Repeated-batch fermentation in biofilm reactors with plastic-composite supports for lactic acid production

Novel plastic supports consisting of polypropylene blended with oat hulls/soybean flour or oat hulls/zein were evaluated as supports for mixed- and pure-culture, repeated-batch, lactic acid fermentations in biofilm reactors. Streptomyces viridosporus T7A (ATCC 39115) was used to form a biofilm for mixed-culture fermentations, and Lactobacillus casei subsp. rhamnosus (ATCC 11443) was used for L-lactic acid production. The pure- and mixed-culture biofilm reactors were operated as repeated-batch fermentors with pH controlled at 5 for more than 2 months in which each reactor's medium was changed every 3 days for 24 batches. The plastic-composite supports performed better than polypropylene-alone supports. Significantly (P<0.05) higher concentrations of lactic acid were produced by the mixed- and pure-culture biofilm bioreactors with corresponding plastic-composite supports (55 g/l and 60 g/l respectively) than with polypropylene-alone supports (48 g/l for both mixed and pure culture). However, the percentage yields, maximum productivity, glucose consumption rates, and growth rates (based on the mass of suspended cells only) were not significantly different between reactors. Maximum lactic acid concentration was consistently greater for the plastic-composite support biofilm reactors. In the suspension culture at pH 5 without plastic supports, maximum lactic acid concentration at days 3 and 5 was 48 g/l and 60 g/l, respectively. These results confirm that the use of plastic-composite supports is recommended for pure-culture lactic acid production in long-term repeated-batch fermentation, and that cell immobilization was occurring.Journal Paper No. J-15813 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Projects No. 3253 and 0178     

Ingredient selection for plastic composite supports for L-(+)-lactic acid biofilm fermentation by Lactobacillus casei subsp. rhamnosus.

Plastic composite supports containing 50% agricultural products (oat hulls, soybean hulls, yeast extract, soybean flour, dried bovine erythrocytes, bovine albumin, and/or mineral salts) and 50% (wt/wt) polypropylene were produced by high-temperature twin-screw extrusion. The research employed two half sets of a five-factorial fractional design (2(5 - 1)) to evaluate the effects of different agricultural components on the properties of the plastic composite supports and to select the best plastic composite support formulation for lactic acid fermentation. The biofilm population was affected by the contact angle and relative hydrophobicity of the supports (r = 0.79 to 0.82). Lactic acid was produced by the suspended cells (r = 0.96) and the biofilm on the plastic composite support discs (r = 0.85). Incorporation of yeast extract into plastic composite supports enhanced growth of free and attached cells in minimal medium (P < 0.0001). The presence of soybean hulls, yeast extract, or mineral salts in plastic composite supports produced less hydrophobic supports (P < 0.0001) and enhanced cell attachment (P < 0.03). Under all conditions, suspended-cell and polypropylene disc controls gave negligible lactic acid production and cell density. Plastic composite supports containing soybean hulls, yeast extract, soybean flour, bovine albumin, and mineral salts gave the highest biofilm population (2.3 x 10(9) CFU/g of support), cell density (absorbance of 1.8 at 620 nm), and lactic acid concentration (7.6 g/liter) in minimal medium.     

Efficient acetic acid production by repeated fed-batch fermentation using two fermentors

Summary In acetic acid fermentation, the number of viable cells decrease as the acetic acid concentration increases to more than about 40 g/l, which means that the productivity attainable by conventional fed-batch and repeated fed-batch operations using one fermentor is limited. In this paper, based on a fed-batch experiment using Acetobacter aceti 2096, a mathematical model was developed. The optimization carried out showed the superiority of repeated fed-batch operation using two fermentors. The performance evaluation was made with respect to productivity and product concentration. It was shown to be attractive in practice to use multiple fermentors, in particular for high product concentrations. Experiments were then conducted to ascertain the simulation results. Offprint requests to: T. Kobayashi     

Enhancement of lactic acid production with ram horn peptone by Lactobacillus casei

