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.     

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.     

Anaerobic thermophilic fermentation for acetic acid production from milk permeate

Fermentation of milk permeate to produce acetic acid under anaerobic thermophilic conditions (approximately 60 degrees C) was studied. Although none of the known thermophilic acetogenic bacteria can ferment lactose, it has been found that one strain can use galactose and two strains can use lactate. Moorella thermoautotrophica DSM 7417 and M. thermoacetica DSM 2955 were able to convert lactate to acetate at thermophilic temperatures with a yield of approximately 0.93 g g(-1). Among the strains screened for their abilities to produce acetate and lactate from lactose, Clostridium thermolacticum DSM 2910 was found precisely to produce large amounts of lactate and acetate. However, it also produced significant amounts of ethanol, CO2 and H2. The lactate yield was affected by cell growth. During the exponential phase, acetate, ethanol, CO2 and H2 were the main products of fermentation with an equimolar acetate/ethanol ratio, whereas during the stationary phase, only lactic acid was produced with a yield of 4 mol per mol lactose, thus reaching the maximal theoretical value. When this bacterium was co-cultured with M. thermoautotrophica, lactose was first converted mainly to lactic acid, then to acetic acid, with a zero residual lactic acid concentration and an overall yield of acetate around 80%. Under such conditions, only 13% of the fermented lactose was converted to ethanol by C. thermolacticum.     

Low-cost propionate salt as road deicer: evaluation of cheese whey and other media constituents

Propionate and acetate salts are environmentally friendly, effective road deicer substitutes for widely used sodium chloride. A low-cost medium, using raw cheese whey and hydrolyzed whey permeate/whey permeate powder as substrates, and corn-steep liquor as a nutrient supplement, was studied for lactic acid production, replacing synthetic lactose and other high-cost nutrients. A non-sterile stage-I fermentation process for improved lactate productivity using an inexpensive commercial medium was performed at a 20-L fermenter level. A lactate yield of 0.98 g/g lactose and a productivity of 1.1 g/L/h was obtained with complete lactose utilization. When synthetic lactate and glucose were used as substrates in propionate and acetate fermentation, a total acid yield of 0.55 g/g glucose and lactate consumed and a batch productivity of 0.22 g/L/h was obtained. A stage-II fermentation process to produce propionate and acetate salts from cheese whey-derived lactate (stage-I fermentation broth) resulted in 1.6%( w/v) propionate after a total of 161 h (stages I and II).     

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.     

Acetic acid production from whey lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum

Summary Acetic acid was produced from anaerobic fermentation of lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum at 35° C and pHs between 7.0 and 7.6. Lactose was converted to lactic acid, and then to acetic acid in this mixed culture fermentation. The overall acetic acid yield from lactose was about 95% at pH 7.6 and 90% at pH 7.0. The fermentation rate was also higher at pH 7.6 than at pH 7.0. In batch fermentation of whey permeate containing about 5% lactose at pH 7.6, the concentration of acetic acid reached 20 g/l within 20 h. The production rate then became very slow due to end-product inhibition and high Na⁺ concentration. About 30 g/l acetate and 20 g/l lactate were obtained at a fermentation time of 80 h. However, when diluted whey permeate containing 2.5% lactose was used, all the whey lactose was converted to acetic acid within 30 h by this mixed culture.     

Continuous propionate production from whey permeate using a novel fibrous bed bioreactor

