Effect of pH control on lactic acid fermentation of starch by Lactobacillus manihotivorans LMG 18010T

Lactic acid fermentation of starch by Lactobacillus manihotivorans LMG 18010T, a new amylolytic L(+) lactic acid producer, was investigated and compared with starch fermentation by Lact. plantarum A6. At non-controlled pH, growth and lactic acid production from starch by Lact. manihotivorans LMG 18010T lasted 25 h. Specific growth and lactic acid production rates continuously decreased from the onset of the fermentation, unlike Lact. plantarum A6 which was able to grow and convert starch product hydrolysis into lactic acid more rapidly and efficiently at a constant rate up to pH 4.5. In spite of complete and rapid starch hydrolysis by Lact. manihotivorans LMG 18010T during the first 6 h, only 45% of starch hydrolysis products were converted to lactic acid. When pH was maintained at 6.0, lactic acid, amylase and final biomass production by Lact. manihotivorans LMG 18010T increased markedly and the fermentation time was reduced by half. Under the same conditions, an increase only in amylase production was observed with Lact. plantarum A6. When grown on glucose or starch at pH 6.0, Lact. manihotivorans LMG 18010T had an identical maximum specific growth rate (0.35 h(-1)), whereas the maximum rate of specific lactic acid production was three times higher with glucose as substrate. Lactobacillus manihotivorans LMG 18010T did not produce amylase when grown on glucose. Based on the differences in the physiology between the two species and other amylolytic lactic acid bacteria, different applications may be expected.     

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.     

Simultaneous liquefaction,saccharification, and fermentation of l-lactic acid using aging paddy rice with hull by an isolated thermotolerant Enterococcus faecalis DUT1805

Simultaneous liquefaction, saccharification, and fermentation (SLSF) has attracted much attention for the production of bio-based chemicals, including l-lactic acid, due to its high efficiency and low cost. In this study, a lactic acid-producing bacterium with high tolerance of temperature up to 55 °C was isolated and characterized as Enterococcus faecalis DUT1805. Various strategies of stepwise controlled temperature were proposed and investigated for glucose utilization. The results indicated that E. faecalis DUT 1805 exhibited an optimal temperature at 50 °C, which could achieve temperature compatibility of enzyme, saccharification, and fermentation, and decrease the possibility of contamination by the other microorganisms during the large-scale fermentation. To reduce the cost of raw material and operation for lactic acid production, aging paddy rice with hull (APRH) was used in l-lactic acid production by simultaneous liquefaction, saccharification, and fermentation (SLSF). An open SLSF operation at 50 °C and pH 6.5, and 17% (w/v) solid loading in 5 L bioreactors was demonstrated with the lactic acid titer, yield, and productivity of 73.75 g/L, 87% to initial starch, and 2.17 g/(L h), respectively.

     

Electrotransformation of Lactobacillus manihotivorans LMG 18010T and LMG 18011

An electroporation procedure was developed for the genetic transformation of intact cells of Lactobacillus manihotivorans , a new Lactobacillus species isolated from cassava sour starch fermentation in Colombia. Transformation efficiency of Lact. manihotivorans strains LMG 18010^T and LMG 18011 was measured and compared with electroporability of Lact. plantarum strains NCIMB 8299 and LMG 9211, by investigating the effect of changes in various parameters. For Lact. manihotivorans strain LMG 18010^T, the composition of the culture medium, such as the type of peptone and the presence of Tween-80, was found to be the most critical parameter, as well as the aeration conditions of the culture; better electroporation was obtained without air. The presence of MgCl₂ in the recovery medium was favourable to regeneration of electroporated cells. Plasmid-curing of the cells did not improve their electroporability. Transformants were obtained with Lact. manihotivorans strain LMG 18010^T and the plasmids pLZ12 and pGK13, whereas Lact. manihotivorans strain LMG 18011 was transformable with plasmids pLP825 and pLZ12, with different electroporation conditions.     

L-乳酸调控乳酸产生菌产物光学纯度的分析

发酵初期在米根霉菌发酵培养基中添加L-乳酸可以调控发酵产物乳酸的光学纯度。随着L-乳酸添加量的增加,所产L-乳酸的光学纯度随之增加,当L-乳酸的添加量≥1.5g/L时,D-乳酸不再产生。同时,L-乳酸的产量、生物量、糖转化率也随之降低。该调控方法对乳酸菌调控产L-乳酸光学纯度影响不大,对大肠杆菌发酵调控产D-乳酸光学纯度没有效果。     

<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.     

