Biocycle Fermentation Based on Lactic Acid Bacteria and Yeast for the Production of Natural Ethyl Lactate.

Ethyl lactate is widely used in food and pharmaceutical industries, but the complexity of the synthesis process, in particular, involving the addition of organic solvents, hinders its application. Here, we report a natural green strategy to produce ethyl lactate by exploiting the synergistic fermentation of lactic acid bacteria and ester-producing microbes using biomass as a substrate. Interestingly, it is worth noting that the conjugate fermentation has a higher ethyl lactate yield (3.05 g/L) compared to the mixed fermentation (1.32 g/L). The ester production capacity was increased by 2.3 times. These entire processes require only the addition of biomass without introducing any organic solvent. In addition, the obtained catalytic esterification system can reuse the ester-producing microbes by simple centrifugation and maintain over seven cycles of catalysis while it retained a high activity. We firmly believe that the results of this study will provide new ideas for achieving sustainable green production of natural ethyl lactate.

Introduction

Driven by environmental concerns and concept of sustainability, an increasing demand to use biorenewable materials instead of petroleum-based feedstocks for chemical production has been noticed.[1] As is well known, ethyl lactate is an agrochemical solvent defined as generally recognized as safe and due to its low toxicity it has been approved by Food and Drug Administration (FDA) and European Food Safety Authority (EFSA) as a pharmaceutical ingredient and food additives.[1−4] At present, the research on ethyl lactate as a green solvent in the food industry has become a hot spot because of its characteristics, such as ease of recycle and reuse, no toxicity, and harmlessness.[2] It has been reported that ethyl lactate has been used as a green solvent to extract sterols, astaxanthin, and other food nutrition additives from plants.[5,6] In addition, ethyl lactate can also be dissolved/dispersed in various pharmaceutical active excipients in the applied pharmaceutical industry without destroying the pharmacological activity of the active ingredient.[1,7]

Despite these numerous applications, it still remains a considerable security challenge to apply ethyl lactate in food and pharmaceutical industry due to the limitations of existing technologies and the residual organic solvents. As a common technique, acid including sulfuric acid, phosphoric acid, and anhydrous hydrogen chloride, as a catalyst for the homogeneous catalysis, have been discovered or engineered to synthesize ethyl lactate.[8,9] This technology, nevertheless, suffers from equipment corrosion and high operational cost due to the inherent shortcomings of liquids (their relatively discharge of acid-containing waste and the occurrence of a higher proportion of side reactions).[1] As an alternative technology, some researchers have used zeolites, ion-exchange resins, and simulated moving bed membrane reactor (PermSMBR) to immobilize lipases for the preparation of recyclable catalytic reaction systems, those strategies are critical for green sustainable reaction process because of their inbuilt advantages such as noncorrosion property and less environmental impacts.[10−13] Despite impressive advances, process complexity and economic costs have increased significantly due to the extra addition of the carrier.[14] In addition, immobilized enzymes are generally applied to heterogeneous catalysis, where the introduction of organic solvents is bound to pose a potential hazard to the biosafety of the product.[15,16] Therefore, it is of great importance to develop efficient strategies with simple, green, and recyclable superiority for producing ethyl lactate.

However, a great challenge is the design and implementation of completely green products and processes. There is no systematic and reliable way to ensure that the chemistry implemented is green, since the number of chemical synthesis pathways is vast, and in general it is only possible to verify if a proposed manufacturing process is “greener” than other alternatives.[1] Biocatalysis is a relatively green and safe method because it is derived from microbial fermentation of biomass (such as starch) and has advantages in terms of reducing costs and simplifying operation.[17] Our research aims to develop a green biological reaction process for fermentation via lactic acid bacteria and ester-producing microbes. The lactic acid bacteria produce lactic acid by fermentation using rice saccharification solution as a raw material, and an esterase produced by the ester-producing microbes itself catalyzes the conversion of lactic acid and alcohol produced by yeast into ethyl lactate. The entire process is safe and simple as well as does not involve the addition of any organic solvents. Furthermore, the recycling of the ester-producing yeast can effectively reduce the cost and meet the production needs.

