Optimization of nutritional supplements for enhanced lactic acid production utilizing sugar refinery by-products

The present study investigated the synergistic effect of nutritional supplements (amino acid and Tween 80) on lactic acid production by Lactobacillus delbruckii utilizing a sugar refinery by product (cane molasses) in a submerged fermentation process. Initially, the effect of individual factors on lactic acid yield was studied by supplementing amino acids and their combinations, Tween 80 and cane molasses at varying concentrations in production medium. A combination of l-phenylalanine and l-lysine gave a maximum lactic acid yield of 47.89?±?0.1 g/L on a dry cell weight basis at individual factor level. Similarly, maximum lactic acid yield was obtained by supplementing the production medium with 40.0 g/L and 2.0 g/L Tween 80 and cane molasses, respectively, at individual factor level. In order to further improve the lactic acid yield, nutritional supplements were optimized by central composite rotatable design (CCRD) using Minitab 15 software. Shake flask cultivation under optimized conditions, i.e., cane molasses (32.40 g/L), Tween 80 (2.0 g/L) and l-phenylalanine and l-lysine (34.0 mg/L) gave a lactic acid yield of 64.86?±?0.2 g/L, corresponding to 95.0 % of the predicted yield of 67.78?±?0.3 g/L. Batch cultivation performed in 7.5 L bioreactor (working volume: 3.0 L) under optimized conditions gave maximum lactic acid yield and productivity of 79.12?±?0.2 g/L and 3.40 g/L·h, which is higher than previous studies with reduced fermentation time. Screening of lactic acid producing bacteria and characterization of lactic acid was also done.     

Integrated production of lactic acid and biomass on distillery stillage

The possibilities of parallel lactic acid and biomass production in batch and fed-batch fermentation on distillery stillage from bioethanol production were studied. The highest lactic acid yield and productivity of 92.3 % and 1.49 g L^?1 h^?1 were achieved in batch fermentation with initial sugar concentration of 55 g L^?1. A significant improvement of the process was achieved in fed-batch fermentation where the concentration of lactic acid was increased to 47.6 % and volumetric productivity for 21 % over the batch process. A high number of Lactobacillus rhamnosus ATCC 7469 viable cells of 10⁹ CFU ml^?1 was attained at the end of fed-batch fermentation. The survival of 92.9 % of L. rhamnosus cells after 3 h of incubation at pH 2.5 validated that the fermentation media remained after lactic acid removal could be used as a biomass-enriched animal feed thus making an additional value to the process.     

Homo-fermentative production of d-lactic acid by Lactobacillus sp. employing casein whey permeate as a raw feed-stock

Casein whey permeate (CWP), a lactose-enriched dairy waste effluent, is a viable feed stock for the production of value-added products. Two lactic acid bacteria were cultivated in a synthetic casein whey permeate medium with or without pH control. Lactobacillus lactis ATCC 4797 produced d-lactic acid (DLA) at 12.5 g l^?1 in a bioreactor. The values of Leudking–Piret model parameters suggested that lactate was a growth-associated product. Batch fermentation was also performed employing CWP (35 g lactose l^?1) with casein hydrolysate as a nitrogen supplement in a bioreactor. After 40 h, L. lactis produced 24.3 g lactic acid l^?1 with an optical purity >98 %. Thus CWP may be regarded as a potential feed-stock for DLA production.     

Lactic Acid Production in a Mixed-Culture Biofilm Reactor

Novel solid supports, consisting of polypropylene blended with various agricultural materials (pp composite), were evaluated as supports for pure- and mixed-culture continuous lactic acid fermentations in biofilm reactors. Streptomyces viridosporus T7A (ATCC 39115) was used to form a biofilm, and Lactobacillus casei subsp. rhamnosus (ATCC 11443) was used for lactic acid production. For mixed-culture fermentations, a 15-day continuous fermentation of S. viridosporus was performed initially to establish the biofilm. The culture medium was then inoculated with L. casei subsp. rhamnosus. For pure-culture fermentation, L. casei subsp. rhamnosus was inoculated directly into the reactors containing sterile pp composite chips. The biofilm reactors containing various pp composite chips were compared with a biofilm reactor containing pure polypropylene chips and with a reactor containing a suspension culture. Continuous fermentation was started, and each flow rate (0.06 to 1.92 ml/min) was held constant for 24 h; steady state was achieved after 10 h. Lactic acid production was determined throughout the 24-h period by high-performance liquid chromatography. Production rates that were two to five times faster than those of the suspension culture (control) were observed for the pure- and mixed-culture bioreactors. Both lactic acid production rates and lactic acid concentrations in the culture medium were consistently higher in mixed-culture than in pure-culture fermentations. Biofilm formation on the chips was detected at harvest by chip clumping and Gram staining.     

