Lactic acid production from enzyme-thinned corn starch usingLactobacillus amylovorus

Summary An alternative process for industrial lactic acid production was deveooped using a starch degrading lactic acid producing organism,Lactobacillus amylovorus B-4542. In this process, saccharification takes place during the fermentation, eliminating the need for complete hydrolysis of the starch to glucose prior to fermentation. The cost savings of this alternative are substantial since it eliminates the energy input, separate reactor tank, time, and enzyme associated with the typical pre-fermentation saccharification step. The only pre-treatment was gelatinization and enzyme-thinning of the starch to overcome viscosity problems associated with high starch concentrations and to make the starch more rapidly degradable. This fermentation process was optimized for temperature, substrate level, nitrogen source and level, mineral level, B-vitamins, volatile fatty acids, pH, and buffer source. The rate of the reaction and the final level of lactic acid obtained in the optimized liquefied starch process was similar to that obtained withL. delbrueckii B-445 using glucose as the substrate.     

Lactic acid production by aggregated continuous cultures of Lactobacillus acidophilus under aerobic conditions

Summary The ability of Lactobacillus acidophilus to aggregate, to produce lactic acid for a long term continuous fermentation process and to exist as aggregate cell cultures in a gas-lift reactor under aerobic conditions was studied. The main product of fermentation was lactic acid and only the traces of other end-products were determined. The highest fermentation efficiency of lactic acid was 98.6% and the highest productivity was 9.6 g.l^–1.h^–1 of lactic acid.     

Homo-d-lactic acid production from mixed sugars using xylose-assimilating operon-integrated Lactobacillus plantarum

In order to achieve efficient D-lactic acid fermentation from a mixture of xylose and glucose, the xylose-assimilating xylAB operon from Lactobacillus pentosus (PXylAB) was introduced into an L-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (ΔldhL1-xpk1::tkt-Δxpk2) strain in which the phosphoketolase 1 gene (xpk1) was replaced with the transketolase gene (tkt) from Lactococcus lactis, and the phosphoketolase 2 (xpk2) gene was deleted. Two copies of xylAB introduced into the genome significantly improved the xylose fermentation ability, raising it to the same level as that of ΔldhL1-xpk1::tkt-Δxpk2 harboring a xylAB operon-expressing plasmid. Using the two-copy xylAB integrated strain, successful homo-D-lactic acid production was achieved from a mixture of 25 g/l xylose and 75 g/l glucose without carbon catabolite repression. After 36-h cultivation, 74.2 g/l of lactic acid was produced with a high yield (0.78 g per gram of consumed sugar) and an optical purity of D-lactic acid of 99.5%. Finally, we successfully demonstrated homo-D-lactic acid fermentation from a mixture of three kinds of sugar: glucose, xylose, and arabinose. This is the first report that describes homo-D-lactic acid fermentation from mixed sugars without carbon catabolite repression using the xylose-assimilating pathway integrated into lactic acid bacteria.     

Lactic acid fermentation by cells of Lactobacillus rhamnosus immobilized in polyacrylamide gel

Summary The process of lactic acid fermentation of lactose to lactic acid by Lactobacillus rhamnosus ATCC 7469 has been studied. The following processes have been explored: growth kinetics, as well as lactose utilization, production of lactic acid and further degradation of lactic acid. The immobilization experiments were conducted with microbial cells entrapped in polyacrylamide gels. Gels with different ratios of the monomer (acrylamide) and the cross-linking agent (N,N′-methylene-bis-acrylamide) have been tested. These were used in a repeat-batch process. The current processes inside and outside the gel particles were subjects of examination. The evolution of the activity of immobilized cells with repeated use showed that the particles served mainly as a donor of cells for the free culture. In all experiments a very high degree of conversion, 85–90% was observed. After several runs however, the particles were exhausted for microbial cells. A kinetic model of the process of lactic acid production was developed. This model allowed the evaluation of the effect of microbial growth and diffusion limitations inside the gel particles on the process rate and the separate contribution of the free and immobilized cells to the overall fermentation process upon multiple use.     

Lactic acid production from submerged fermentation of broken rice using undefined mixed culture

The present work aimed to characterize and optimize the submerged fermentation of broken rice for lactic acid (LA) production using undefined mixed culture from dewatered activated sludge. A microorganism with amylolytic activity, which also produces LA, Lactobacillus amylovorus, was used as a control to assess the extent of mixed culture on LA yield. Three level full factorial designs were performed to optimize and define the influence of fermentation temperature (20–50?°C), gelatinization time (30–60 min) and broken rice concentration in culture medium (40–80 g L^?1) on LA production in pure and undefined mixed culture. LA production in mixed culture (9.76 g L^?1) increased in sixfold respect to pure culture in optimal assessed experimental conditions. The optimal conditions for maximizing LA yield in mixed culture bioprocess were 31?°C temperature, 45 min gelatinization time and 79 g L^?1 broken rice concentration in culture medium. This study demonstrated the positive effect of undefined mixed culture from dewatered activated sludge to produce LA from culture medium formulated with broken rice. In addition, this work establishes the basis for an efficient and low-cost bioprocess to manufacture LA from this booming agro-industrial by-product.     