Herein, a biocatalytic production of ethyl lactate was developed to solve the complex process and biosafety issues encountered in the production of ethyl lactate. To this end, we first screened lactobacilli and ester-producing yeasts that produce lactic acid and ethyl lactate. Then, the ester-producing ability of the mixed fermentation (mixing two kinds of bacteria) and the conjugate fermentation (using lactic acid fermentation broth as a source of lactic acid instead of directly adding lactic acid bacteria) was examined. A fermentation method with a high yield of ester is selected for cyclic fermentation to develop a safe and economical production method. To the best of our knowledge, this work is the first attempt on the biofermentation and solvent-free production of ethyl lactate, which is characterized by simplicity, greenness, and recyclability. The excellent performance makes us believe that this system will attract widespread interest from both fundamental and industrial researchers because it unites some key advantages in terms of green and sustainable chemistry.

Results and Discussion

For the biosynthesis of ethyl lactate, one-pot and a two-step procedure, as depicted in Figure , was used. Considering that the synthesis of ethyl lactate is based on lactic acid and ethanol as substrate, we choose lactic acid bacteria to ferment biomass to produce lactic acid and ester-producing microbes (ester-producing yeast) to produce alcohol. On this basis, the esterase secreted by the ester-producing microbes is used to convert alcohol and lactic acid into ethyl lactate. As can be seen from part a of Figure , the lactic acid bacteria and yeast cocultured the transformed biomass to ethyl lactate. For another, a two-step procedure involved first the transformation of biomass into lactic acid by lactic acid bacteria; the second step is to add lactic acid to the ester-producing microbe fermentation broth to produce ethyl lactate (Figure b). After fermentation, the ethyl lactate was separate by simple centrifugation. Also, the recovered microbe (precipitation after centrifugation) is used for the next fermentation.

Figure 1

Schematic illustration of mixed fermentation (a) and conjugated fermentation (b) to produce ester.

Evaluation of Lactic Acid Bacteria for Lactic Acid Production

It is worth mentioning that lactic acid, as a precursor for the synthesis of ethyl lactate, was obtained by the fermentation of lactic acid bacteria. A satisfactory titer of lactic acid was obtained via screening the acidogenic capacity of four different lactobacilli (Lactobacillus fermentum (LF), Lactobacillus casei (LC), Lactobacillus rhamnosus (LR), and Lactobacillus plantarum (LP)). As shown in Figure , the four lactic acid bacteria are all rod-shaped Gram-positive bacteria (Figure a–d, Gram staining micrographs), which is consistent with the results of previous researchers.[18] After 72 h of incubation for lactic acid bacteria, the acid production of the four lactic acid bacteria reached a steady level. Notably, the lactic acid production was greatly improved with further extending of the culture time, which may be due to the saturation of the acid production inhibiting the activity of lactic acid bacteria and enzymes. Interestingly, LR had the highest acid yield (29.01 g/L) after being cultured for 72 h (Figure f). Comparison of the yields with previous studies (in a similar system) confirmed that the production of lactic acid was satisfactory.[19] In addition, the larger calcium lysate further demonstrates that LR has a higher acid production capacity than the other three lactic acid bacteria (Figure a1–a4). This may be due to the higher proliferative capacity of L. rhamnosus during culture (Figure e, maximum bacterial OD600 value of 6.8%). Thus, LR was selected as the candidate acid-producing bacteria, and the culture time was 72 h, taking into account the simplification of the process and the economic cost.

Figure 2

(a–d) Microscopic examination of LF (a), LC (b), LR (c), and LP (d). OD600 value of four lactic acid bacteria as a function of time. (e) Amount of acid produced by four lactic acid bacteria at different times (f).

Evaluation of Ester-Producing Yeast for Ethyl Lactate Production

It is generally accepted that the formation of ethyl lactate consists of two parts: (1) secretion of esterase by esterogenic microorganisms and (2) esterification of alcohol and lactate to ethyl lactate.[20] Therefore, an effective ester-producing strain is the key to obtaining high-yield ethyl lactate (a schematic representation of ethyl lactate production is shown in Figure h). Herein, we selected four common ester-producing strains (Saccharomyces cerevisiae (SC), Wickerhamomyces anomalus (WA), Candida antarctic (AC), and Monacus purpureus (AP)) to produce ethyl lactate. Optical microscopy image showed that all yeast cells presented elliptical (Figure a–c), and the buds were clearly visible.[21] As for the mycelium of AP, they basically have the same size.[22] Moreover, its branches and septum were obvious (Figure d). As shown in Figure e,f, the increase of the lactic acid content in the system significantly inhibited the proliferation of the ester-producing microbes and the production of ethanol, since led to the deviation of optimum pH of ester-producing microbes (optimal pH is generally neutral) and the reduction of esterase activity. It must also be mentioned that WA has a higher ethanol yield (Figure f) and dry cell weight (Figure e) than other yeasts (SC, AC, and AP). At 3% lactic acid concentration, the remarkable ester-producing ability of WA is as high as 2.73 g/L (Figure g, gas chromatography-mass spectrometer (GC-MS) further identified the product after fermentation; Figure S1), higher cell dry weight (3.2 g/L; Figure e) and ethanol yield (8.6 g/L, Figure f) further indicates that the WA has higher lactic acid tolerance, which provides the possibility of its high production of ethyl lactate. Therefore, we selected WA as an ester-producing strain.

