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.     

Direct fermentation of starch to L-(+)-lactic acid using Lactobacillus amylophilus

Summary Fermentation of L-(+)-lactic acid from soluble starch by Lactobacillus amylophilus was studied. The bacterium produced about 30 g of L-(+)-lactic acid from 50 g of soluble starch when the pH of the culture was ranging from pH 5 to pH 6.8 at 28°C. 53.4 g of L-(+)-lactic acid was produced when 100 g of starch was added in the medium. The fermentation procedures will reduce the cost of complete hydrolysis of starch to glucose prior to fermentation.     

Selection of mutants of Aspergillus niger showing enhanced productivity of citric acid from starch in shaking culture

Mutants with enhanced citric acid production from soluble starch were induced from Aspergillus niger WU-2223L. After UV-irradiation of a conidial suspension of strain WU-2223L, mutants were selected on modified starch-methyl red agar plates on the basis of higher amylolytic activity and acid productivity. The 8 mutants selected showed enhanced citric acid production from soluble starch in shaking culture. Among them, a representative mutant strain, 2M-43, produced 48.0gg/l of citric acid from 120 g/l of soluble starch in 9 d of cultivation in shaking culture, whereas strain WU-2223L produced 35.1 g/l. Glucoamylase activities in the culture filtrates of strains 2M-43 and WU-2223L reached maximum levels of 3.62 U/ml and 2.11 U/ml, respectively, both at 3 d of cultivation, and thereafter decreased.     

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.     

Display of α-Amylase on the Surface of Lactobacillus casei Cells by Use of the PgsA Anchor Protein, and Production of Lactic Acid from Starch

We developed a new cell surface engineering system based on the PgsA anchor protein from Bacillus subtilis. In this system, the N terminus of the target protein was fused to the PgsA protein and the resulting fusion protein was expressed on the cell surface. Using this new system, we constructed a novel starch-degrading strain of Lactobacillus casei by genetically displaying α-amylase from the Streptococcus bovis strain 148 with a FLAG peptide tag (AmyAF). Localization of the PgsA-AmyA-FLAG fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. The lactic acid bacteria which displayed AmyAF showed significantly elevated hydrolytic activity toward soluble starch. By fermentation using AmyAF-displaying L. casei cells, 50 g/liter of soluble starch was reduced to 13.7 g/liter, and 21.8 g/liter of lactic acid was produced within about 24 h. The yield in terms of grams of lactic acid produced per gram of carbohydrate utilized was 0.60 g per g of carbohydrate consumed at 24 h. Since AmyA was immobilized on the cells, cells were recovered after fermentation and used repeatedly. During repeated utilization of cells, the lactic acid yield was improved to 0.81 g per g of carbohydrate consumed at 72 h. These results indicate that efficient simultaneous saccharification and fermentation from soluble starch to lactic acid were carried out by recombinant L. casei cells with cell surface display of AmyA.     

<Emphasis Type="Italic">Rhizopus arrhizus</Emphasis> – a producer for simultaneous saccharification and fermentation of starch waste materials to l(+)-lactic acid

Rhizopus arrhizus, strain DAR 36017, produced L(+)-lactic acid in a simultaneous saccharification and fermentation process using starch waste effluents. Lactic acid at 19.5-44.3 g l(-1) with a yield of 0.85-0.96 g g(-1) was produced in 40 h using 20-60 g starch l(-1). Supplementation of nitrogen source may be unnecessary if potato or corn starch waste effluent was used as a production medium.     

Lactic acid production directly from starch in a starch-controlled fed-batch operation using Lactobacillus amylophilus

Based on the batch results, we constructed a simplified simultaneous saccharification and fermentation (SSF) model for the simulation of lactic acid production directly from unhydrolyzed potato starch using Lactobacillus amylophilus. The results of batch operation at different initial starch concentrations (20, 40 and 60 g/l) indicated that a higher initial starch concentration would lead to a slightly lower productivity, but would largely decrease the yield. Among that, the batch with 20 g/l of initial starch had the maximum productivity and the maximum yield, which would be 0.31 g/(l h) and 98% (g/g), respectively. In view of increasing the productivity and the final lactic acid concentration, a starch-controlled fed-batch operation with 20 g/l of initial starch was performed. It showed the fed-batch operation with starch controlled at 8 ± 1 g/l by adjusting the starch-feeding rate led to the maximum productivity of 0.75 g/(l h) and the yield of 69%.     

Direct fermentative production of lactic acid on cassava and other starch substrates

Lactobacillus amylovorus utilized raw corn, rice and wheat starch medium to produce lactic acid with a productivity of 10.1, 7.9 and 7.8 g lactic acid/L, but had lower productivities of 4.8 g/L and 4.2 g/L on cassava and potato starch in basal medium respectively. When peptone (1%) is added to basal medium with cassava starch as substrate, conversion rate increased from 43% conversion to 70% conversion (7.7 g lactic acid/L). The availability of some components of protein in corn starch is assumed to be the reason for high lactic acid production as compared to that of cassava starch.     

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     

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.     

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.     