Continuous production of propionate from whey lactose by Propionibacterium acidipropionici immobilized in a novel fibrous bed bioreactor was studied. In conventional batch propionic acid fermentation, whey permeate without nutrient supplementation was unable to support cell growth and failed to give satisfactory fermentation results for over 7 days. However, with the fibrous bed bioreactor, a high fermentation rate and high conversion were obtained with plain whey permeate and de-lactose whey permeate. About 2% (wt/vol) propionic acid was obtained from a 4.2% lactose feed at a retention time of 35 to 45 h. The propionic acid yield was approximately 46% (wt/vol) from lactose. The optimal pH for fementation was 6.5, and lower fermentation rates and yields were obtained at lower pH values. The optimal temperature was 30 degrees C, but the temperature effect was not dramatic in the range of 25 to 35 degrees C. Addition of yeast extract and trypticase to whey permeate hastened reactor startup and increased the fermentation rate and product yields, but the addition was not required for long-term reactor performance. The improved fermentation results with the immobilized cell bioreactor can be attributed to the high cell density, approximately 50 g/L, attained in the bioreactor, Cells were immobilized by loose attachement to fiber surfaces and entrapment in the void spaces within the fibrous matrix, thus allowing constant renewal of cells. Consequently, this bioreactor was able to operate continuously for 6 months without encountering any clogging, degeneration, or contamination problems. Compared to conventional batch fermentors, the new bioreactor offers many advantages for industrial fermentation, including a more than 10-fold increase in productivity, acceptance of low-nutrient feedstocks such as whey permeate, and resistance to contamination. (c) 1994 John Wiley & Sons, Inc.     

Improvement of acetate production from lactose by growing Clostridium thermolacticum in mixed batch culture

AIMS: The objective of this study was to increase the acetate production by Clostridium thermolacticum growing on lactose, available as a renewable resource in the milk and whey permeate from the cheese industry. METHODS AND RESULTS: Experiments for increased acetate productivity by thermophilic anaerobes grown on lactose were carried out in batch cultures. Lactose at concentration of 30 mmol l(-1) (10 g l(-1)) was completely degraded by Cl. thermolacticum and growth rate was maximal. High concentrations of by-products, ethanol, lactate, hydrogen and carbon dioxide were generated. By using an efficient hydrogenotroph, Methanothermobacter thermoautotrophicus, in a defined thermophilic anaerobic consortium (58 degrees C) with Cl. thermolacticum and the acetogenic Moorella thermoautotrophica, the hydrogen partial pressure was dramatically lowered. As a consequence, by-products concentrations were significantly reduced and acetate production was increased. CONCLUSION: Through efficient in situ hydrogen scavenging in the consortium, the metabolic pattern was modified in favour of acetate production, at the expense of reduced by-products like ethanol. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of this thermophilic anaerobic consortium opens new opportunities for the efficient valorization of lactose, the main waste from the cheese industry, and production of calcium-magnesium acetate, an environmentally friendly road de-icer.     

A kinetic model for methanogenesis from whey permeate in a packed bed immobilized cell bioreactor

The fermentation kinetics of methane production from whey permeate in a packed bed immobilized cell bioreactor at mesophilic temperatures and pHs around neutral was studied. Propionate and acetate were the only two major organic intermediates found in the methanogenic fermentation of lactose. Based on this finding, a three-step reaction mechanism was proposed: lactose was first degraded to propionate, acetate, CO(2), and H(2) by fermentative bacteria; propionate was then converted to acetate by propionate-degrading bacteria; and finally, CH(4) and CO(2) were produced from acetate, H(2), and CO(2) by methanogenic bacteria. The second reaction step was found to be the rate-limiting step in the overall methanogenic fermentation of lactose. Monod-type mathematical equations were used to model these three step reactions. The kinetic constants in the models were sequentially determined by fitting the mathematical equations with the experimental data on acetate, propionate, and lactose concentrations. A mixed-culture fermentation model was also developed. This model simulates the methanogenic fermentation of whey permeate very well.     

Alcohol production from cheese whey permeate using genetically modified flocculent yeast cells.

Alcoholic fermentation of cheese whey permeate was investigated using a recombinant flocculating Saccharomyces cerevisiae, expressing the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus enabling for lactose metabolization. Data on yeast fermentation and growth on cheese whey permeate from a Portuguese dairy industry is presented. For cheese whey permeate having a lactose concentration of 50 gL(-1), total lactose consumption was observed with a conversion yield of ethanol close to the expected theoretical value. Using a continuously operating 5.5-L bioreactor, ethanol productivity near 10 g L(-1) h(-1) (corresponding to 0.45 h(-1) dilution rate) was obtained, which raises new perspectives for the economic feasibility of whey alcoholic fermentation. The use of 2-times concentrated cheese whey permeate, corresponding to 100 gL(-1) of lactose concentration, was also considered allowing for obtaining a fermentation product with 5% (w/v) alcohol.     

