Efficient Production Of L-lactic Acid Research Articles

Combinatorial metabolic engineering and process optimization enables highly efficient production of L-lactic acid by acid-tolerant Saccharomyces cerevisiae

L-lactic acid (L-LA) is widely used in the food, pharmaceutical, and cosmetic industries. In recent years, the production of L-LA using microbial fermentation has been favored. Herein, a Saccharomyces cerevisiae TAM strain tolerant to pH 2.4, was used as the starting strain. Exogenous L-lactate dehydrogenase expressing S. cerevisiae TAM strain with downregulated glycerol and ethanol synthesis pathways produced an L-LA titer of 29.8 g/L, and it increased to 50.5 g/L after carboxylic acid transport pathway modulation at the shake-flask level. Subsequently, increased energy supply and redox balancing increased the L-LA titer to 72.7 g/L in shake-flask fermentation without a neutralizer, with the yield of 0.66 g/g. Finally, optimization of the fermentation conditions, such as the seed quantity, oxygen level, and pH in a 15-L bioreactor, increased the L-LA titer to 192.3 g/L at pH 4.5, with a yield of 0.78 g/g. Overall, this study proposes an efficient L-LA bioproduction method.

Combination of Cell Growth Dependent and Independent Modes for Efficient L-Lactic Acid Production by Lactobacillus paracasei

In this study,on the basis of L-lactic acid production by Lactobacillus paracasei,a high cell density strategy with the combination of cell growth dependent and independent modes was developed through investigating the optimal initial glucose concentration and the metabolic capacity of resting cells. The results indicated that the cells grew well under 140 g/L of initial glucose concentration,and all the glucose could be rapidly exhausted to produce L-lactic acid. Moreover,the cells could still maintain high metabolic activity within 6~7 h after the end of fermentation. Finally,based on the above results,the L-lactic acid productivity of high cell density phase was enhanced by 1.4-fold compared with that of normal batch fermentation,and the L-lactic acid yield was also improved significantly to 0.985 g/g. This study extended the highly efficient production period so as to improve the production efficiency of L-lactic acid. Otherwise,the auxiliary time required in the fermentation process could be effectively reduced,including seed preparation and cell growth. Thus,it would be potentially applied for future industrial lactic acid production.

Efficient production of L-lactic acid from corn straw hydrolysate

In this study, L-lactic acid was produced from corn straw hydrolysate by the strain Lactobacillus sp. L47, the fermentation conditions such as nitrogen sources, mineral salts, metal ions, incubation periods, pH values and temperatures were investigated, and the optimal incubation conditions were obtained: corn straw hydrolysate of 243.1 ml/l, (NH4)2SO4 10 g/l, yeast extract 10 g/l, KH2PO4 0.5 g/l, ZnSO4 • 7H2O 0.3 g/l, MgSO4• 7H2O 0.1 g/l, MnSO4• H2O 0.01 g/l, CaCO3 60 g/l, initial medium pH 6.0, controlled fermentation pH 6.0, 20 ml of seed, incubation temperatrure 46 °C. Under the optimal fermentation conditions, the highest L-lactic acid production was 99.8 g/l in 40 h.

Efficient l-lactic acid production from corncob residue using metabolically engineered thermo-tolerant yeast

Lactic acid is an important industrial product and the production from inexpensive and renewable lignocellulose can reduce the cost and environmental pollution. In this study, a Kluyveromyces marxianus strain which produced lactic acid efficiently from corncob was constructed. Firstly, two of six different lactate dehydrogenases, which from Plasmodium falciparum and Bacillus subtilis, respectively, were proved to be effective for l-lactic acid production. Then, five single genetic modifications were conducted. The overexpression of Saccharomyces cerevisiae proton-coupled monocarboxylate transporter, K. marxianus 6-phosphofructokinase, or disruption of K. marxianus putative d-lactate dehydrogenase enhanced the l-lactic acid accumulation. Finally, the strain YKX071, obtained via combination of above effective genetic engineering, produced 103.00 g/L l-lactic acid at 42 °C with optical purity of 99.5% from corncob residue via simultaneous saccharification and co-fermentation. This study first developed an effective platform for high optical purity l-lactic acid production from lignocellulose using yeast with inexpensive nitrogen sources.