Lactic acid production from whey permeate by immobilized Lactobacillus casei

Two matrices have been assessed for their ability to immobilize Lactobacillus casei cells for lactic acid fermentation in whey permeate medium. Agar at 2% concentration was found to be a better gel than polyacrylamide in its effectiveness to entrap the bacterial cells to carry out batch fermentation up to three repeat runs. Of the various physiological parameters studied, temperature and pH were observed to have no significant influence on the fermentation ability of the immobilized organism. A temperature range of 40–50°C and a pH range of 4.5–6.0 rather than specific values, were found to be optimum when fermentation was carried out under stationary conditions. In batch fermentation ～90% conversion of the substrate (lactose) was achieved in 48 h using immobilized cell gel cubes of 4 × 2 × 2 mm size, containing 400 mg dry bacterial cells per flask and 4.5% w/v (initial) whey lactose content as substrate. However, further increase in substrate levels tested (>4.5% w/v) did not improve the process efficiency. Supplementation of Mg²⁺ (1 mM) and agricultural by-products (mustard oil cake, 6%) in the whey permeate medium further improved the acid production ability of the immobilized cells under study.     

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%.     

Lactic acid production from molasses utilizing Lactobacillus delbrueckii and invertase together

Conclusion The prices of the process substrates such as glucose, sucrose and molasses (as $/ton) are 1500, 1600 and 24, respectively. For molasses plus invertase, the price increases to 46 $/ton. Thus compared with the other possible substrates, the lactic acid production procedure used in this study does not cause any appreciable increase in the pruduction cost due to the utilization of invertase, while enhancing the yield of product.     

Elucidation of the mechanism of lactic acid growth inhibition and production in batch cultures of Lactobacillus rhamnosus

Batch cultures of Lactobacillus rhamnosus were carried out at different pH values in order to study the limitation of growth and lactic acid production by the hydrogen ion, non-dissociated lactic acid and internal lactate concentrations. The effect of pH between 5 and 6.8 was studied at non-limiting concentrations of glucose; this is more significant for the lactic acid fermentation rate than for the maximum specific growth rate, as shown by the incomplete substrate consumption at lower values of medium pH and by the constant maximum cell mass obtained within the range of pH values studied. To check whether these results were a direct consequence of the different concentrations of the non-dissociated form of lactic acid at different external pH values, specific growth rates and lactic acid productions rates were calculated for each external pH value. The same specific growth rates were observed at the same non-dissociated lactic acid concentrations only at pH values of 5 and 5.5. For higher values of pH (pH > 6) the specific growth rate falls to zero as the non-dissociated lactic acid concentration decreases. This shows that generalisations made from studies performed within very narrow ranges of pH are not valid and that the non-dissociated form of lactic acid is not the only inhibiting species. The internal pH was measured experimentally for each external pH value in order to calculate the internal lactate ion concentration. This form is described to be the inhibitory one. The results obtained confirmed that the specific growth rate reached zero at approximately the same lactate concentration for all the pH values studied. Received: 31 January 1997 / Received revision: 15 May 1997 / Accepted: 19 May 1997     

Kinetic studies of corn stover saccharification using sulphuric acid

The kinetics of crystalline cellulose and hemicellulose hydrolysis in corn stover were studied with a nonisothermal technique. Reactions were arrested at temperatures between 160 and 240 degrees C and product sugars were analyzed using a Bio-Rad HPX-85 liquid chromatographic column. A simple first-order series reaction model was used for both cellulose and hemicellulose hydrolysis reactions. Kinetic parameters were obtained for three different sulphuric acid concentrations (0.49, 0.92, and 1.47 wt %). Activation energies remained constant over this acid concentration range but the preexponential factors showed an increase with acid concentration. Relationships were obtained between the preexponential factors and acid concentrations. Cellulose hydrolysis and glucose degradation reactions were observed to be of higher order with respect to acid concentration in comparison with the previous studies with other raw materials.     

