Production of L(+)-lactic acid from glucose and starch by immobilized cells of Rhizopus oryzae in a rotating fibrous bed bioreactor

A rotating fibrous-bed bioreactor (RFB) was developed for fermentation to produce L(+)-lactic acid from glucose and cornstarch by Rhizopus oryzae. Fungal mycelia were immobilized on cotton cloth in the RFB for a prolonged period to study the fermentation kinetics and process stability. The pH and dissolved oxygen concentration (DO) were found to have significant effects on lactic acid productivity and yield, with pH 6 and 90% DO being the optimal conditions. A high lactic acid yield of 90% (w/w) and productivity of 2.5 g/L.h (467 g/h.m(2)) was obtained from glucose in fed-batch fermentation. When cornstarch was used as the substrate, the lactic acid yield was close to 100% (w/w) and the productivity was 1.65 g/L.h (300 g/h.m(2)). The highest concentration of lactic acid achieved in these fed-batch fermentations was 127 g/L. The immobilized-cells fermentation in the RFB gave a virtually cell-free fermentation broth and provided many advantages over conventional fermentation processes, especially those with freely suspended fungal cells. Without immobilization with the cotton cloth, mycelia grew everywhere in the fermentor and caused serious problems in reactor control and operation and consequently the fermentation was poor in lactic acid production. Oxygen transfer in the RFB was also studied and the volumetric oxygen transfer coefficients under various aeration and agitation conditions were determined and then used to estimate the oxygen transfer rate and uptake rate during the fermentation. The results showed that the oxygen uptake rate increased with increasing DO, indicating that oxygen transfer was limited by the diffusion inside the mycelial layer.     

Development of a continuous cell-recycle fermentation system for production of lactic acid by Lactobacillus paracasei

Experimental devices for stimulating productivity of lactic acid fermentation were installed and an electromagnetic flow-meter and a pneumatic diaphragm pump were employed to alleviate the fouling of the membrane and to improve the durability of membrane cell-recycle bioreactors (MCRBs). In this case, the continuous fermentation using a 5 L automatic fermentor lasted stably over 150 h, and could repeat periodically with simple intermittent on-line cleaning and sterilization of the membrane filtration system. Maximum value of OD₆₂₀ of 98.7 was obtained, six times greater than that of the fed-batch fermentation. Meanwhile, maximum productivity of 31.5 g/(L h) was recorded, 10 times greater than that occurred in the fed-batch fermentation. Compared with conventional MCRBs, the MCRB system with a diaphragm pump and tangential flow-rate controlling was more stable and durable. The tangential velocity of membrane module could be monitored and controlled on-line, and the possibility of contamination due to hose rupture could be eliminated.     

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.     

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。     

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.     

Acetic acid production from lactose by an anaerobic thermophilic coculture immobilized in a fibrous-bed bioreactor

An anaerobic thermophilic coculture consisting of a heterofermentative bacterium (Clostridium thermolacticum) and a homoacetogen (Moorella thermoautotrophica) was developed for acetic acid production from lactose and milk permeate. The fermentation kinetics with free cells in conventional fermentors and immobilized cells in a recycle batch fibrous-bed bioreactor were studied. The optimal conditions for the cocultured fermentation were found to be 58 degrees C and pH 6.4. In the free-cell fermentation, C. thermolacticum converted lactose to acetate, ethanol, lactate, H(2) and CO(2), and the homoacetogen then converted lactate, H(2), and CO(2) to acetate. The overall acetate yield from lactose ranged from 0.46 to 0.65 g/g lactose fermented, depending on the fermentation conditions. In contrast, no ethanol was produced in the immobilized-cell fermentation, and the overall acetate yield from lactose increased to 0.8-0.96 g/g lactose fermented. The fibrous-bed bioreactor also gave a higher final acetate concentration (up to 25. 5 g/L) and reactor productivity (0.18-0.54 g/L/h) as compared to those from the free-cell fermentation (final acetate concentration, 15 g/L; productivity, 0.06-0.08 g/L/h). The superior performance of the fibrous-bed bioreactor was attributed to the high cell density (20 g/L) immobilized in the fibrous-bed and adaptation of C. thermolacticum cells to tolerate a higher acetate concentration. The effects of yeast extract and trypticase as nutrient supplements on the fermentation were also studied. For the free-cell fermentation, nutrient supplementation was necessary for the bacteria to grow in milk permeate. For the immobilized-cell fermentation, plain milk permeate gave a high acetate yield (0.96 g/g), although the reactor productivity was lower than those with nutrient supplementation. Balanced growth and fermentation activities between the two bacteria in the coculture are important to the quantitative conversion of lactose to acetic acid. Lactate and hydrogen produced by C. thermolacticum must be timely converted to acetic acid by the homoacetogen to avoid inhibition by these metabolites.     