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.     

Production of succinic acid and lactic acid by Corynebacterium crenatum under anaerobic conditions

A new succinic acid and lactic acid production bioprocess by Corynebacterium crenatum was investigated in mineral medium under anaerobic conditions. Corynebacterium crenatum cells with sustained acid production ability and high acid volumetric productivity harvested from the glutamic acid fermentation broth were used to produce succinic acid and lactic acid. Compared with the first cycle, succinic acid production in the third cycle increased 120% and reached 43.4 g/L in 10 h during cell-recycling repeated fermentations. The volumetric productivities of succinic acid and lactic acid could maintain above 4.2 g/(L·h) and 3.1 g/(L·h), respectively, for at least 100 h. Moreover, wheat bran hydrolysates could be used for succinic acid and lactic acid production by the recycled C. crenatum cells. The final succinic acid concentration reached 43.6 g/L with a volumetric productivity of 4.36 g/(L·h); at the same time, 32 g/L lactic acid was produced.     

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.     

In situ separation of lactic acid from fermentation broth using ion exchange resins

Lactic acid fermentation is an end product inhibited reaction. In situ separation of lactic acid from fermentation broth using ion exchange resins was investigated and compared with conventional fermentation system. Amberlite resin (IRA-400, Cl⁻) was used to separate lactic acid from fermentation broth and pH was controlled online with an automatic pH controller. The effect of process variables on lactic acid production by Lactobacillus casei in whey permeate was studied. The maximum productivity was obtained at pH = 6.1, T = 37 °C and impeller speed = 200 rpm. The maximum concentration of lactic acid at optimum condition was found to be 37.4 g/L after 38 h of fermentation using in situ separation system. The productivity of in situ separation system was five times increased in comparison with conventional system.     

Conversion of acid hydrolysate of oil palm empty fruit bunch to L-lactic acid by newly isolated Bacillus coagulans JI12

Cost-effective conversion of lignocellulose hydrolysate to optically pure lactic acid is commercially attractive but very challenging. Bacillus coagulans JI12 was isolated from natural environment and used to produce L-lactic acid (optical purity?>?99.5 %) from lignocellulose sugars and acid hydrolysate of oil palm empty fruit bunch (EFB) at 50 °C and pH 6.0 without sterilization of the medium. In fed-batch fermentation with 85 g/L initial xylose and 55 g/L xylose added after 7.5 h, 137.5 g/L lactic acid was produced with a yield of 98 % and a productivity of 4.4 g/L?h. In batch fermentation of a sugar mixture containing 8.5 % xylose, 1 % glucose, and 1 % L-arabinose, the lactic acid yield and productivity reached 98 % and 4.8 g/L?h, respectively. When EFB hydrolysate was used, 59.2 g/L of lactic acid was produced within 9.5 h at a yield of 97 % and a productivity of 6.2 g/L?h, which are the highest among those ever reported from lignocellulose hydrolysates. These results indicate that B. coagulans JI12 is a promising strain for industrial production of L-lactic acid from lignocellulose hydrolysate.     

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.     

Effective conversion of industrial starch waste to L-Lactic acid by Lactococcus lactis in a dialysis sac bioreactor

We describe here a simple technological process based on the direct fermentation of potato starch waste (PSW), an inexpensive agro-processing industrial waste, by a potential probiotic strain, Lactococcus lactis subsp. lactis, for enhancing L-lactic acid production. To maximize bioconversion and increase cell stability, we designed and tested a novel dialysis sac-based bioreactor. Shake flask fermentation (SFF) and fed batch fermentation in the dialysis sac bioreactor were compared for L-lactic acid production efficiency. The results showed that the starch (20 g/L) in the PSW-containing medium was completely consumed within 24 h in the dialysis sac bioreactor, compared with 48 h in the SFF. The maximum lactic acid concentration (18.9 g/L) and lactic acid productivity (0.79 g/L·h) obtained was 1.2- and 2.4-fold higher in the bioreactor than by SFF, respectively. Simultaneous saccharification and fermentation was effected at pH 5.5 and 30 °C. L. lactis cells were viable for up to four cycles in the fed batch fermentation compared to only one cycle in the SFF.     

