An economic approach for L-(+) lactic acid fermentation by Lactobacillus amylophilus GV6 using inexpensive carbon and nitrogen sources

AIMS: Development of cost-effective production medium by applying statistical designs for single-step fermentation of starch (corn flour - CF) to L-(+) lactic acid, using inexpensive nitrogen sources as substitutes for peptone and yeast extract in MRS medium by amylolytic Lactobacillus amylophilus GV6. METHODS AND RESULTS: A two-level Plackett-Burman design was employed for screening various available crude starches (flours) for L-(+) lactic acid production by Lact. amylophilus GV6 using red lentil flour (RL) and bakers yeast cells (YC) as substitutes for commercial peptone and yeast extract in MRS medium in anaerobic submerged fermentation. Of all the tested flours, CF was found to be the most significant. Central composite rotatable design was employed to determine maximum production of L-(+) lactic acid at optimum values of process variables, CF, RL, YC, CaCO(3) and incubation period (IP). minitab analyses showed that lactic acid production was significantly affected by the linear terms CF, RL, CaCO(3) and IP. The interactions of CF-RL, CF-YC, CF-CaCO(3), RL-YC and RL-CaCO(3) and the square terms CF and IP were significant. The maximum lactic acid production of 29 g/37 g of starch present in 50 g of CF was obtained at optimized concentrations of CF 5%, RL 0.7%, YC 0.8%, CaCO(3) 0.8% and IP 2.9 days. CONCLUSIONS: Successful application of Plackett-Burman design helped in identifying CF as the best carbon source among the tested flours for L-(+) lactic acid production using inexpensive nitrogen sources. Further optimization of the process variables by response surface methods (RSMs) led to maximum production of lactic acid (29 g lactic acid from 37 g of starch present in 50 g of flour). SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus amylophilus GV6 showed 78.4% lactic acid production efficiency (g lactic acid produced/g starch taken) and 96% lactic acid yield efficiency (g lactic acid produced/g starch utilized). Information from the present studies provides a better understanding on production of L-(+) lactic acid on fermentation of CF using inexpensive nitrogen sources and on changes in the production as a response from interaction of factors. Use of inexpensive nitrogen sources and starch as substrate in MRS medium for single-step fermentation of lactic acid can become an efficient, economic and viable process. This report is on optimization of inexpensive nitrogen sources completely replacing peptone and yeast extract in single-step submerged fermentation of starch (present in CF) to lactic acid with high production efficiency.     

Selection of medium components by Plackett-Burman design for production of L(+) lactic acid by Lactobacillus amylophilus GV6 in SSF using wheat bran

Plackett-Burman design was employed for screening 15 parameters for production of L(+) lactic acid from wheat bran, an inexpensive substrate and solid support, by Lactobacillus amylophilus GV6 in solid state fermentation (SSF). Eleven nutrients belonging to two categories viz.; nitrogen sources and salt sources along with three physical parameters and a buffer were screened. This design screens n variables in n + 1 number of experiments. Coefficients and sum of squares ratio in percentage (SS%) of these variables were calculated by subjecting the experimental data to statistical analysis. The nitrogen sources peptone, yeast extract and tri-ammonium citrate, along with NaH2PO4.2H2O and Tween 80, were found to influence productivity, which can be further optimized for increased lactic acid production. Use of this design is scarce in solid state fermentation and has not been attempted previously for single step conversion of starch to L(+) lactic acid using a bacterial system.     