Enhanced isomer purity of lactic acid from the non-sterile fermentation of kitchen wastes

In order to improve the purity of lactic acid isomers, the effects of pH, temperature, fermentation time and their interactions on l(+) or d(-)-lactic acid production were evaluated during lactic acid fermentation of the non-sterile kitchen wastes. The results showed that l(+)-lactic acid was the main isomeric form. The isomer purity was much higher at acidic or alkalic pH (non-controlled pH, pH 5 and pH 8) than neutral pH (pH 6 and pH 7). Increasing the fermentation temperature from 35 degrees C to 45 degrees C at pH 7 enhanced the isomer purity from 60:40 to 83:17. The optimal fermentation time for the purity of lactic acid isomers was found to depend on the corresponding pH and temperature. From the response surface analysis, the optimized combination of pH and temperature could obviously increase the l(+)-isomer concentration. It is confirmed that the variation of the isomer purity with pH, temperature and fermentation time change resulted from the substitution of microbial community composition. The lactic acid bacteria and Clostridium sp. dominated the fermentation of non-sterile kitchen wastes, and the emergence and disappearance of lactic acid bacteria which produced l(+)-isomer and Clostridium sp. resulted in the variations of the isomer purity.     

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.     

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.     

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.     

Simultaneous saccharification and L-(+)-lactic acid fermentation of protease-treated wheat bran using mixed culture of lactobacilli

Protease-treated wheat bran (20% w/v) of particle size less than 300 μm containing 65% (w/w) starch was used for the simultaneous saccharification and l-(+)-lactic acid fermentation by the mixed cultures of Lactobacillus casei and Lactobacillus delbrueckii. Maximum lactate yield after various process optimizations was 123 gl⁻¹ with a productivity of 2.3 gl⁻¹ h⁻¹ corresponding to a conversion of 0.95 g lactic acid per gram starch after 54 h at 37°C. By using protease-treated wheat bran around tenfold decrease in supplementation of the costly medium component, like yeast extract, was achieved together with a considerable increase in the production level.     

Direct conversion of starch to L(+) lactic acid by amylase producing Lactobacillus amylophilus GV6

Lactobacillus amylophilus strain GV6, isolated from corn starch processing industrial wastes, was amylolytic and produced 0.96?g L(+) lactic acid per gram of soluble starch. The optimum temperature and pH for growth and L(+) lactic acid production were 37?°C and 6.5, respectively. At low substrate concentrations, the lactic acid production on corn starch was almost similar to soluble starch. The strain is fermenting various naturally available starches directly to lactic acid. The total amylase activity of the strain is 0.59?U/ml/min. The strain produced 49 and 76.2?g/l L(+) lactic acid from 60?g/l corn starch and 90?g/l soluble starch, respectively. This is the highest L(+) lactic acid among the wild strains of L. amylophilus reported so far.     

Chitin and L(+)-lactic acid production from crab (Callinectes bellicosus) wastes by fermentation of Lactobacillus sp. B2 using sugar cane molasses as carbon source

Crab wastes are employed for simultaneous production of chitin and L(+)-lactic acid by submerged fermentation of Lactobacillus sp. B2 using sugar cane molasses as carbon source. Response surface methodology was applied to design the culture media considering demineralization. Fermentations in stirred tank reactor (2L) using selected conditions produced 88% demineralization and 56% deproteinization with 34% yield of chitin and 19.5 gL(-1) of lactic acid (77% yield). The chitin purified from fermentation displayed 95% degree of acetylation and 0.81 and 1 ± 0.125% of residual ash and protein contents, respectively.     

Use of inexpensive nitrogen sources and starch for L(+) lactic acid production in anaerobic submerged fermentation

L(+) Lactic acid fermentation was studied by Lactobacillus amylophilus GV6 under the influence of inexpensive nitrogen sources (red lentil-RL, and Baker's yeast cells-YC) and starch by response surface methodology (RSM). Central composite rotatable design (CCRD) was employed to determine maximum lactic acid production at optimum values for process variables RL, YC and incubation period (IP) and a satisfactory fit model was realized. Lactic acid production was significantly affected by RL and IP interactions as well as by independent variables RL and YC. Maximum lactic acid production of 13.5 g/15.2g starch was obtained with RL 0.8%, YC 1% and IP of 48 h, with 92% lactic acid yield efficiency (g lactic acid produced/g substrate utilized) and 40% increase (from 50 g to 92 g/100 g starch utilized) in lactic acid production. This is the first report on response optimization in direct fermentation of starch to lactic acid using inexpensive nitrogen sources substituting peptone and yeast extract in anaerobic submerged fermentation by amylolytic lactic acid bacteria (LAB).     