Continuous production of ammonium lactate by Streptococccus cremoris in a three-stage reactor

Batch and continuous fermentation studies were performed to optimize the production of ammonium lactate from whey to optimize the production of ammonium lactate from whey permeate. The product known as fermented ammoniated condensed whey permeate (FACWP) is a very promising animal feed. After an initial screening of four strains which produce predominantly L(+)- lactic acid, the desired isomer [D(-)-lactic acid is toxic], Streptococcus cremoris 2487 was chosen for further study. In batch mode, pH between 6.0 and 6.5 and 35 degrees C provided optimum incubation conditions. To stimulate a plug flow reactor, three CSTRs (continuous stirred tank reactors) were connected in tandem. For a 7.5-h retention time, 1.6-fold and 1.3-fold higher productivities were obtained for three-stage than for the single- and two-stage reactors, respectively. Various retentions times were examined (5, 7.5, and 10 h; 5g/L yeast extract). Although maximum lactate productivity occurred at a 5-h residence time (5.38 g/L H. 75% lactose utilization), lactose utilization was more complete at 7.5 h (4.38 g/L h productivity, 91% lactose utilization and a productivity, 91% lactose utilization). Retention time was increased to 15 h to obtain 95.9% lactose utilization and a productivity of 2.42g/L h for 2g/L yeast extract. Based on this lower yeast extract concentration, it was determined that ammonium lactate production and subsequent concentration by 11-fold would yield a product (FACWP) 17% more than soybean meal (crude protein contents are equivalent, 44%) at current market prices.     

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.     

固定化米根霉发酵制L-乳酸

采用海藻酸钙包埋法固定化米根霉（Ｒｈｉｚｏｐｕｓｏｒｙｚａｅ）,菌体在颗粒表面形成一层菌丝膜,有利于氧气和其它营养物质的传递;三相流化床生物反应器结构简单、动力消耗低、反应器内物质混合均匀、氧传递量大于固定化米根霉的需氧量,非常适合好氧的固定化米根霉发酵。利用它进行重复使用固定化米根霉的间歇发酵或连续发酵制备Ｌ 乳酸,整个过程一般可持续两周以上。固定化米根霉的产酸速率达１６～１８ｇ／Ｌ ｂｅａｄ．ｈｒ,得率为７０～８０％,反应器生产能力约为传统搅拌罐的３倍。采用海藻酸钙包埋法固定化米根霉在三相流化床生物反应器中进行发酵可以有效地提高Ｌ 乳酸的生产效率,具有良好的工业应用前景。     

A novel recycle batch immobilized cell bioreactor for propionate production from whey lactose

Recycle batch fermentations using immobilized cells of Propionibacterium acidipropionici were studied for propionate production from whey permeate, de-lactose whey permeate, and acid whey. Cells were immobilized in a spirally wound fibrous sheet packed in a 0.5-L column reactor, which was connected to a 5-L stirred tank batch fermentor with recirculation. The immobilized cells bioreactor served as a breeder for these recycle batch fermentations. High fermentation rates and conversions were obtained with these whey media without nutrient supplementation. It took approximately 55 h to ferment whey permeate containing approximately 45 g/L lactose to approximately 20 g/L propionic acid. Higher propionate concentrations can be produced with various concentrated whey media containing more lactose. The highest propionic acid concentration obtained with the recycle batch reactor was 65 g/L, which is much higher than the normal maximum concentration of 35 to 45 g/L reported in the literature. The volumetric productivity ranged from 0.22 g/L . h to 0.47 g/L . h, depending on the propionate concentration and whey medium used. The corresponding specific cell productivity was 0.033 to 0.07 g/L . g cell. The productivity increased to 0.68 g/L . h when whey permeate was supplemented with 1% (w/v) yeast extract. Compared with conventional batch fermentation, the recycle batch fermentation with the immobilized cell bioreactor allows faster fermentation, produces a higher concentration of product, and can be run continually without significant downtime. The process also produced similar fermentation results with nonsterile whey media. (c) 1995 John Wiley & Sons, Inc.     