Sugar-cane pressmud as a novel and inexpensive substrate for production of lactic acid in a solid-state fermentation system

Lactobacillus casei subsp. casei CFTRI 2022 produced a higher concentration of lactic acid (5.27 g/100 g dry sugar-cane pressmud) in a solid-state fermentation (SSF) system as compared to L. helveticus CFTRI 2026 and Streptococcus thermophilus CFTRI 2034. The lactic acid production by L. casei subsp. casei CFTRI 2022 was found to be significantly influenced by the initial moisture content, initial pH and initial sugar concentration of the medium. Studies on four inert materials to reduce the initial sugar concentration in the medium showed the high potential of microcrystalline cellulose whereas the use of diatomaceous earth, acid-washed river sand and washed pith bagasse posed problems. The data indicate the potential of lactic acid production from sugar-cane pressmud in an SSF system.     

Effect of lactic acid on diacetyl and acetoin production byLactobacillus casei subsp.rhamnosus ATCC 7469

Lactic acid or its acidity apparently play an important role in the regulation of the biosynthesis of flavor compounds inLactobacillus casei subsp.rhamnosus ATCC 7469. In pyruvate-containing media,L. casei produces lactic acid, acetoin, and diacetyl. A specific pH-dependent system is necessary for both the use of pyruvate and the induction of acetoin and diacetyl production. In cell extracts ofL. casei, lactic acid inhibits the enzymatic activity of acetolactate decarboxylase (ALD) and acetolactate synthetase (ALS); this effect does not occur in whole cells under standard physiological conditions. Lactic acid prevents the use of pyruvate, and the induction of acetoin and diacetyl production. When pyruvate-containing media are used, the pH must be kept close to 6.0 in order to obtain the best production of acetoin and diacetyl.     

Repeated-batch fermentation in biofilm reactors with plastic-composite supports for lactic acid production

Novel plastic supports consisting of polypropylene blended with oat hulls/soybean flour or oat hulls/zein were evaluated as supports for mixed- and pure-culture, repeated-batch, lactic acid fermentations in biofilm reactors. Streptomyces viridosporus T7A (ATCC 39115) was used to form a biofilm for mixed-culture fermentations, and Lactobacillus casei subsp. rhamnosus (ATCC 11443) was used for L-lactic acid production. The pure- and mixed-culture biofilm reactors were operated as repeated-batch fermentors with pH controlled at 5 for more than 2 months in which each reactor's medium was changed every 3 days for 24 batches. The plastic-composite supports performed better than polypropylene-alone supports. Significantly (P<0.05) higher concentrations of lactic acid were produced by the mixed- and pure-culture biofilm bioreactors with corresponding plastic-composite supports (55 g/l and 60 g/l respectively) than with polypropylene-alone supports (48 g/l for both mixed and pure culture). However, the percentage yields, maximum productivity, glucose consumption rates, and growth rates (based on the mass of suspended cells only) were not significantly different between reactors. Maximum lactic acid concentration was consistently greater for the plastic-composite support biofilm reactors. In the suspension culture at pH 5 without plastic supports, maximum lactic acid concentration at days 3 and 5 was 48 g/l and 60 g/l, respectively. These results confirm that the use of plastic-composite supports is recommended for pure-culture lactic acid production in long-term repeated-batch fermentation, and that cell immobilization was occurring.Journal Paper No. J-15813 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Projects No. 3253 and 0178     

Bioprocess engineering to produce 9-(nonanoyloxy) nonanoic acid by a recombinant <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>-based biocatalyst

Here, Corynebacterium glutamicum ATCC13032 expressing Baeyer–Villiger monooxygenase from Pseudomonas putida KT2440 was designed to produce 9-(nonanoyloxy) nonanoic acid from 10-ketostearic acid. Diverse parameters including cultivation and reaction temperatures, type of detergent, and pH were found to improve biotransformation efficiency. The optimal temperature of cultivation for the production of 9-(nonanoyloxy) nonanoic acid from 10-ketostearic acid using whole cells of recombinant C. glutamicum was 15 °C, but the reaction temperature was optimal at 30 °C. Enhanced conversion efficiency was obtained by supplying 0.05 g/L of Tween 80 at pH 7.5. Under these optimal conditions, recombinant C. glutamicum produced 0.28 mM of 9-(nonanoyloxy) nonanoic acid with a 75.6% (mol/mol) conversion yield in 2 h. This is the first report on the biotransformation of 10-ketostearic acid to 9-(nonanoyloxy) nonanoic acid with a recombinant whole-cell C. glutamicum-based biocatalyst and the results demonstrate the feasibility of using C. glutamicum as a whole-cell biocatalyst.     