Effects of Cultivation Parameters on the Morphology of Rhizopus arrhizus and the Lactic Acid Production in a Bubble Column Reactor

The use of filamentous Rhizopus for lactic acid production is facing a challenge due to its low yield mainly caused by the difficulty to control its morphology in submerged fermentation processes. This study was aimed at investigating the impacts of cultivation parameters on the morphology of Rhizopus arrhizus DAR 36017 and lactic acid production using waste potato starch in a laboratory scale bubble column reactor (BCR). The fungal morphology was significantly influenced by carbon sources, process pH, starch concentrations, sparger designs and aeration rates. The favorable morphology for lactic acid production was a freely dispersed small pellet, which was achieved under operation conditions at pH 5.0–6.0, starch concentrations of 60–120 g/L and aeration rates of 0.2–0.8 vvm using a sintered stainless steel disc sparger. Optimal cultivation conditions at pH 6.0 and an aeration rate of 0.4 vvm resulted in the formation of freely dispersed small pellets and 103.8 g/L lactic acid with a yield of 87 % from 120 g/L liquefied potato starch in 48 h. The overall results in terms of lactic acid yield and productivity are comparable to those reported in previous studies using immobilized Rhizopus cells in batch fermentations.     

Production of D-lactic acid from defatted rice bran by simultaneous saccharification and fermentation

Production of d-lactic acid from rice bran, one of the most abundant agricultural by-products in Japan, is studied. Lactobacillus delbrueckii subsp. delbrueckii IFO 3202 and defatted rice bran powder after squeezing rice oil were used for the production. Since the rice bran contains polysaccharides as starch and cellulose, we coupled saccharification with amylase and cellulase to lactic acid fermentation. The indigenous bacteria in the rice bran produced racemic lactic acid in the saccharification at pH 6.0-6.8. Thus the pH was controlled at 5.0 to suppress the growth of the indigenous bacteria. L. delbrueckii IFO 3202 produced 28 kgm(-3) lactic acid from 100 kgm(-3) rice bran after 36 h at 37 degrees C. The yield based on the amount of sugars soluble after 36-h hydrolysis of the bran by amylase and cellulase (36 kgm(-3) from 100 kgm(-3) of the bran) was 78%. The optical purity of produced d-lactic acid was 95% e.e.     

Simultaneous saccharification and fermentation of cellulose for lactic acid production

Lactic acid production from α-cellulose by simultaneous saccharification and fermentation (SSF) was studied. The cellulose was converted in a batch SSF using cellulase enzyme Cytolase CL to produce glucose sugar andLactobacillus delbrueckii to ferment the glucose to lactic acid. The effects of temperature, pH, yeast extract loading, and lactic acid inhibition were studied to determine the optimum conditions for the batch processing. Cellulose was converted efficiently to lactic acid, and enzymatic hydrolysis was the rate controlling step in the SSF. The highest conversion rate was obtained at 46°C and pH 5.0. The observed yield of lactic acid from α-cellulose was 0.90 at 72 hours. The optimum pH of the SSF was coincident with that of enzymatic hydrolysis. The optimum temperature of the SSF was chosen as the highest temperature the microorganism could withstand. The optimum yeast extract loading was found to be 2.5 g/L. Lactic acid was observed to be inhibitory to the microorganisms’ activity.     

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.     

Optimization of L(+)-lactic acid production employing statistical experimental design

Summary An orthogonal 2³-factorial experimental design was used to optimize L(+)-lactic acid production byLactobacillus casei. With a 22 % (v/v) inoculum the optimum concentration of yeast extract for maximum lactic acid concentration and yield was about 2 % (w/v) and that of the initial glucose 9 to 11 %.     

Design and evaluation of a Lactobacillus manihotivorans species-specific rRNA-targeted hybridization probe and its application to the study of sour cassava fermentation

Based on 16S rRNA sequence comparison, we have designed a 20-mer oligonucleotide that targets a region specific to the species Lactobacillus manihotivorans recently isolated from sour cassava fermentation. The probe recognized the rRNA obtained from all the L. manihotivorans strains tested but did not recognize 56 strains of microorganisms from culture collections or directly isolated from sour cassava, including 29 species of lactic acid bacteria. This probe was then successfully used in quantitative RNA blots and demonstrated the importance of L. manihotivorans in the fermentation of sour cassava starch, which could represent up to 20% of total lactic acid bacteria.