Studies on immobilized Saccharomyces cerevisiae. I. Analysis of continuous rapid ethanol fermentation in immobilized cell reactor

Rapid fermentation of cane molasses into ethanol has been studied in batch, continuous (free-cell and cell-immobilized systems) by a strain of Saccharomyces cerevisiae at temperature 30 degrees C and pH 5.0. The maximum productivity of ethanol obtained in immobilized system was 28.6 g L(-1) h(-1). The cells were immobilized by natural mode on a carrier of natural origin and retention of 0.132 g cells/g carrier was achieved. The immobilized-cell column was operated continuously at steady state over a period of 35 days. Based on the parameter data monitored from the system, mathematical analysis has been made and rate equations proposed, and the values of specific productivity of ethanol and specific growth rate for immobilized cells computed. It has been established that immobilized cells exhibit higher specific rate of ethanol formation compared to free cells but the specific growth rate appears to be comparatively low. The yield of ethanol in the immobilized-cell system is also higher than in the free-cell system.     

Ethanol production from concentrated whey permeate using a fed-batch culture ofKluyveromyces fragilis

Summary Fed-batch fermentation of non-supplemented concentrated whey permeate resulted in high ethanol productivity for feeds of lactose for which batch fermentation had a poor performance. At an initial lactose concentration of 100 g/L and a constant lactose feeding rate of 18 g/h we have obtained: ethanol concentration 64 g/L, ethanol productivity 3.3 g/Lh, lactose consumption 100%, ethanol yield 0.47 g/g, and biomass yield 0.058 g/g.Nomenclature St total lactose fed per medium volume in the bioreactor, g/L - Si initial lactose concentration, g/L - F lactpse feeding rate, g/h - P final ethanol concentration, g/L - Y_p/s ethanol yield, g ethanol/g lactose - Y_x/s biomass yield, g biomass/g lactose - X_S lactose consumption, % - Qp overall ethanol volumetric productivity, g/Lh - m maximum specific growth rate, h - qsm maximum specific lactose consumption rate, g/gh - qpm maximum specific ethanol production rate, g/gh     

Acetate production from lactose by Clostridium thermolacticum and hydrogen-scavenging microorganisms in continuous culture--effect of hydrogen partial pressure

The effect of the addition of hydrogen-consuming microorganisms on the metabolism of Clostridium thermolacticum was studied. By growing this bacterium in continuous culture at 58 degrees C, on 29 mmol lactose l(-1) (10 gl(-1)) in the feed, with the H2-consuming microorganisms Methanothermobacter thermoautotrophicus and Moorella thermoautotrophica, the volumetric productivity of acetate was increased up to 3.9 mmol l(-1)h(-1) at a dilution rate of 0.058 h(-1). This was about three times higher than the maximal acetate volumetric productivity quantified when C. thermolacticum was cultivated alone. In the consortium, C. thermolacticum was the only species able to metabolize lactose; it produced not only acetate, but also hydrogen, carbon dioxide and lactate. The other species of the consortium were growing on these by-products. Meth. thermoautotrophicus played an important role as a very efficient hydrogen scavenger and decreased the hydrogen partial pressure drastically: hydrogen was converted to methane. Moor. thermoautotrophica converted lactate as well as hydrogen and carbon dioxide into acetate. As a consequence, lactose was efficiently consumed and the only organic product in the liquid phase was acetate.     

Effect of inoculum composition and low KCl supplementation on the biological and rheological stability of an immobilized-cell system for mixed mesophilic lactic starter production.

Two strains of Lactococcus lactis subsp. lactis (L. lactis KB and KBP) and one of L. lactis subsp. lactis biovar. diacetylactis (L. diacetylactis MD) were immobilized separately in kappa-carrageenan-locust bean gum gel beads. Continuous fermentations were carried out in supplemented whey permeate in a 1-L pH-controlled stirred tank reactor inoculated with a 30% (v/v) bead inoculum and a bead ratio of 55:30:15 for KB, KBP, and MD, respectively. The process demonstrated a high productivity and microbial stability during the 7-week continuous culture. Compared with previous experiments carried out with an inoculum bead ratio of 33:33:33 for KB, KBP, and MD beads, respectively, the modification of the inoculum bead ratio had apparently little effect on free and immobilized, total and specific populations. A dominant behavior of L. diacetylactis MD over the other strains of the mixed culture was observed both with free-cell populations in the effluent and with immobilized-cell populations. Additional experiments were carried out with other strain combinations for continuous inoculation-prefermentation of milk. The data also confirmed the dominance of L. diacetylactis during long-term continuous immobilized-cell fermentations. This dominance may be tentatively explained by the local competition involved in the development of the bead cross-contamination and in citrate utilization by L. diacetylactis strains. The gel beads demonstrated a high rheological stability during the 7-week continuous fermentation even at low KCl supplementation of the broth medium (25 mM KCl).     