Figure 3

(a–d) Optical microscopy image of SC (a), WA (b), AC (c), and AP (d). Dry cell weight (e) and ethanol yield (f) and ethyl lactate yield (g) of the yeast at different lactic acid concentrations, with culturing at 30 °C for 120 h. A schematic representation of ethyl lactate production (h).

Optimization of Ester Production Process

For the production of green ethyl lactate with safety in fermentation, here we proposed to use a one-pot way, that is, mixed fermentation (mixed fermentation of lactic acid bacteria and yeast, lactic acid from the production of lactic acid bacteria, without additional lactic acid) to produce ethyl lactate. As shown in Figure a, the amount of yeast increased quickly at the initial stage, gradually slowed down with further fermentation time, and finally became steady. Most interestingly, the amount of yeast in mixed fermentation (lactic acid bacteria and yeast) was basically the same as that in yeast fermentation (no lactic acid bacteria added). After 72 h of culture, the proliferation of the WA reached its maximum and became stable (9.8 × 107 cfu/mL). In addition, optical microscopy image showed that yeast grew well and maintained a highly viable cell rate during fermentation. In the early stage of culture, there were fewer number of cells, and the cells exhibited budding reproduction (inset image in Figure a). With further increase in incubation time up to 72 h, the number of cells increased rapidly and the number of viable cells was maintained (higher transparent yeast and few blue yeast). As the incubation time increased, the number of dead cells significantly increased. This is consistent with the observation from the results of dry cell weight. These results indicate that the addition of lactic acid bacteria does not significantly affect yeast reproduction.

Figure 4

(a) Effect of lactic acid bacteria on yeast reproduction: transparent is the live yeast (marked by the yellow arrow), the blue appearance is the dead yeast stained with the methylene reagent (marked by the red arrow), and the linear form in the inset is the lactic acid bacteria. (b) Effect of different temperatures on the fermentation of mixed bacteria.

To explore the ester production efficiency of mixed fermentation, WA and LR (highest lactate producers) were mixed (WA/LR, a viable cell count ratio of 1:1) at different temperature (26 to 38 °C). Figure b shows that the system had the largest ester yield (1.32 g/L) for 72 h at 30°C. Moreover, higher and lower culture temperatures resulted in a significant decrease in ester production. In particular, as the culture temperature increased to 38 °C, the amount of ethyl lactate produced was rapidly decreased (0.33 g/L), which accounted for only 1/4 of the ester yield at 30 °C. Although we optimized the optimum ethyl lactate yield at 30 °C, this yield is still unsatisfactory. To further explore the reasons for the low yield, we investigated the acid production of lactic acid bacteria at different temperatures (from 26 to 38 °C). As shown in Figure S2 (see the Supporting Information), the acid production of lactic acid bacteria (culture temperature: 26°C) is only about 16% of the acid production at 38 °C, and its reproductive capacity (yield of lactic acid, 4.67 g/L) is less than 20% at 38 °C (yield of lactic acid, 29.01 g/L). In addition, it is generally considered that the optimum temperature of the yeast is lower (less than 37 °C). Therefore, we speculate that the difference in the optimal temperature between WA and LR may be result in the low ester titer.

Given that the temperature difference in the mixed fermentation results in low ester production, using conjugated fermentation may be an effective means to solve the problem. That is, lactic acid is first produced by using sole LR and then the fermentation broth from which the lactic acid bacteria are removed is added to the yeast culture solution to produce ethyl lactate. Figure a clarifies the esterification kinetics of WA (the culture temperature was 30 °C). Over time (0–1 days), the content of ethyl lactate increased slowly because the fermentation broth contained less ester-producing yeast at this time (inset image of Figure a), which was in the logarithmic phase of reproduction. After 1 day of culture, the amount of ethyl lactate in the culture solution rose sharply and reached a maximum value (2.94 g/L) at 3 days. Conversely, by prolonging the incubation time, the total amount of ethyl lactate was slightly reduced, which was due to the decline in the viability of the ester-producing yeast (Figure a). In addition, the ethyl lactate consumed by itself was larger than the amount of ester produced. As shown in Figure b, the yeast had a higher ester yield (3.05 g / L, for 3 days) at 26 °C; besides higher and lower culture temperatures, the yeast had a lower ester yield. Compared with mixed fermentation, conjugated fermentation is carried out at the optimum temperature of yeast, which is beneficial for the increase in esterase activity and can significantly increase the total ester content of the fermentation broth, thereby obtaining a high ethyl lactate of the natural fermentation source.