Optimization of lactic acid production from beet molasses by Lactobacillus delbrueckii NCIMB 8130

Production of lactic acid from beet molasses by Lactobacillus delbrueckii NCIMB 8130 in static and shake flask fermentation was investigated. Shake flasks proved to be a better fermentation system for this purpose. Substitution of yeast extract with other low cost protein sources did not improve lactic acid production. The maximum lactic acid concentration was achieved without treatment of molasses. A Central Composite Design was employed to determine the maximum lactic acid concentration at optimum values for the process variables (sucrose, yeast extract, CaCO₃). A satisfactory fit of the model was realized. Lactic acid production was significantly affected both by sucrose–yeast extract and sucrose–CaCO₃ interactions, as well as by the negative quadratic effects of these variables. Sucrose and yeast extract had a linear effect on lactic acid production while the CaCO₃ had no significant linear effect. The maximum lactic acid concentration (88.0 g/l) was obtained at concentrations for sucrose, yeast extract and CaCO₃ of 89.93, 45.71 and 59.95 g/l, respectively.     

Biotechnological production of lactic acid integrated with fishmeal wastewater treatment by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis>

Fishmeal wastewater, a seafood processing waste, was utilized for production of lactic acid and fungal biomass by Rhizopus oryzae AS 3.254 with the addition of sugars. The 30 g/l exogenous glucose in fishmeal wastewater was superior to starch in view of productivities of lactic acid and fungal biomass, and COD reduction. Fishmeal wastewater can be a replacement for peptone which was the most suitable nitrogen source for lactic acid production among the tested organic or inorganic nitrogen sources. Exogenous NaCl (12 g/l) completely inhibited the production of lactic acid and fungal growth. In the medium of COD 5,000 mg/l fishmeal wastewater with the addition of 30 g/l glucose, the maximum productivity of lactic acid was 0.723 g/l h corresponding to productivity of fungal biomass 0.0925 g/l h, COD reduction 84.9% and total nitrogen removal 50.3% at a fermentation time of 30 h.     

Production of lactic acid and byproducts from waste potato starch by <Emphasis Type="Italic">Rhizopus arrhizus</Emphasis>: role of nitrogen sources

Summary Lactic acid was produced by Rhizopus arrhizus using waste potato starch as the substrate. The aim of this study was to identify the role of nitrogen sources and their impact on the formation of lactic acid and associated byproducts. Ammonium sulphate, ammonium nitrate, urea, yeast extract and peptone were assessed in conjunction with various ratios of carbon to nitrogen (C:N). Fermentation media with a low C:N ratio enhanced the production of lactic acid, biomass and ethanol, while a high C:N ratio favoured the production of fumaric acid. Ammonium nitrate appeared to be the most suitable nitrogen source for achieving a high and stable lactic acid yield, and minimizing the production of byproducts such as biomass and ethanol, while urea proved to be the least favourable nitrogen source. Yeast extract and peptone appeared to improve fungal cell growth. The kinetics data revealed that a high concentration of ammonium nitrate enhanced the lactic acid productivity. The maximum lactic acid concentration of 36.4 g/l, representing a yield of 91%, was obtained with addition of 0.909 g/l ammonium nitrate in 32 h.     

Optimisation of media and cultivation conditions for L(+)(S)-lactic acid production by Lactobacillus casei NRRL B-441

Process variables and concentration of carbon in media were optimised for lactic acid production by Lactobacillus casei NRRL B-441. Lactic acid yield was inversely proportional to initial glucose concentration within the experimental area (80-160 g l(-1)). The highest lactic acid concentration in batch fermentation, 118.6 g l(-1), was obtained with 160 g 1(-1) glucose. The maximum volumetric productivity, 4.4 g 1(-1) h(-1) at 15 h, was achieved at an initial glucose concentration of 100 g l(-1). Similar lactic acid concentrations were reached with a fedbatch approach using growing cells, in which case the fermentation time was much shorter. Statistical experimental design and response surface methodology were used for optimising the process variables. The temperature and pH optima for lactic acid production were 35 degrees C, pH 6.3. Malt sprout extract supplemented with yeast extract (4 g l(-1)) appeared to be an economical alternative to yeast extract alone (22 g l(-1)) although the fermentation time was a little longer. The results demonstrated both the separation of the growth and lactic acid production phases and lactic acid production by non-growing cells without any nutrient supplements. Resting L. casei cells converted 120 g l(-1) glucose to lactic acid with 100% yield and a maximum volumetric productivity of 3.5 g l(-1) h(-1).     