Production of carboxylic acids from hydrolyzed corn meal by immobilized cell fermentation in a fibrous-bed bioreactor

Corn meal hydrolyzed with amylases was used as the carbon source for producing acetic, propionic, and butyric acids via anaerobic fermentations. In this study, corn meal, containing 75% (w/w) starch, 20% (w/w) fibers, and 1.5% (w/w) protein, was first hydrolyzed using amylases at 60 degrees C. The hydrolysis yielded approximately 100% recovery of starch converted to glucose and 17.9% recovery of protein. The resulting corn meal hydrolyzate was then used, after sterilization, for fermentation studies. A co-culture of Lactococcus lactis and Clostridium formicoaceticum was used to produce acetic acid from glucose. Propionibacterium acidipropionici was used for propionic acid fermentation, and Clostridium tyrobutylicum was used for butyric acid production. These cells were immobilized on a spirally wound fibrous matrix packed in a fibrous-bed bioreactor (FBB) developed for multi-phase biological reactions or fermentation. The bioreactor was connected to a stirred-tank fermentor that provided pH and temperature controls via medium circulation. The fermentation system was operated at the recycle batch mode. Temperature and pH were controlled at 37 degrees C and 7.6, respectively, for acetic acid fermentation, 32 degrees C and 6.0, respectively, for propionic acid fermentation, and 37 degrees C and 6.0, respectively, for butyric acid production. The fermentation demonstrated a yield of approximately 100% and a volumetric productivity of approximately 1 g/(1 h) for acetic acid production. The propionic acid fermentation achieved an approximately 60% yield and a productivity of 2.12 g/(1 h), whereas the butyric acid fermentation obtained an approximately 50% yield and a productivity of 6.78 g/(1 h). These results were comparable to, or better than those fermentations using chemically defined media containing glucose as the substrate, suggesting that these carboxylic acids can be efficiently produced from direct fermentation of corn meal hydrolyzate. The corn fiber present as suspended solids in the corn meal hydrolyzate did not cause operating problem to the immobilized cell bioreactor as is usually encountered by conventional immobilized cell bioreactor systems. It is concluded that the FBB technology is suitable for producing value-added biochemicals directly from agricultural residues or commodities such as corn meal.     

Bifidobacterium longum ATCC 15707 cell production during free- and immobilized-cell cultures in MRS-whey permeate medium

Bifidobacterium longum ATCC 15707 cell production was studied in MRS medium supplemented with whey permeate (MRS-WP) during free-cell batch fermentations and continuous immobilized-cell cultures. Very high populations were measured after 12 h batch cultures in MRS-WP medium controlled at pH 5.5 (1.7+/-0.5x10(10) cfu/ml), approximately 2-fold higher than in non-supplemented MRS. Our study showed that WP is a low-cost source of lactose and other components that can be used to increase bifidobacteria cell production in MRS medium. Continuous fermentation in MRS-WP of B. longum immobilized in gellan gum gel beads produced the highest cell concentrations in the effluent (4.9+/-0.9x10(9) cfu/ml) at a dilution rate (D) of 0.5 h(-1). However, maximal volumetric productivity (6.9+/-0.4x10(9) cfu ml(-1)h(-1)) during continuous cultures was obtained at D =2.0 h(-1), and was approximately 9.5-fold higher than during free-cell batch cultures at an optimal pH of 5.5 (7.2x10(8) cfu ml(-1)h(-1)).     