Figure 5

(a) Kinetics of ester production by conjugated fermentation: transparent is the live yeast (marked by the yellow arrow) and the blue appearance is the dead yeast stained with the methylene reagent (marked by the red arrow). (b) Effect of different fermentation temperatures on ester production.

Cyclic Stability of Biofermentation

Considering that biocatalysts with good cycle stability further reduce the cost of bioconversion, WA isolated by centrifugation was used to catalyze the synthesis of ethyl lactate in a continuous batch. It is worth noting that ester-producing yeast maintains a high survival rate (more than 50%) and a complete morphology during the entire fermentation cycle (Figure a). Most surprisingly, WA retained more than 95% of its activity after seven biotransformation cycles (examined after reactivation) (Figure b), showing high reusability. This indicates that the whole process has little effect on yeast cells, which is the key to the whole study.

Figure 6

(a) Microscopy of ester-producing bacteria during recycling (first (a1), third (a2), and seventh (a3) cycle after activation; first (b1), third (b2), and seventh (b3) cycle after the cycle). (b) Cell viability of ester-producing bacteria after activation. Recycling results of the esterification of ethyl lactate by ester-producing bacteria in aqueous phase.

Conclusions

In summary, we have proposed a viable and relatively simple method for producing ethyl lactate by fermentation from ester-producing yeast and lactic acid bacteria. The strain obtained after simple centrifugation can be recycled to the next fermentation, which produces a green sustainable catalytic reaction and product separation in the reaction vessel. This process saves resources for clean production and catalytic recycling of ethyl lactate. Its circulation highlights more than seven cycles. With this successful ester bacteria separation and recycling system, the overall efficiency of the chemical process may be significantly improved and the work-up method may be simplified. This biocatalysis platform could be readily extended to other catalytic esterification systems, such as the production of ethyl acetate, ethyl hexanoate, and even ethyl butyrate, the essence of which is the replacement of acidogenic strains. In addition, the key advantage of this strategy is that it is a green process without any organic solvent, which provides the possibility of the catalytic production of products such as food and medicines, in accordance with the concept of green and sustainable chemistry. Therefore, this strategy may open interesting avenues for establishing green and sustainable platforms with synthetic natural esters.

Experimental Section

Materials

L. fermentum (LF), Lactobacillus delbrueckii spp. (LD), L. rhamnosus (LR), and L. plantarum (LP) were gifts from South China Agricultural University (Guangdong, China). S. cerevisiae (SC, CGMCC2.3853), Wickerhamomyces anomalus (WA, CGMCC2.470), and Candida antarctica (CA, CGMCC2.3605) were obtained from China General Microbiological Culture Collection Center (Beijing, China). Monascus purpureus (MP, GIM3.239) was purchased from Guangdong Institute of Microbiology (Guangdong, China).

Growth Conditions

LF, LC, LR, and LP were respectively inoculated into 10 mL of rice saccharification medium at 2% (v/v) and cultured in at 37 °C in an incubator. At the intervals of 12 h, the samples were taken to determine the OD600 and lactic acid yield.[23] The growth curve and lactic acid concentration curve of the lactic acid bacteria were plotted to compare the growth of four lactic acid bacteria and the yield of lactic acid. In addition, after culturing for 3 days, 100 μL of the fermentation broth diluted 6 times was applied to a rice saccharification solution plate to which 2% calcium carbonate was added and cultured in a 37 °C incubator until the single bacteria colony was observed. The colony trait, such as color, edge, surface morphology, etc., and the size of dissolved calcium circle were observed.[24] Then, a smear was prepared and the morphology of the lactic acid bacteria was observed using an optical microscope (Olympus bx50, Japan).