Production of bakers' yeast in cheese whey ultrafiltrate

A process for the production of bakers' yeast in whey ultrafiltrate (WU) is described. Lactose in WU was converted to lactic acid and galactose by fermentation. Streptococcus thermophilus was selected for this purpose. Preculturing of S. thermophilus in skim milk considerably reduced its lag. Lactic fermentation in 2.3x-concentrated WU was delayed compared with that in unconcentrated whey, and fermentation could not be completed within 60 h. The growth rate of bakers' yeast in fermented WU differed among strains. The rate of galactose utilization was similar for all strains, but differences in lactic acid utilization occurred. Optimal pH ranges for galactose and lactic acid utilization were 5.5 to 6.0 and 5.0 to 5.5, respectively. The addition of 4 g of corn steep liquor per liter to fermented WU increased cell yields. Two sources of nitrogen were available for growth of Saccharomyces cerevisiae: amino acids (corn steep liquor) and ammonium (added during the lactic acid fermentation). Ammonium was mostly assimilated during growth on lactic acid. This process could permit the substitution of molasses by WU for the industrial production of bakers' yeast.     

A media design program for lactic acid production coupled with extraction by electrodialysis

The aim of this study was to investigate industrial media for lactic acid fermentation to reduce the cost of nitrogen sources. Corn steep liquor (CSL) was successfully used at 5% (v/v) in batch fermentations. Use of soluble CSL improved the productivity approximately 20% with an advantage of clearer fermentation broth. Yeast extract (YE)-complemented CSL media further increased the productivity. It was found that 3.1 g L(-1) yeast extract and 5% CSL could be an effective substitute for 15 g L(-1) yeast extract in 10% glucose medium. Spent brewery yeast was also used as a sole nitrogen source equivalent to 5% CSL. Lactic acid was recovered by electrodialysis from the cell free broth. Depleted cell free broth supplemented with 5 g L(-1) of yeast extract performed reasonably in batch cultures. Reuse of the fermentation broth may reduce the cost of raw materials as well as minimize the fermentation wastes.     

高糖和氮源对鼠李糖乳杆菌(Lactobacillus rhamnosus)L-乳酸发酵的影响

鼠李糖乳杆菌经实验室耐高糖高酸选育,能够在高糖浓度下高效高产L-乳酸。以酵母粉为氮源和生长因子,葡萄糖初始浓度分别为120 g/L和146 g/L,摇瓶培养120h,L-乳酸产量分别为104g/L和117.5g/L,L-乳酸得率分别为86.7%和80.5%。高葡萄糖浓度对菌的生长和乳酸发酵有一定的抑制。增加接种量,在高糖浓度发酵条件下,可以缩短发酵时间,但对增加乳酸产量效果不明显。乳酸浓度对鼠李糖乳杆菌生长和产酸有显著的影响。初始乳酸浓度到达70g/L以上时,鼠李糖乳杆菌基本不生长和产酸,葡萄糖消耗也被抑制。酵母粉是鼠李糖乳杆菌的优良氮源,使用其它被测试的氮源菌体生长和产酸都有一定程度的下降。用廉价的黄豆粉并补充微量维生素液,替代培养基中的酵母粉,可以使产酸浓度和碳源得率得以基本维持。     

