate fermentation by promoting the cell growth of LAB and regulating the activities of the key enzymes involved in metabolism . To elucidate the influence of trace elements in terms of Mn2+, Mg2+, P3+ and Fe3+ on L. delbrueckii, the individual constituents were excluded one-by-one in synthetic medium with four B vitamin and YE3 addition. D-lactate accumulation and optical purity had no fluctuations in the absence of Mn2+, P3+ and Fe3+, but significantly decreased in the absence of Mg2+, indicating that Mg2+ was necessary for D-lactate production by L. delbrueckii (Table 1). Moreover, the optimum Mg2+ concentration for fermentation was also studied. The maximum D-lactate titer of 47.9 g/l and optical purity of 99.5% were achieved under Mg2+ concentration of 38.5 mg/l. Nevertheless, the relatively high dose of Mg2+ decreased the lactate titer, which might be contributed to the inhibitory influence on cell growth.
Optimization of vitamin supplementation in biomass hydrolysate
To determine the optimal dose of four B vitamins for D-lactic acid production by L. delbrueckii, fermentations were carried out in RSH medium with YE3 and varied enrichment factors of B vitamins (α = 0.5, 1.0, 1.5 and 2.0), respectively (Fig. 3 and Table 2). Fermentation in YE3 medium (α = 0) was conducted as the performance reference. The results showed that the increasing amount of B vitamins continuously stimulated the growth of L. delbrueckii, finally reaching the maximum cell mass of 5.61 g/l at α 2.0. Glucose was unable to be completely converted into lactate at α 0.5 after 18 h fermentation, resulting in a poor concentration of 30.7 g/l. When B vitamin addition was improved to α 1.0, the glucose was fully depleted, achieving the maximum lactate titer, productivity, yield and optical purity of 47.6 g/l, 2.64 g/l/h, 0.95 g/g and 99.5%, respectively. The lactate concentration was enhanced by 134.5% in comparison to that of control medium. Nevertheless, additional B vitamin supplementation led to slight decrease in D-lactate concentration and optical purity. Singhvi et al.  reported that lactate dehydrogenase expression in lactic acid producing bacteria was influenced by nitrogen sources, resulting in the enhancement of D-lactic acid production. Therefore, it was speculated that the increased supplementation of B vitamin in medium might cause the variation of lactate dehydrogenase involved in the isomerization reaction, consequently improving the amount of L-lactic acid during fermentation. However, the hypothesis needed further investigation. In conclusion, α 1.0 was considered as the optimal amount of B vitamin for the subsequent experiments.
Membrane integrated continuous fermentation
Continuous fermentation with and without cell recycling was conducted by RSH using four B vitamin together with YE3 as nutrient sources. To assess the optimal dilution rate for fermentation, the experiments were performed under dilution rates range of 0.1 h−1 to 0.7 h−1. The concentration of cell mass, residual sugar, D-lactic acid and productivity as a function of dilution rate were depicted in Fig. 4. In the case of MICF system (Fig 4B), the initial glucose was thoroughly converted to D-lactic acid under the dilution rate 0.3 h−1, reaching D-lactate titer of 46.9 g/l. The cell mass progressively increased with the change of dilution rate and the cell density of 13.45 g/l was attained at the dilution rate of 0.7 h−1.The maximum productivity of 18.61 g/l/h was achieved when the dilution rate was set at 0.4 h−1, and then decreased with the increased dilution rate. In the case of fermentation without cell-recycle, the cell mass presented a liner decreasing trend with the variation of dilution rate, suggesting serious cell loss during fermentation (Fig 4A). Accordingly, more residual sugars and a decrease in lactate concentration were observed. The maximum cell density and productivity were 4.98 g/l and 9.43 g/l/h, which were almost half of those obtained from MICF system. The observations indicated that the specific growth rate of cells was equal to the dilution rate during the lactate fermentation with cell recycling. In comparison to a free cell continuous fermentation, the membrane integrated fermentation effectively prevented cell loss via medium exchange and maintained cells growth at its maximum rate, thereby greatly enhancing the D-lactic acid concentration and productivity. Moreover, although the increasing dilution rate promoted the progressive cell growth due to the rapid delivery of fresh medium, but the more unconsumed sugar detected in fermenter because the fermentation broth had insufficient time to ferment. Overall, taking account the few residual sugars, high cell density and D-lactic acid concentration and productivity, a dilution rate of 0.4 h−1 was considered applicable for subsequent experiments.