Modeling of continuous L(+)-lactic acid production with immobilized R. oryzae in an airlift bioreactor

Continuous L(+)-lactic acid production was carried out in an airlift bioreactor with immobilized R. oryzae in polyurethane foam cubes. In a pseudo-steady state, the productivity of lactic acid increased with increasing dilution rate or feeding glucose concentration. A double-layer reaction-diffusion model for the pseudo-steady state process was developed to describe the bioreaction system. Using independently determined model parameters, the model prediction agreed well with the experimental results. Therefore, the model can be employed to understand the fermentation behavior, and for the process design and optimization.     

Lactic acid production from sugarcane bagasse hydrolysates by Lactobacillus pentosus: Integrating xylose and glucose fermentation

Lactic acid, traditionally obtained through fermentation process, presents numerous applications in different industrial segments, including production of biodegradable polylactic acid (PLA). Development of low cost substrate fermentations could improve economic viability of lactic acid production, through the use of agricultural residues as lignocellulosic biomass. Studies regarding the use of sugarcane bagasse hydrolysates for lactic acid production by Lactobacillus spp. are reported. First, five strains of Lactobacillus spp. were investigated for one that had the ability to consume xylose efficiently. Subsequently, biomass fractionation was performed by dilute acid and alkaline pretreatments, and the hemicellulose hydrolysate (HH) fermentability by the selected strain was carried out in bioreactor. Maximum lactic acid concentration and productivity achieved in HH batch were 42.5 g/L and 1.02 g/L h, respectively. Hydrolyses of partially delignified cellulignin (PDCL) by two different enzymatic cocktails were compared. Finally, fermentation of HH and PDCL hydrolysate together was carried out in bioreactor in a hybrid process: saccharification and co-fermentation with an initial enzymatic hydrolysis. The high fermentability of these process herein developed was demonstrated by the total consumption of xylose and glucose by Lactobacillus pentosus, reaching at 65.0 g/L of lactic acid, 0.93 g/g of yield, and 1.01 g/L h of productivity. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2718, 2019     

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.     

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.     

Acetate production from whey lactose using co-immobilized cells of homolactic and homoacetic bacteria in a fibrous-bed bioreactor

Acetate was produced from whey lactose in batch and fed-batch fermentations using co-immobilized cells of Clostridium formicoaceticum and Lactococcus lactis. The cells were immobilized in a spirally wound fibrous sheet packed in a 0.45-L column reactor, with liquid circulated through a 5-L stirred-tank fermentor. Industrial-grade nitrogen sources, including corn steep liquor, casein hydrolysate, and yeast hydrolysate, were studied as inexpensive nutrient supplements to whey permeate and acid whey. Supplementation with either 2.5% (v/v) corn steep liquor or 1.5 g/L casein hydrolysate was adequate for the cocultured fermentation. The overall acetic acid yield from lactose was 0.9 g/g, and the productivity was 0.25 g/(L h). Both lactate and acetate at high concentrations inhibited the homoacetic fermentation. To overcome these inhibitions, fed-batch fermentations were used to keep lactate concentration low and to adapt cells to high-concentration acetate. The final acetate concentration obtained in the fed-batch fermentation was 75 g/L, which was the highest acetate concentration ever produced by C. formicoaceticum. Even at this high acetate concentration, the overall productivity was 0.18 g/(L h) based on the total medium volume and 1.23 g/(L h) based on the fibrous-bed reactor volume. The cells isolated from the fibrous-bed bioreactor at the end of this study were more tolerant to acetic acid than the original culture used to seed the bioreactor, indicating that adaptation and natural selection of acetate-tolerant strains occurred. This cocultured fermentation process could be used to produce a low-cost acetate deicer from whey permeate and acid whey.     