The production of mixed cultures containing strains of Lactococcus lactis, Leuconostoc cremoris and Lactobacillus rhamnosus, on commercial starter media

Mixed starters containing Lactococcus lactis, Leuconostoc cremoris and Lactobacillus rhamnosus strains were produced on commercial starter media (MB Complete, Thermolac, Marlac), as well as on milk. With the exception of Marlac, the starters were cultured under pH control. The effect of media and incubation temperature (22 or 32°C) on population ratios, on specific acidifying activities (SAA) of the cultures as well as on their ability to produce aroma compounds in milk was studied. The starters had higher contents in lactobacilli when they were produced at 32°C, whereas a tendency to obtain higher Leuconostoc populations was observed at 22°C. With respect to the lactococci, there was a significant interaction between temperature and growth medium for both strains. Thus, Le. cremoris T2 reached higher populations at 32°C if grown in MB complete and Thermolac, whereas in Marlac and skim milk, viable counts were higher at 22°C. The lactococci represented 50% of the total population of the culture at the beginning of the incubation, but they composed between 80% and 99% of the total population following fermentation. The best medium for growth of Leuconostoc was milk, but populations of only 10⁸ cfu/ml were reached. The lactobacilli did not grow well in MB Complete, and their development was best in the low-phosphate Marlac medium. The cultures grown on Marlac had the highest SAA values, whereas those grown on MB complete had the lowest. Overall, more ethanol and diacetyl were detected in the fermented milks when the starters used to inoculate them were produced at 22°C. Journal of Industrial Microbiology & Biotechnology (2000) 25, 288–297. Received 23 June 2000/ Accepted in revised form 22 September 2000     

Exopolysaccharide production during batch cultures with free and immobilized Lactobacillus rhamnosus RW-9595M

AIMS: Exopolysaccharides (EPS) were produced by Lactobacillus rhamnosus RW-9595M during pH-controlled batch cultures with free cells and repeated-batch cultures with cells immobilized on solid porous supports (ImmobaSil). METHODS AND RESULTS: Cultures were conducted in supplemented whey permeate (SWP) medium containing 5 or 8% (w/w) whey permeate. For free-cell batch cultures in 8% SWP medium, very high maximum cell counts (1.3 x 10(10) CFU ml(-1)) and EPS production (2350 mg l(-1)) were measured. A high EPS production (1750 mg l(-1)) was measured after four cycles for a short incubation period of only 7 h. Several methods for immobilized biomass determination based on analysis of biomass components (proteins, ATP and DNA) were tested. The DNA analysis method proved to be the most appropriate under these circumstances. This method revealed a high maximum immobilized biomass of 8.5 x 10(11) CFU ml(-1) support during repeated immobilized cell cultures in 5% SWP. The high immobilized biomass increased maximum EPS volumetric productivity (250 mg l(-1) h(-1) after 7 h culture) compared with free-cell batch cultures (110 mg l(-1) h(-1) after 18 h culture). CONCLUSIONS: High EPS productions were achieved during batch cultures of Lact. rhamnosus RW-9595M in SWP medium, exceeding 1.7 g EPS per litre. Repeated-batch cultures with immobilized cells resulted in increased EPS productivity compared with traditional free-cell cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The study clearly shows the high potential of the strain Lact. rhamnosus RW-9595M and immobilized cell technology for production of EPS as a functional food ingredient.     

Lactic acid production from sugar-cane juice by a newly isolated Lactobacillus sp.

A newly isolated sucrose-tolerant, lactic acid bacterium, Lactobacillus sp. strain FCP2, was grown on sugar-cane juice (125 g sucrose l⁻¹, 8 g glucose l⁻¹ and 6 g fructose l⁻¹) for 5 days and produced 104 g lactic acid l⁻¹ with 90% yield. A higher yield (96%) and productivity (2.8 g l⁻¹ h⁻¹) were obtained when strain FCP2 was cultured on 3% w/v (25 g sucrose l⁻¹, 2 g glucose l⁻¹ and 1 g fructose l⁻¹) sugar-cane juice for 10 h. Various cheap nitrogen sources such as silk worm larvae, beer yeast autolysate and shrimp wastes were also used as a substitute to yeast extract.     

Continuous cultivation of Lactobacillus brevis NCL912 for production of gamma-aminobutyric acid

A continuous cultivation method for Lactobacillus brevis NCL912 to synthesize gamma-aminobutyric acid was developed in this work. Different dilution rates were evaluated for obtaining steady state in continuous cultivation. The results showed that steady state could be achieved at dilution rates from 0.08 to 0.12 h^?1. The highest gamma-aminobutyric acid productivity (5.11 g L^?1?h^?1) was obtained at dilution rate of 0.09 h^?1. The kinetic models were established for continuous gamma-aminobutyric acid production by using the Monod equation for microbial growth, and the Luedeking–Piret equation for product formation. The microbial growth and product formation can be described by equations $ \mu = {{{0.1234{C_S}}} \left/ {{\left( {0.9338+{C_S}} \right)}} \right.} $ and $ {Q_P}=6.86\,\mathrm{g}\,{{\mathrm{g}}^{-1 }}\mathrm{cell}\,{{\mathrm{h}}^{-1 }} $ , respectively. The production of gamma-aminobutyric acid by L. brevis NCL912 was non-growth-associated.     