SC, WA, CA, and MP were inoculated in rice saccharification solution (12° Bx) containing different concentrations of lactic acid (0, 1, 2, 3, and 4%). The cells were cultured at a constant temperature incubator at 30 °C for 5 days, and the dry weight of the cells and the total ethyl lactate content were measured to compare the growth of the four ester-producing strains under different lactic acid concentrations and the production of ethyl lactate.[25] Methylene blue staining was used to count the alive cells and, according to the count result, to draw a cellular growth curve. Specifically, a drop of normal saline was added to a clean glass slide. A single colony was then picked with an inoculation ring and applied to the center of normal saline about 1 cm2, uniformly coated, and stained with methylene blue. The agent was stained. After the dyeing was completed, the yeast morphology was observed by an optical microscope (magnified, 40×). Finally, the stained yeast was placed on a hemocytometer to count the number of cells and the viable cell rate.

Mixed Fermentation

The activated ester-producing strain and lactic acid bacteria were inoculated at a concentration (in a 1:1 ratio of viable cell count) in a rice saccharification solution and cultured in a constant temperature incubator at 26, 30, 34, and 38 °C for 5 days. The lactic acid content and ethyl lactate content in the fermentation broth were measured every 12 h.

Conjugated Fermentation

The ester-producing yeast was inoculated into rice saccharification solution (sugar degree 12° Bx, pH 6.0) at a ratio of 4 and 3% lactic acid was added (extracted from fermentation broth, which was concentrated by vacuum freeze-dryer (Ningbo Xinzhi Biotechnology Co., Ltd., China) after removing lactic acid bacteria by centrifugation). The final volume of the solution was kept at 100 mL. The cells were cultured in a constant temperature incubator at 26, 30, 34, and 38 °C for 5 days, and the content of lactic acid and ethyl lactate in the fermentation broth was measured every 12 h.

Ester-Producing Microbes Recycling

To evaluate the reusability of the biocatalyst, the ester-producing microbes were isolated by centrifugation at 6000g for 5 min after each batch of reaction. Then, the recovered ester-producing microbes were used for the new reaction under the same conditions. The operational stability in each batch cycle was characterized by measuring the relative ethyl lactate yield compared to the first reaction.

High-Performance Liquid Chromatography (HPLC) Analysis

The synthesized ethyl lactate was analyzed in the following method.[26] A sample (2 μL) was injected onto the column (COSMOSIL 5 C 18 -MS-II, 4.6 × 150 mm2; Nacalai Tesque, Kyoto, Japan) and eluted with 20% (v/v) methanol containing 0.08% trifluoroacetic acid at 40 °C. The concentration of ethyl lactate was determined from the chromatographic data monitored at UV 210 nm processed by LC Solution software (Shimadzu).

Lactic acid concentration was determined using high-performance liquid chromatography (HPLC).[16] A Shimadzu LC-20AD liquid chromatograph (Shimadzu, U.K.) equipped with a Shimadzu SPD-20A UV–vis detector, a Shimadzu SIL-20A HT auto sampler, and a CTO-10AS VP column oven were used. The samples were eluted with 0.005 N H2SO4 at a flow rate of 0.6 mL/min from an organic acid analysis column (300 × 7.8 mm2 inner diameter, Rezex-ROA organic acid column, Phenomenex Inc., U.K.) at 60 °C. Bioculture medium samples were centrifuged and filtered as described above. Thirty microliters were injected into the HPLC and the concentration of lactic acid was determined by interpolating from a previously established lactic acid calibration curve. The coefficient of variation for four samples was 0.9% for a concentration level of 0.5 M lactic acid.

Gas Chromatography Analysis

Gas chromatography (GC) was employed for the determination of ethanol concentration in the samples from four ester strain fermentations. Shimadzu GC-2014 (Shimadzu, U.K.) equipped with a flame ionization detector and a 30 m long Zebron ZB-5 capillary column (Phenomenex, U.K.) with 0.25 mm internal diameter was used. The mobile phase used was nitrogen, while the stationary phase of the column was 5% phenyl and 95% dimethylpolysiloxane. Aqueous samples were centrifuged for 5 min at 13 000g, and the supernatant was filtered through 0.2 μm filters. Ethanol was extracted into hexane by vigorous vortexing 1 mL of the filtered sample with 2 mL of hexane for 1 min at room temperature. One microliter of the extract was injected into the GC, and the temperature of the column was kept constant at 40 °C for 3 min. The concentration of ethanol was calculated by interpolating from a previously established ethanol calibration curve.

Statistical Analysis

Each test was conducted in triplicate. Analyses of variance for all the treatments were carried out by Duncan’s multiple-range test (p < 0.05) using SPSS (SPSS Inc., Chicago, IL, version 13.0).