Extractive fermentation for butyric acid production from glucose by Clostridium tyrobutyricum

A novel extractive fermentation for butyric acid production from glucose, using immobilized cells of Clostridium tyrobutyricum in a fibrous bed bioreactor, was developed by using 10% (v/v) Alamine 336 in oleyl alcohol as the extractant contained in a hollow-fiber membrane extractor for selective removal of butyric acid from the fermentation broth. The extractant was simultaneously regenerated by stripping with NaOH in a second membrane extractor. The fermentation pH was self-regulated by a balance between acid production and removal by extraction, and was kept at approximately pH 5.5 throughout the study. Compared with conventional fermentation, extractive fermentation resulted in a much higher product concentration (>300 g/L) and product purity (91%). It also resulted in higher reactor productivity (7.37 g/L. h) and butyric acid yield (0.45 g/g). Without on-line extraction to remove the acid products, at the optimal pH of 6.0, the final butyric acid concentration was only approximately 43.4 g/L, butyric acid yield was 0.423 g/g, and reactor productivity was 6.77 g/L. h. These values were much lower at pH 5.5: 20.4 g/L, 0.38 g/g, and 5.11 g/L. h, respectively. The improved performance for extractive fermentation can be attributed to the reduced product inhibition by selective removal of butyric acid from the fermentation broth. The solvent was found to be toxic to free cells in suspension, but not harmful to cells immobilized in the fibrous bed. The process was stable and provided consistent long-term performance for the entire 2-week period of study.     

Enhanced propionic acid production from Jerusalem artichoke hydrolysate by immobilized Propionibacterium acidipropionici in a fibrous-bed bioreactor

Propionic acid is an important chemical that is widely used in the food and chemical industries. To enhance propionic acid production, a fibrous-bed bioreactor (FBB) was constructed and Jerusalem artichoke hydrolysate was used as a low-cost renewable feedstock for immobilized fermentation. Comparison of the kinetics of immobilized-cell fermentation using the FBB with those of fed-batch free-cell fermentation showed that immobilized-cell fermentation gave a much higher propionic acid concentration (68.5 vs. 40.6 g/L), propionic acid yield (0.434 vs. 0.379 g/g) and propionic acid productivity (1.55 vs. 0.190 g/L/h) at pH 6.5. Furthermore, repeated batch fermentation, carried out to evaluate the stability of the FBB system, showed that long-term operation with a high average propionic acid yield of 0.483 g/g, high productivity of 3.69 g/L/h and propionic acid concentration of 26.2 g/L were achieved in all eight repeated batches during fermentation for more than 200 h. It is thus concluded that the FBB culture system can be utilized to realize the economical production of propionic acid from Jerusalem artichoke hydrolysate during long-term operation.     

Improved oxygen transfer and increased l-lactic acid production by morphology control of Rhizopus oryzae in a static bed bioreactor

Rhizopus oryzae was immobilized on a cotton matrix in a static bed bioreactor. Compared with free cells in a stirred tank bioreactor, immobilized R. oryzae in this bioreactor gave higher lactic acid production but lower ethanol production. The highest lactic acid production rate (2.09 g/L h) with the final concentration of 37.83 g/L from 70 g/L glucose was achieved when operating the bioreactor at 700 rpm and 0.5 vvm air. To better understand the relationship between shear effects (agitation and aeration) and R. oryzae morphology and metabolism, oxygen transfer rate, fermentation kinetics, and lactate dehydrogenase activity were determined. In immobilized cell culture, higher oxygen transfer rate and lactic acid production were achieved but lower lactate dehydrogenase activity was found as compared with those in free cell culture operated at the same conditions. These results clearly imply that mass transport was the rate controlling step in lactic acid fermentation by R. oryzae.     

Enhancement of pilot scale production of l(+)-lactic acid by fermentation coupled with separation using membrane bioreactor

Traditional batch fermentation leads to a higher energy consumption and lower production capability because of longer culture time. In this work, a pilot scale bioreactor composed of a 3000 L fermentor and external ceramic microfiltration equipment was used to perform cell-recycle fermentation. Repeat feeding medium was also used to relieve the substrate inhibition. In such pilot system, the maximum yield and productivity of l(+)-lactic acid production reached 157.22 ± 3.42 g/L and 8.77 ± 0.15 g/L/h which were 4.23% and 315.64% higher than those of batch fermentation, respectively, when equal amount of sugar was consumed. The cost of l(+)-lactic acid production was successfully reduced about two-thirds by the increase of yield and productivity. 12 rounds of cell-recycle fermentations were successfully achieved in the pilot system. The membrane filtration productivity reached to 61.27 ± 2.74 L/m²/h which increased 172.80%, while the cell damaging rate dropped to 3.88 ± 0.18% which decreased 85.77%, compared with those of the ultrafiltration. Furthermore, the ceramic microfiltration membrane showed advantages in tolerance for the temperature, pressure and acid, compared with the organic ultrafiltration membrane. The experimental results indicated that the method could give a reference for low cost production of l(+)-lactic acid in an industrial scale.     

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.