Evaluation of biofilm reactor solid support for mixed-culture lactic acid production

A combination of lactobacilli and biofilm-forming bacteria were evaluated in continuous fermentations for lactic acid production using various supports. Twelve different bacteria, including species of Bacillus, Pseudomonas, Streptomyces, Thermoactinomyces, and Thermomonospora were tested for biofilm-forming capabilities. Solid supports that were evaluated in either batch or continuous fermentations were pea gravels, 3M-macrolite ceramic spheres, and polypropylene mixed with 25% of various agricultural materials (e.g. corn starch, oat hulls) and extruded to form chips (pp-composite). Biofilm formation was evaluated by the extent of clumping of solid supports, weight gain and (in some instances) Gram stains of the supports after drying overnight at 70° C. The supports consistently producing the best biofilm were pp-composite chips followed by 3M-Macrolite spheres then by pea gravels. The best biofilm formation was observed with P. fragi (ATCC 4973), S. viridosporus T7A (ATCC 39115), and Thermoactinomyces vulgaris (NRRL B-5790), grown optimally at 25, 37, and 45° C, respectively, on various pp-composite chips. Lactic acid bacteria used in the fermentations were Lactobacillus amylophilus (NRRL B-4437), L. casei (ATCC 11443), and L. delbrueckii mutant DP3; these grow optimally at 25, 37 and 45° C, respectively. Lactic acid and biofilm bacteria with compatible temperature optima were inoculated into 50-ml reactors (void volume 25 ml) containing sterile pp-composite supports. Lactic acid production and glucose consumption were determined by HPLC at various flow rates from 0.06 to 1.92 ml/min. Generally, mixed-culture biofilm reactors produced higher levels of lactic acid than lactic acid bacteria alone. S. viridosporus T7A and L. casei on pp-composite chips were the best combination of those tested, and produced 13.0 g/l lactic acid in the reactors without pH control. L. casei produced 10.3 g/l lactic acid under similar conditions.Journal paper no. J-14840 of the Iowa Agriculture and Home Economics Experiment Station, Ames Iowa. Project nos. 2889 and 0178 Correspondence to: A. L. Pometto     

Kinetic model-based feed-forward controlled fed-batch fermentation of Lactobacillus rhamnosus for the production of lactic acid from Arabic date juice

Arabic date is overproduced in Arabic countries such as Saudi Arabia and Iraq and is mostly composed of sugars (70–80 wt%). Here we developed a fed-batch fermentation process by using a kinetic model for the efficient production of lactic acid to a high concentration from Arabic date juice. First, a kinetic model of Lactobacillus rhamnosus grown on date juice in batch fermentation was constructed in EXCEL so that the estimation of parameters and simulation of the model can be easily performed. Then, several fed-batch fermentations were conducted by employing different feeding strategies including pulsed feeding, exponential feeding, and modified exponential feeding. Based on the results of fed-batch fermentations, the kinetic model for fed-batch fermentation was also developed. This new model was used to perform feed-forward controlled fed-batch fermentation, which resulted in the production of 171.79 g l^?1 of lactic acid with the productivity and yield of 1.58 and 0.87 g l^?1 h^?1, respectively.     

Lactic acid fermentation on barley flour without additional nutrients

Summary An amylolytic lactic acid producing Lactobacillus amylovorus produced 36 g/l of lactic acid in mixed cultures with L. casei without additional nutrients at 37 °C in 48 h, when barley flour concentration was 180 g/l (appr. 108 g/l starch) and barley malt quantity 0.8% of flour weight. This represented an improvement of up to 20% in comparison to the fermentation with L. amylovorus or L. casei alone. By simultaneous glucoamylase addition lactic acid production yield was about doubled. With L. casei the lactic acid yield was from 580 g in 72 h to 667 g in 144 h per kg barley flour.     

Expression of a Cloned Cyclopropane Fatty Acid Synthase Gene Reduces Solvent Formation in Clostridium acetobutylicum ATCC 824

The cyclopropane fatty acid synthase gene (cfa) of Clostridium acetobutylicum ATCC 824 was cloned and overexpressed under the control of the clostridial ptb promoter. The function of the cfa gene was confirmed by complementation of an Escherichia coli cfa-deficient strain in terms of fatty acid composition and growth rate under solvent stress. Constructs expressing cfa were introduced into C. acetobutylicum hosts and cultured in rich glucose broth in static flasks without pH control. Overexpression of the cfa gene in the wild type and in a butyrate kinase-deficient strain increased the cyclopropane fatty acid content of early-log-phase cells as well as initial acid and butanol resistance. However, solvent production in the cfa-overexpressing strain was considerably decreased, while acetate and butyrate levels remained high. The findings suggest that overexpression of cfa results in changes in membrane properties that dampen the full induction of solventogenesis. The overexpression of a marR homologous gene preceding the cfa gene in the clostridial genome resulted in reduced cyclopropane fatty acid accumulation.