Medium optimization for production of gamma-aminobutyric acid by Lactobacillus brevis NCL912

Production of gamma-aminobutyric acid (GABA) was carried out in Erlenmeyer flasks by Lactobacillus brevis NCL912. Traditional methods were first adopted to select the key factors that impact the GABA production to preliminarily determine the suitable concentration ranges of the key factors. It was found that glucose, soya peptone, Tween-80 and MnSO₄·4H₂O were the key factors affecting GABA production. Then, response surface methodology was applied to analyze the optimum contents of the four key factors in the medium, and the production of GABA was predicted as 349.69 mM under the optimized conditions with this model. Afterward, the experiment was performed under the optimized conditions, and the yield of GABA reached 345.83 mM, which was 130% higher than the initial medium. The results showed that experimental yield and predicted values of GABA yield were in good agreement.     

Genome shuffling enhanced L-lactic acid production by improving glucose tolerance of Lactobacillus rhamnosus

Genome shuffling is a powerful strategy for rapid engineering of microbial strains for desirable industrial phenotypes. Here we applied the genome shuffling to improve the glucose tolerance of Lactobacillus rhamnosus ATCC 11443 while simultaneously enhancing the L-lactic acid production. The starting population was generated by ultraviolet irradiation and nitrosoguanidine mutagenesis and then subjected for the recursive protoplast fusion. The positive colonies from library created by fusing the inactivated protoplasts were more likely to be screened on plates containing different concentrations of high glucose and 2% CaCO(3). Characterization of all mutants and wild-type strain in the shake flask indicated the compatibility of two optimal phenotypes of glucose tolerance and lactic acid enhancement. The lactic acid production, cell growth and glucose consumption of the best performing strain from the second round genome shuffled populations were 71.4%, 44.9% and 62.2% higher than those of the wild type at the initial glucose concentration of 150 g/l in the 16l bioreactor. Furthermore, the higher lactic acid concentrations were obtained when the initial glucose concentrations increased to 160 and 200 g/l in batch fermentation.     

Lactic acid production from agriculture residues

Various agriculture feedstock residues were evaluated for lactic acid production by simultaneous saccharification and fermentation (SSF) using Lactobacillus delbrueckii and Lactobacillus plantarum, without any additional nutrients. Lactic acid production was higher in alfalfa fiber and soya fiber compared to corncob (soft) and wheat straw. In Lactobacillus plantarum, the amount of lactic acid obtained from alfalfa fiber and soya fiber was 46 and 44 g/100 g fiber, respectively. However, in Lactobacillus delbrueckii, the lactic acid production in soya fiber was 44 g/100 g fiber and that of alfalfa was 32 g/100 g fiber. Small amounts of acetic acid were also produced from SSF of agricultural feedstocks residues. During SSF of alfalfa fiber, lactic acid production in both L. delbrueckii and L. plantarum was enhanced by adding pectinases and cellulases. Lactic acid production from alfalfa fiber did not change with increasing O₂ transfer rates in the fermentation medium, whereas acetic acid production in both Lactobacillus cultures increased with increasing O₂ transfer rates.     

Cellulase production from the corn stover fraction based on the organ and tissue

In this study, the biodegradation of fractionated corn stover in solid-state fermentation by Trichoderma reesei YG3 was investigated. Fractions of miscellaneous cells (MC) and fasciculi (FC) tissue from leaf, shell or core were separated using the combined pretreatment method of carding classification after steam explosion. The highest enzyme activities including the filter paper activity, endoglucanase, exoglucanase and β-glucosidase activities, weight loss rate of dry material and biodegradation rate were all observed in the MC tissue fraction from the leaf, which was more nutritious, while lowest activity was observed in the FC tissue fraction from the shell. The maximum filter paper activity and weight loss rate of the dry material were 4.56 and 1.89 times the minimum and the cellulose and hemicellulose biodegradation rates were 51.22 and 39.38% versus 23.85 and 26.51%, respectively. These variances maybe attributed to the heterogeneity of the component in the fractions. A higher weight loss rate corresponded to higher enzymatic activities, whereas cellulose biodegradation was not proportional to cellulase activities. Hemicellulose biodegradation was much slower than cellulose degradation. Here, we demonstrated the importance of fractionation in component biodegradation and utilization of straw.     

Lactic acid production by Lactobacillus delbreuckii in a hollow fiber fermenter

Summary Lactic acid was produced by viable Lactobacillus delbreuckii NRRL-B445 in a hollow fiber fermenter. Final cell densities in the fluid surrounding the fibers in the fermenter were apparently as high as 480 gms DW/L, and volumetric productivities reached 100 gms/L-hr lactic acid. The observed cell yields were appreciably lower than batch cell yields.