Production of Bakers' Yeast in Cheese Whey Ultrafiltrate

A process for the production of bakers' yeast in whey ultrafiltrate (WU) is described. Lactose in WU was converted to lactic acid and galactose by fermentation. Streptococcus thermophilus was selected for this purpose. Preculturing of S. thermophilus in skim milk considerably reduced its lag. Lactic fermentation in 2.3×-concentrated WU was delayed compared with that in unconcentrated whey, and fermentation could not be completed within 60 h. The growth rate of bakers' yeast in fermented WU differed among strains. The rate of galactose utilization was similar for all strains, but differences in lactic acid utilization occurred. Optimal pH ranges for galactose and lactic acid utilization were 5.5 to 6.0 and 5.0 to 5.5, respectively. The addition of 4 g of corn steep liquor per liter to fermented WU increased cell yields. Two sources of nitrogen were available for growth of Saccharomyces cerevisiae: amino acids (corn steep liquor) and ammonium (added during the lactic acid fermentation). Ammonium was mostly assimilated during growth on lactic acid. This process could permit the substitution of molasses by WU for the industrial production of bakers' yeast.     

The effect of supplementation by different nitrogen sources on the production of lactic acid from date juice by Lactobacillus casei subsp. rhamnosus

Production of lactic acid from date juice by fermentation has been studied using Lactobacillus casei subsp. rhamnosus as the producer organism. The optimum substrate concentration, expressed in its glucose content, was 60 g l(-1). Various nitrogen sources were compared with yeast extract in terms of their efficiency for lactic acid production. None of these nitrogen sources gave lactic acid concentrations as high as that obtained with yeast extract. As yeast extract supplementation was not economically attractive, different proportions of (NH4)2SO4 and yeast extract were used. When the elemental nitrogen ratio of(NH4)2SO4 to yeast extract was 4:1, the substrate use and efficiency of lactic acid production were the same as in date juice supplemented with 20 g l(-1) yeast extract (0:5).     

Lactic acid production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing a <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lactate dehydrogenase gene

This work demonstrates the first example of a fungal lactate dehydrogenase (LDH) expressed in yeast. A L(+)-LDH gene, ldhA, from the filamentous fungus Rhizopus oryzae was modified to be expressed under control of the Saccharomyces cerevisiae adh1 promoter and terminator and then placed in a 2μ-containing yeast-replicating plasmid. The resulting construct, pLdhA68X, was transformed and tested by fermentation analyses in haploid and diploid yeast containing similar genetic backgrounds. Both recombinant strains utilized 92 g glucose/l in approximately 30 h. The diploid isolate accumulated approximately 40% more lactic acid with a final concentration of 38 g lactic acid/l and a yield of 0.44 g lactic acid/g glucose. The optimal pH for lactic acid production by the diploid strain was pH 5. LDH activity in this strain remained relatively constant at 1.5 units/mg protein throughout the fermentation. The majority of carbon was still diverted to the ethanol fermentation pathway, as indicated by ethanol yields between 0.25–0.33 g/g glucose. S. cerevisiae mutants impaired in ethanol production were transformed with pLdhA68X in an attempt to increase the lactic acid yield by minimizing the conversion of pyruvate to ethanol. Mutants with diminished pyruvate decarboxylase activity and mutants with disrupted alcohol dehydrogenase activity did result in transformants with diminished ethanol production. However, the efficiency of lactic acid production also decreased. Electronic Publication     

Production of penicillic acid by Aspergillus sclerotiorum CGF

The production of penicillic acid by Aspergillus sclerotiorum CGF for the biocontrol of Phytophthora disease was investigated in submerged fermentation using media composed of different nutrients. Soluble starch was found to be the most effective substrate among the carbon sources used, and produced the highest penicillic acid concentration of 2.98 mg ml(-1). When organic nitrogen sources were used, pharmamedia, yeast extract, and polypeptone-S were found to be suitable organic nitrogen sources (2.46-2.71 mg ml(-1)). The production of penicillic acid was not detected in when inorganic nitrogen sources were used. Only Na2HPO4, among the metal ions and phosphate salts tested, increased the production of penicillic acid (approximately 20%). When A. sclerotiorum CGF was cultured in optimal medium [8.0% (w/v) soluble starch, 0.6% (w/v) yeast extract, and 0.3% (w/v) Na2HPO4], maximum penicillic acid concentration (approximately 9.40 mg ml(-1)) and cell mass (approximately 17.4 g l(-1)) were obtained after 12 days.     