In addition, the optimal nutrient conditions determined in batch fermentation were proven to be suitable for MICF system using RSH as substrate. With the supplementation of YE3 and four B vitamin, the D-lactic acid concentration attained at dilution rate of 0.4 h−1 was 46.4 g/l, slightly higher than that obtained in synthetic medium with YE10 (45.5 g/l) under batch fermentation mode.
A 350 h continuous fermentation with cell recycling was conducted by gradually reducing YE from initial 3 g/l (phase I) to 0.5 g/l (phase III). As Fig. 5 exhibited, the cell mass, D-lactate concentration and productivity presented a sharp increase during the first 18 h, then D-lactate production and productivity entered a steady state while the cell mass increased until fermentation was completed. Reducing the YE content in mixed nutrient source at different phases gave no influence on cell growth and lactate accumulation, the average D-lactate titer and productivity obtained under the three phases were almost identical, at approximately 46.6 g/l and 18.56 g/l/h, respectively. Such stable fermentation performance further demonstrated the positive joint effect of B vitamin supplementation and MICF on YE reduction and improvement in fermentation efficiency. The continuous fermentation with cell recycling under appropriate dilution rate could greatly reduce the cell washout and maintain the high cell density by B vitamin supplementation during the long term operation, thereby, the constant lactate titer and productivity could be achieved even with the continuous reduction of YE. In addition, no signs membrane fouling was observed in our fermentation system. The concentration of main byproducts in terms of acetic acid and ethanol examined after fermentation was < 0.7 g/l. The low concentration of byproducts led to a high chemical purity of D-lactate.
Table 3 summarized the recently published works on microbial D-lactic acid production from lignocellulosic wastes. Except for this study, all other reports were conducted in batch or fed-batch fermentation mode with different microbes. Regarding the nutrient sources, some agricultural wastes such as barley extract, whey protein hydrolysate, ME, soybean meal, cottonseed meal were used as single nutrients or supplemented with YE or YE and PEP. It was notable that the fermentation efficiencies in these studies were unsatisfied. The highest productivity value of 2.80 g/l/h was reported in the literature of fermentation from pulp mill residue using a mixed nutrient, which was far lower than that of our study of 18.56 g/l/h. Although various studies had been performed using MICF to improve L-lactic acid productivity, its application for D-lactic acid fermentation from lignocellulosic biomass was little. Hence, our experimental results provided important reference information for developing an economically efficient process for D-lactic acid production. Furthermore, the enantiomeric purity of D-lactate obtained in our work was 99.5%, which was preferred since high enantiomeric purities of more than 99.0% were needed to form heat-resistant PLA.
Finally, the YE content in our mixed nutrient source was 3 g/l in batch fermentation and an average of 1.5 g/l in continuous fermentation, far less than other studies, in which at least 5 g/l YE was required. Additionally, the supplementation amount of four B vitamins was minor, 13.8 mg/l in total. The unit prices of YE, VB1, VB2, VB3 and VB5 were $3.5, $36.6, $10.9, $9.1 and $9.3 per kilogram (≥99% purity, www. 1688.com, China). Under batch fermentation, when YE10 was used as nutrient source, 222 kg YE was necessary to produce one ton of D-lactic acid product, adding $777.0 in cost. By substitution of YE10 with YE3 and four B vitamins, the YE cost could be reduced to $233.3; together with the cost of $3.5 of four B vitamins, the total nutrient cost could become $236.8, yielding a saving of 69.5%. During 350 h continuous fermentation with a membrane, the YE content was further reduced to 1 g/l and 0.5 g/l at phase II and phase III, respectively, which resulted in nutrient cost of $109.0, saving up to 86.0%.The results clearly defined the suggested strategy of four B vitamins supplementation combined with MICF system could significantly reduce the nutrient cost in fermentation by L. delbrueckii while also greatly improving the fermentation efficiency.
In conclusion, the research of using inexpensive nutrient sources to substitute YE provided a new insight into economical D-lactic acid production from lignocellulosic feedstock. However, there were still challenges required to be addressed to improve the process. One of the focuses for future work was to develop an effective pretreatment method to obtain the inherent B vitamin in lignocellulosic feedstocks, which would reduce the use of commercial B vitamin and thus further reducing the nutrient cost. The other important aspect was the selection of new D-lactic acid producing bacterial strains with the characteristics of highly productive, substrate and lactic acid resistant as well as capable of consuming all kinds of carbon sources presented in cheap feedstocks. Additionally, the improvement of lactic acid purification process e.g. simplifying the operation steps and eliminating the generation of byproduct to achieve production cost reduction was also expected.