Efficient production of l-lactic acid using co-feeding strategy based on cane molasses/glucose carbon sources

L-Lactic acid is an important platform chemical, which ought to be produced under cost control to meet its huge demand. Cane molasses, a waste from sugar manufacturing processes, is hopeful to be utilized as a cheap carbon source for L-lactic acid fermentation. Considering that cane molasses contains nutrients and hazardous substances, efficient production of L-lactic acid was developed by using a co-feeding strategy based on the utilization of cane molasses/glucose carbon sources. Based on the medium optimization with response surface method, 168.3g/L L-lactic acid was obtained by a Bacillus coagulans strain H-1 after 78h fed-batch fermentation, with a productivity of 2.1g/Lh and a yield of 0.88g/g. Since cane molasses is a feasible carbon source, the co-feeding fermentation might be a promising alternative for the economical production of L-lactic acid.

Bacillus sp. strain P38: An efficient producer of l-lactate from cellulosic hydrolysate, with high tolerance for 2-furfural

In this study, efficient polymer-grade L-lactic acid production was achieved with the strain Bacillus sp. P38 by using cellulosic hydrolysate as the sole carbon source. In fed-batch fermentation, 180 g L(-1)L-lactic acid was obtained with a volumetric productivity of 2.4 g L(-1)h(-1) and a yield of 0.96 g g(-1) total reducing sugars. No D-isomer of lactic acid was detected in the broth. Strain P38 tolerated up to 10 g L(-1) 2-furfural, and lactate production was sharply inhibited only when the 2-furfural concentration was higher than 6 g L(-1). Moreover, strain P38 also tolerated high concentrations (>6 g L(-1)) of other fermentation inhibitors in cellulosic hydrolysate, such as vanillin and acetic acid, although it was slightly sensitive to formic acid. The efficient L-lactic acid production, combined with high inhibitor tolerance and efficient pentose utilization, indicate that Bacillus sp. P38 is a promising producer of polymer-grade L-lactic acid from cellulosic biomass.

Efficient production of l-lactic acid by an engineered Thermoanaerobacterium aotearoensewith broad substrate specificity

BackgroundEfficient conversion of lignocellulosic biomass to optically pure lactic acid is a key challenge for the economical production of biodegradable poly-lactic acid. A recently isolated strain, Thermoanaerobacterium aotearoense SCUT27, is promising as an efficient lactic acid production bacterium from biomass due to its broad substrate specificity. Additionally, its strictly anaerobic and thermophilic characteristics suppress contamination from other microoragnisms. Herein, we report the significant improvements of concentration and yield in lactic acid production from various lignocellulosic derived sugars, achieved by the carbon flux redirection through homologous recombination in T. aotearoense SCUT27.ResultsT. aotearoense SCUT27 was engineered to block the acetic acid formation pathway to improve the lactic acid production. The genetic manipulation resulted in 1.8 and 2.1 fold increase of the lactic acid yield using 10 g/L of glucose or 10 g/L of xylose as substrate, respectively. The maximum l-lactic acid yield of 0.93 g/g glucose with an optical purity of 99.3% was obtained by the engineered strain, designated as LA1002, from 50 g/L of substrate, which is very close to the theoretical value (1.0 g/g of glucose). In particular, LA1002 produced lactic acid at an unprecedented concentration up to 3.20 g/L using 10 g/L xylan as the single substrate without any pretreatment after 48 h fermentation. The non-sterilized fermentative production of l-lactic acid was also carried out, achieving values of 44.89 g/L and 0.89 g/g mixed sugar for lactic acid concentration and yield, respectively.ConclusionsBlocking acetic acid formation pathway in T. aotearoense SCUT27 increased l-lactic acid production and yield dramatically. To our best knowledge, this is the best performance of fermentation on lactic acid production using xylan as the sole carbon source, considering the final concentration, yield and fermentation time. In addition, it should be mentioned that the performance of non-sterilized simultaneous fermentation from glucose and xylose was very close to that of normal sterilized cultivation. All these results used the mutant strain, LA1002, indicated that it is a new promising candidate for the effective production of optically pure l-lactic acid from lignocellulosic biomass.