Characterization of ethanol fermentation waste and its application to lactic acid production by Lactobacillus paracasei

In this study, an ethanol fermentation waste (EFW) was characterized for use as an alternative to yeast extract for bulk fermentation processes. EFW generated from a commercial plant in which ethanol is produced from cassava/rice/wheat/barley starch mixtures using Saccharomyces cerevisiae was used for lactic acid production by Lactobacillus paracasei. The effects of temperature, pH, and duration on the autolysis of an ethanol fermentation broth (EFB) were also investigated. The distilled EFW (DEFW) contained significant amounts of soluble proteins (2.91 g/l), nitrogen (0.47 g/l), and amino acids (24.1 mg/l). The autolysis of the EFB under optimum conditions released twice as much amino acids than in the DEFW. Batch fermentation in the DEFW increased the final lactic acid concentration, overall lactic acid productivity, and lactic acid yield on glucose by 17, 41, and 14 %, respectively, in comparison with those from comparable fermentation in a lactobacillus growth medium (LGM) that contained 2 g/l yeast extract. Furthermore, the overall lactic acid productivity in the autolyzed then distilled EFW (ADEFW) was 80 and 27 % higher than in the LGM and DEFW, respectively.     

Integrated process of starch ethanol and cellulosic lactic acid for ethanol and lactic acid production

The sequential production of bioethanol and lactic acid from starch materials and lignocellulosic materials was investigated as ethanol fermentation broth (EFB) can provide nutrients for lactic acid bacteria. A complete process was developed, and all major operations are discussed, including ethanol fermentation, broth treatment, lactic acid fermentation, and product separation. The effect of process parameters, including ethanol fermentation conditions, treatment methods, and the amount of EFB used in simultaneous saccharification and fermentation (SSF), is investigated. Under the selected process conditions, the integrated process without additional chemical consumption provides a 1.08 acid/alcohol ratio (the broth containing 22.4 g/L ethanol and 47.6 g/L lactic acid), which corresponds to a polysaccharide utilization ratio of 86.9 %. Starch ethanol can thus promote cellulosic lactic acid by providing important nutrients for lactic acid bacteria, and in turn, cellulosic lactic acid could promote starch ethanol by improving the profit of the ethanol production process. Two process alternatives for the integration of starch ethanol and cellulosic lactic acid are compared, and some suggestions are given regarding the reuse of yeast following the cellulosic SSF step for lactic acid production.     

Utilization of Molasses Sugar for Lactic Acid Production by Lactobacillus delbrueckii subsp. delbrueckii Mutant Uc-3 in Batch Fermentation

Efficient lactic acid production from cane sugar molasses by Lactobacillus delbrueckii mutant Uc-3 in batch fermentation process is demonstrated. Lactic acid fermentation using molasses was not significantly affected by yeast extract concentrations. The final lactic acid concentration increased with increases of molasses sugar concentrations up to 190 g/liter. The maximum lactic acid concentration of 166 g/liter was obtained at a molasses sugar concentration of 190 g/liter with a productivity of 4.15 g/liter/h. Such a high concentration of lactic acid with high productivity from molasses has not been reported previously, and hence mutant Uc-3 could be a potential candidate for economical production of lactic acid from molasses at a commercial scale.     