Formation of autodiploid strains in Aspergillus niger and their application to citric acid production from starch

Autodiploid strains were induced by colchicine treatment of Aspergillus niger WU-2223L, a citric acid-producing strain. In shaking culture, a representative autodiploid strain, L-d1, yielded higher citric acid than the parental strain, WU-2223L. When glucose was used as a carbon source, L-d1 and WU-2223L produced 67.2 g/l and 62.0 g/l of citric acid, respectively, from 120 g/l of glucose in 9 d-cultivation. Furthermore, the autodiploid strain L-d1 produced 49.6 g/l of citric acid, 1.4 times as much as that produced by WU-2223L from 120 g/l of soluble starch. During the whole period of cultivation with starch, the extracellular glucoamylase activity of L-d1 was on the same level as that of WU-2223L, but the extracellular acid-protease activity of L-d1 was much higher. The addition of pepstatin, an inhibitor of acid protease, to the culture broth at 2 d greatly increased the extracellular glucoamylase activity, and citric acid production by L-d1 reached a level of 59.0 g/l. During several subcultivations on both minimal and complete agar media, the autodiploid strains were genetically stable since they formed diploid conidia in their uniform colonies without producing sectors, and maintained citric acid productivity. However, when cultivated on minimal and complete agar media containing benomyl as a haploidizing reagent, the autodiploid strains readily formed sectors of haploid segregants. The properties of the haploid strains obtained by the benomyl treatment of the autodiploid strains were similar in morphology and citric acid productivity to those of the parental strain, WU-2223L. These results indicated that the enhanced production of citric acid from soluble starch by the autodiploid strains was due to autodiploid formation but not to gene mutation caused by the colchicine treatment.     

Direct production of L+-lactic acid from starch and food wastes using Lactobacillus manihotivorans LMG18011

This study describes several essential factors for direct and effective lactic acid production from food wastes by Lactobacillus manihotivorans LMG18011, and optimum conditions for simultaneous saccharification and fermentation using soluble starch and food wastes as substrates. The productivity was found to be affected by three factors: (1) initial pH, which influenced amylase production for saccharification of starch, (2) culture pH control which influenced selective production of L(+)-lactic acid, and (3) manganese concentration in medium which improved in production rate and yield of lactic acid. The optimum initial pH was 5.0-5.5, and the fermentation pH for the direct and effective fermentation from starchy substrate was 5.0 based on the yield of L(+)-lactic acid. Under these conditions, 19.5 g L(+)-lactic acid was produced from 200 g food wastes by L. manihotivorans LMG18011. Furthermore, the addition of manganese stimulated the direct fermentation significantly, and enabled complete bioconversion within 100 h.     

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.

     

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.     

Simultaneous saccharification and co-fermentation of crystalline cellulose and sugar cane bagasse hemicellulose hydrolysate to lactate by a thermotolerant acidophilic Bacillus sp

Polylactides produced from renewable feedstocks, such as corn starch, are being developed as alternatives to plastics derived from petroleum. In addition to corn, other less expensive biomass resources can be readily converted to component sugars (glucose, xylose, etc.) by enzyme and/or chemical treatment for fermentation to optically pure lactic acid to reduce the cost of lactic acid. Lactic acid bacteria used by the industry lack the ability to ferment pentoses (hemicellulose-derived xylose and arabinose), and their growth and fermentation optima also differ from the optimal conditions for the activity of fungal cellulases required for depolymerization of cellulose. To reduce the overall cost of simultaneous saccharification and fermentation (SSF) of cellulose, we have isolated bacterial biocatalysts that can grow and ferment all sugars in the biomass at conditions that are also optimal for fungal cellulases. SSF of Solka Floc cellulose by one such isolate, Bacillus sp. strain 36D1, yielded l(+)-lactic acid at an optical purity higher than 95% with cellulase (Spezyme CE; Genencor International) added at about 10 FPU/g cellulose, with a product yield of about 90% of the expected maximum. Volumetric productivity of SSF to lactic acid was optimal between culture pH values of 4.5 and 5.5 at 50 degrees C. At a constant pH of 5.0, volumetric productivity of lactic acid was maximal at 55 degrees C. Strain 36D1 also co-fermented cellulose-derived glucose and sugar cane bagasse hemicellulose-derived xylose simultaneously (SSCF). In a batch SSCF of 40% acid-treated hemicellulose hydrolysate (over-limed) and 20 g/L Solka Floc cellulose, strain 36D1 produced about 35 g/L lactic acid in about 144 h with 15 FPU of Spezyme CE/g cellulose. The maximum volumetric productivity of lactic acid in this SSCF was 6.7 mmol/L (h). Cellulose-derived lactic acid contributed to about 30% of this total lactic acid. These results show that Bacillus sp. strain 36D1 is well-suited for simultaneous saccharification and co-fermentation of all of the biomass-derived sugars to lactic acid.     

Direct fermentation of starch to lactic acid byLactobacillus amylovorus

Summary Fermentation production of lactic acid directly from starch was studied in a batch fermentor usingLactobacillus amylovorus. At an initial concentration of 120 g/L starch, 96.2 g/L of lactic acid was produced from liquefied starch in 20 hours while 92.5 g/L of lactate was produced from the raw starch in 39 hours. High initial glucose levels (100 g/L) in the medium inhibited the organism, unless it had been adapted by growing it in a low-glucose medium. The direct production of lactic acid from starch could reduce overall production costs significantly.     

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.     

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.     

Direct fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of direct fermentation for lactic acid production by fungal species of Rhizopus arrhizus 3,6017 and Rhizopus oryzae 2,062 was studied with respect to growth pH, temperature and substrate. The direct fermentation was characterized by starch hydrolysis, accumulation of reducing sugar, and production of lactic acid and fungal biomass. Starch hydrolysis, reducing sugar accumulation, biomass formation and lactic acid production were affected with the variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30°C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.87–0.97 g/g starch associated with 1.5–2.0 g/l fungal biomass produced in 36 h fermentation. R. arrhizus 3,6017 had a higher capacity to produce lactic acid, while R. oryzae 2,062 produced more fungal biomass under similar conditions.