Efficient production of l-lactic acid by newly isolated thermophilic Bacillus coagulans WCP10-4 with high glucose tolerance

A thermophilic Bacillus coagulans WCP10-4 with tolerance to high concentration of glucose was isolated from soil and used to produce optically pure L-lactic acid from glucose and starch. In batch fermentation at pH 6.0, 240 g/L of glucose was completely consumed giving 210 g/L of L-lactic acid with a yield of 95 % and a productivity of 3.5 g/L/h. In simultaneous saccharification and fermentation at 50 °C without sterilizing the medium, 200 g/L of corn starch was completely consumed producing 202.0 g/L of L-lactic acid. To the best of our knowledge, this strain shows the highest osmotic tolerance to glucose among the strains ever reported for lactic acid production. This is the first report of simultaneous saccharification and fermentation of starch for lactic acid production under a non-sterilized condition.

Highly efficient production of l-lactic acid from xylose by newly isolated Bacillus coagulans C106

Cost-effective production of optically pure lactic acid from lignocellulose sugars is commercially attractive but challenging. Bacillus coagulans C106 was isolated from environment and used to produce l-lactic acid from xylose at 50°C and pH 6.0 in mineral salts medium containing 1–2% (w/v) of yeast extract without sterilizing the medium before fermentation. In batch fermentation with 85g/L of xylose, lactic acid titer and productivity reached 83.6g/L and 7.5g/Lh, respectively. When fed-batch (120+80+60g/L) fermentation was applied, they reached 215.7g/L and 4.0g/Lh, respectively. In both cases, the lactic acid yield and optical purity reached 95% and 99.6%, respectively. The lactic acid titer and productivity on xylose are the highest among those ever reported. Ca(OH)2 was found to be a better neutralizing agent than NaOH in terms of its giving higher lactic acid titer (1.2-fold) and productivity (1.8-fold) under the same conditions.

Efficient production of l-lactic acid from hydrolysate of Jerusalem artichoke with immobilized cells of Lactococcus lactis in fibrous bed bioreactors

Hydrolysate of Jerusalem artichoke was applied for the production of l-lactic acid by immobilized Lactococcus lactis cells in a fibrous bed bioreactor system. Preliminary experiments had indicated that the high quality hydrolysate, which was derived from the 40min acid treatment at 95°C and pH 1.8, was sufficient to support the cell growth and synthesis of l-lactic acid. With the addition of 5g/l yeast extract, the fermentative performance of free cell system was evidently improved. After the basal settlement of hydrolysate based fermentation, the batch mode and the fed-batch mode fermentation were carried out in the free cell system and the fibrous bed bioreactor system, respectively. In all cases the immobilized cells presented the superior ability to produce l-lactic acid. The comparison of batch mode and fed-batch mode also indicated that the growth-limiting feeding strategy could reduce the lag phase of fermentation process and enhance the production of l-lactic acid. The achieved maximum concentration of l-lactic acid was 142g/l in the fed-batch mode. Subsequent repeated-batch fermentation of the fibrous bed bioreactor system had further exhibited the persistence and stability of this system for the high production of l-lactic acid in a long term. Our work suggested the great potential of the fibrous bed bioreactor system and hydrolysate of J. artichoke in the economical production of l-lactic acid at industrial scale.

Efficient production of l-lactic acid from chicken feather protein hydrolysate and sugar beet molasses by the newly isolated Rhizopus oryzae TS-61

The aim of this study was to investigate production of l-lactic acid from molasses and chicken feather protein hydrolysate (CFP) by the newly isolated Rhizopus oryzae TS-61. R. oryzae TS-61 was capable of utilizing molasses sucrose and CFP as carbon and nitrogen sources, respectively. In contrast to yeast extract and ammonium sulfate, CFP had potential not only to prevent excessive pH changes and foaming but also to provide smaller uniform pellet formation in during fermentation. Thanks to these properties, it was concluded that CFP might have resulted in higher l-lactic acid production than the other two nitrogen sources (yeast extract and ammonium sulfate). At the end of 42-h optimal cultivation period, the highest (38.5g/L) and lowest (28.8g/L) concentrations of l-lactic acid were obtained with CFP and ammonium sulfate, respectively. This is the first report on use of waste chicken feather as a lactic acid production substrate. In addition, a new R. oryzae strain, being capable of using molasses sucrose as carbon source in order to produce l-lactic acid, was isolated.