Efficient production of butyric acid from Jerusalem artichoke by immobilized Clostridium tyrobutyricum in a fibrous-bed bioreactor

Butyric acid is an important specialty chemical with wide industrial applications. The feasible large-scale fermentation for the economical production of butyric acid requires low-cost substrate and efficient process. In the present study, butyric acid production by immobilized Clostridium tyrobutyricum was successfully performed in a fibrous-bed bioreactor using Jerusalem artichoke as the substrate. Repeated-batch fermentation was carried out to produce butyric acid with a high butyrate yield (0.44 g/g), high productivity (2.75 g/L/h) and a butyrate concentration of 27.5 g/L. Furthermore, fed-batch fermentation using sulfuric acid pretreated Jerusalem artichoke hydrolysate resulted in a high butyric acid concentration of 60.4 g/L, with the yield of 0.38 g/g and the selectivity of ∼85.1 (85.1 g butyric acid/g acetic acid). Thus, the production of butyric acid from Jerusalem artichoke on a commercial scale could be achieved based on the system developed in this work.     

Metabolic behavior of immobilized Candida guilliermondii cells during batch xylitol production from sugarcane bagasse acid hydrolyzate

Candida guilliermondii cells, immobilized in Ca-alginate beads, were used for batch xylitol production from concentrated sugarcane bagasse hydrolyzate. Maximum xylitol concentration (20.6 g/L), volumetric productivity (0.43 g/L. h), and yield (0.47 g/g) obtained after 48 h of fermentation were higher than similar immobilized-cell systems but lower than free-cell cultivation systems. Substrates, products, and biomass concentrations were used in material balances to study the ways in which the different carbon sources were utilized by the yeast cells under microaerobic conditions. The fraction of xylose consumed to produce xylitol reached a maximum value (0.70) after glucose and oxygen depletion while alternative metabolic routes were favored by sub-optimal conditions.     

Bioethanol from fermentation of cassava pulp in a fibrous-bed bioreactor using immobilized Δldh, a genetically engineered Thermoanaerobacterium aotearoense

There is an increasing worldwide interest in bioethanol production from agricultural, industrial, and urban residues for both ecological and economic reasons. The acid hydrolysis of cassava pulp to reducing sugars and their fermentation to ethanol were evaluated in a fibrousbed bioreactor with immobilized Δldh, a genetically engineered Thermoanaerobacterium aotearoense. A maximum yield of total reducing sugars of 53.5% was obtained after 8 h of hydrolysis at 85oC in 0.4 mol/L hydrochloric acid with a solid-to-liquid ratio of 1:20, which was optimized by using an orthogonal design based on preliminary experiments. In the FBB, the fed-batch fermentation, using glucose as the sole carbon source, gave a maximum ethanol production of 38.3 g/L with a yield of 0.364 g/g in 100 h; whereas the fed-batch fermentation, using xylose as the sole carbon source, gave 34.1 g/L ethanol with a yield of 0.342 g/g in 135 h. When cassava pulp hydrolysate was used as a carbon source, 39.1 g/L ethanol with a yield of 0.123 g/g cassava pulp in185 h was observed, using the fed-batch fermentation model. In addition, for repeated batch fermentation of cassava pulp hydrolysate carried out in the fibrous-bed bioreactor, long-term operation with high ethanol yield and volumetric productivity were achieved. The above results show that the acid hydrolysate of cassava pulp can be used for ethanol production in a fibrous-bed bioreactor, although some inhibition phenomena were observed during the process of fermentation.     

Semicontinuous production of lactic acid in a bioreactor coupled with nanofiltration membranes

The advantages of nanofiltration membranes coupled with a CSTR were demonstrated for the semicontinuous production of lactic acid from whey permeate. Lactic acid was removed from the growth medium while lactose was kept in the bioreactor with the bacterial cells; moreover, Mg²⁺ ions were also recycled in the bioreactor at 96% and the nanofiltrate color was greatly reduced. The highest volumetric productivity achieved with this device was 7.1 g l⁻¹ h⁻¹ and the lactate concentration was 55 g l⁻¹. The specific productivity was 3.54 h⁻¹. More than 99% of the membrane fouling after 44 h of fermentation was reversible. The initial permeate flux was restored easily by a water rinse. The performance of this type of membrane bioreactor was discussed.