Production of lactic acid by Lactobacillus rhamnosus with vitamin-supplemented soybean hydrolysate

Batch fermentation studies were performed to evaluate the potentials of a complex nitrogen source, soybean, as an alternative to yeast extract for the economical production of lactic acid by Lactobacillus rhamnosus. An enzyme-hydrolysate of soybean meal, Soytone, with an adequate supplementation of vitamins was found to be highly effective in supporting lactic acid production from glucose and lactose. The effects of seven selected vitamins: d-biotin, pyridoxine, p-aminobenzoic acid, nicotinic acid, thiamine, pantothenic acid, and riboflavin, on cell growth and lactic acid production were investigated to provide the basis for the optimization of vitamin supplementation to minimize the cost. Pantothenic acid was the most required compound while the other six vitamins were also essential for high lactic acid productivity. As a result of the optimization, 15 g/l yeast extract could be successfully replaced with 19.3 g/l Soytone supplemented with the vitamins, resulting in a production of 125 g/l lactic acid from 150 g/l glucose. The volumetric productivity and lactate yield were 2.27 g/l/h and 92%, respectively, which were higher than those with 15 g/l yeast extract. The raw material cost was estimated to be 21.4 cent/kg lactic acid, which was only approximately 41% of that with yeast extract.     

Direct fermentation of various pure and crude starchy substrates to L(+) lactic acid using Lactobacillus amylophilus GV6

Lactobacillus amylophilus GV6 fermented a variety of pure and natural starches directly to L(+) lactic acid. Starch to lactic acid conversion efficiency was more than 90% by strain GV6 at low substrate concentrations with all starches. The strain GV6 produced high yields of lactic acid per g of substrate utilized with pure starches such as soluble starch, corn starch, and potato starch, yielding 92–96% at low substrate concentrations in 2 days and 78–89% at high substrate (10%) concentrations in 4–6 days. Strain GV6 also produced high yields of lactic acid per g of substrate utilized with crude starchy substrates such as wheat flour, sorghum flour, cassava flour, rice flour and barley flour yielding 90–93% at low substrate concentrations in 2 days and 80% or more at high substrate concentrations in 6–7 days. Lactic acid yields by L. amylophilus GV6 with pure starches were comparable when low cost crude starchy substrates were used. Lactic acid productivity by strain GV6 is higher than for any other previously reported strains of L. amylophilus.     

Utilization of by-products derived from bioethanol production process for cost-effective production of lactic acid

The by-products of bioethanol production such as thin stillage (TS) and condensed distillers solubles (CDS) were used as a potential nitrogen source for economical production of lactic acid. The effect of those by-products and their concentrations on lactic acid fermentation were investigated using Lactobacillus paracasei CHB2121. Approximately, 6.7 g/L of yeast extract at a carbon source to nitrogen source ratio of 15 was required to produce 90 g/L of lactic acid in the medium containing 100 g/L of glucose. Batch fermentation of TS medium resulted in 90 g/L of lactic acid after 48 h, and the medium containing 10 % CDS resulted in 95 g/L of lactic acid after 44 h. Therefore, TS and CDS could be considered as potential alternative fermentation medium for the economical production of lactic acid. Furthermore, lactic acid fermentation was performed using only cassava and CDS for commercial production of lactic acid. The volumetric productivity of lactic acid [2.94 g/(L·h)] was 37 % higher than the productivity obtained from the medium with glucose and CDS.     

Production of lactic acid and fungal biomass by Rhizopus fungi from food processing waste streams

This study proposed a novel waste utilization bioprocess for production of lactic acid and fungal biomass from waste streams by fungal species of Rhizopus arrhizus 36017 and R. oryzae 2062. The lactic acid and fungal biomass were produced in a single-stage simultaneous saccharification and fermentation process using potato, corn, wheat and pineapple waste streams as production media. R. arrhizus 36017 gave a high lactic acid yield up to 0.94-0.97 g/g of starch or sugars associated with 4-5 g/l of fungal biomass produced, while 17-19 g/l fungal biomass with a lactic acid yield of 0.65-0.76 g/g was produced by the R. oryzae 2062 in 36-48 h fermentation. Supplementation of 2 g/l of ammonium sulfate, yeast extract and peptone stimulated an increase in 8-15% lactic acid yield and 10-20% fungal biomass.