Efficient production of l-lactic acid with high optical purity by alkaliphilic Bacillus sp. WL-S20

Highly efficient polymer-grade l-lactic acid production was achieved by an alkaliphilic strain Bacillus sp. WL-S20 using inexpensive peanut meal as nitrogen source and sodium hydroxide as neutralizing agent. In multi-pulse fed-batch fermentation of Bacillus sp. WL-S20, a l-lactic acid concentration of 225g/l with a yield of 99.3% was obtained. In single-pulse fed-batch fermentation, a concentration of 180g/l was obtained with a yield of 98.6%. No d-isomers of lactic acid were detected. The production of a high concentration of optically pure l-lactic acid by alkaliphilic Bacillus sp. WL-S20, combined with a low-cost nutrient and environment-friendly NaOH-based process, represent a potentially novel way for l-lactic acid production at an industrial scale.

Efficient production of l-lactic acid from xylose by a recombinant Candida utilis strain

Efficient L-lactic acid production from xylose was achieved using a pyruvate decarboxylase-deficient Candida utilis strain expressing an L-lactate dehydrogenase, an NADH-preferring mutated xylose reductase (XR), a xylitol dehydrogenase and a xylulokinase. The recombinant strain showed 53% increased L-lactic acid production compared with the reference strain expressing native XR (NADPH-preferring).

Efficient production of l-lactic acid from cassava powder by Lactobacillus rhamnosus

Cassava is one of the most efficient and rich crops in terms of carbohydrate production, which is a tropical perennial plant that grows on poor or depleted soils. Microbial conversion of such a renewable raw material to useful products is an important objective in industrial biotechnology. l-Lactic acid was efficiently produced from cassava powder by a Lactobacillus rhamnosus strain CASL. The fermentation properties of cassava powder were compared with those of glucose and corn powder. The efficiencies of various fermentation strategies for l-lactic acid production from cassava powder, including simultaneous saccharification and fermentation (SSF), two-step fermentation (TSF) and simultaneous liquefaction, saccharification and fermentation (SLSF), were investigated. The high l-lactic acid concentration (175.4 g/l) was obtained using 275 g/l of cassava powder concentration (total sugar of 222.5 g/l) in SSF batch fermentation. This is the highest l-lactic acid concentration reported, from cassava source, and it provides an efficient l-lactic acid production process with cheap raw bioresources, such as cassava powder.

Efficient production of l-lactic acid from corncob molasses, a waste by-product in xylitol production, by a newly isolated xylose utilizing Bacillus sp. strain

Lignocellulosic biomass-derived sugars are considered nowadays to be an economically attractive carbohydrate feedstock for large-scale fermentations of bulk chemicals such as lactic acid. In the present study, corncob molasses containing a high content of xylose, which is one of the lignocellulosic biomasses and a waste by-product from xylitol production, was used for l-lactic acid production via a newly isolated xylose utilizing Bacillus sp. strain XZL9. Bacillus sp. strain XZL9 can utilize the mixture of sugars including xylose, arabinose, and glucose in corncob molasses for l-lactic acid production. High concentration of l-lactic acid (74.7 g l −1) was obtained from corncob molasses (initial total sugars of 91.4 g l −1) in fed-batch fermentation. This study provides an encouraging means of producing l-lactic acid from lignocellulosic resource such as the low-cost corncob molasses.

Enhancement of L-Lactic Acid Production in Lactobacillus casei from Jerusalem Artichoke Tubers by Kinetic Optimization and Citrate Metabolism

Efficient L-lactic acid production from Jerusalem artichoke tubers by Lactobacillus casei G-02 using simultaneous saccharification and fermentation (SSF) in fed-batch culture is demonstrated. The kinetic analysis in the SSF signified that the inulinase activity was subjected to product inhibition, while the fermentation activity of G-02 was subjected to substrate inhibition. It was also found that the intracellularly NOX activity was enhanced by the citrate metabolism, which increased the carbon flux of Embden-Meyerhof-Parnas (EMP) pathway dramatically, and resulted more ATP production. As a result, when the SSF was carried out at 40 degrees after the initial hydrolysis of 1 h with supplemented sodium citrate of 10g/L, L-lactic acid concentration of 141.5 g/L was obtained in 30 h with a volumetric productivity of 4.7 g/L/h. The conversion efficiency and product yield were 93.6% of the theoretical lactic acid yield and 52.4 g lactic acid/100 g Jerusalem artichoke flour, respectively. Such a high concentration of lactic acid with high productivity from Jerusalem artichoke has not been reported previously, and hence G-02 could be a potential candidate for economical production of L-lactic acid from Jerusalem artichoke at a commercial scale.