Ram horns are a waste material from the meat industry. The use of ram horn peptone (RHP) as a supplement for lactic acid production was investigated using Lactobacillus casei. For this purpose, first, RHP was produced. Ram horns were hydrolysed by treating with acids (3 M H₂SO₄ and 6 M HCl) and neutralizing the solutions to yield ram horn hydrolysate (RHH). The RHH was evaporated to yield RHP. The amounts of protein, nitrogen, ash, some minerals, total sugars, total lipids and amino acids of the RHP were determined and compared with a bacto-tryptone from casein. When the concentrations (1–6% w/v) of the RHP were used in bacterial growth medium as a supplement, 2% RHP (ram horn peptone medium) had a maximum influence on the production of lactic acid by L. casei. The content of lactic acid in the culture broth containing 2% RHP (43 g l^–1) grown for 24 h was 30% higher than that of the control culture broth (33 g l^–1) and 10% higher than that of 2% bacto-tryptone (39 g l^–1). RHP was demonstrated to be a suitable supplement for production of lactic acid. This RHP may prove to be a valuable supplement in fermentation technology.     

Resistance of Lactobacillus casei in plastic-composite-support biofilm reactors during liquid membrane extraction and optimization of the lactic acid extraction system

Lactic acid fermentations were performed with plastic-composite-support (PCS) disks in solvent-saturated media with Lactobacillus casei subsp. rhamnosus (ATCC 11443). The PCS disks contained 50% (w/w) polypropylene, 35% (w/w) ground soybean hulls, 5% (w/w) yeast extract, 5% (w/w) soybean flour, and 5% (w/w) bovine albumin. Bioassays were performed by growing L. casei in solvent-saturated media after soaking the PCS disks. Eighteen different solvent and carrier combinations were evaluated. Overall, L. casei biofilm fermentation demonstrated the same lactic acid production in solvent-saturated medium as suspended cells in medium without solvents (control). To evaluate PCS solvent-detoxifying properties, two bioassays were developed. When solvent-saturated medium in consecutive equal volumes (10 mL then 10 mL) was exposed to PCS, both media demonstrated lactic acid fermentation equal to the control. However, when solvent-saturated medium with two consecutive unequal volumes (10 mL then 90 mL) was exposed to PCS, some degree of toxicity was observed. Furthermore, iso-octane, tributylphosphate (TBP), and Span 80 were optimized for recovery as 91%, 5%, and 4% (v/v), respectively, with a 1:1 ratio of 1.2 M Na(2)CO(3) stripping solution. Also, recovery by emulsion liquid extraction in the hollow-fiber contactor was minimal due to low recovery at pH 5.0 and incompatibility of the solvent and hollow-fiber material. These results suggest that PCS biofilm reactors can benefit lactic acid fermentation by eliminating the toxic effect from solvent leakage into the fermentation medium from liquid-liquid extractive integrated fermentations.     

Production of lactic acid from soybean stalk hydrolysate with Lactobacillus sake and Lactobacillus casei

In order to make full use of soybean stalk produced in large quantity annually in China, a process is proposed for production of lactic acid from soybean stalk hydrolysate with Lactobacillus sake and Lactobacillus casei. Experiments were conducted using the proposed process and experimental results indicate that the potential of 242 mg (g stalk)⁻¹ fermentable sugar is released from hydrolysate through enzymatic saccharication with a saccharication of 51%. The main sugar released from pretreated soybean stalk through enzymatic hydrolysis was a mixture of glucose, xylose and cellobiose at a ratio of 3.9:1.7:1. Fermentation of soybean stalk hydrolysate by L. sake and L. casei yielded the lactic acid conversion of 48% and 56%, respectively, however, lactic acid conversion increased to 71% by co-inoculation of both strains. L. sake and L. casei were able to degrade glucose, but unable to completely assimilate xylose and cellobiose. The proposed process can be used to produce lactic acid from soybean stalk hydrolysate.     

Optimization of the fermentation of whey by Lactobacillus casei

Optimization studies have been carried out for the production of L-lactic acid from the fermentation of whey by Lactobacillus casei. Two Statistical Designs, a full 2⁴ Factorial Design and a full 3² Factorial Design, have been used to determine the most suitable values of the operational variables for obtaining the maximum lactic acid production rate. The variables selected for study were temperature, pH, lactose concentration, and inoculum concentration. Among them only temperature and pH were found to affect the response significantly. A second-order polynomial empirical model relating both the response (lactic acid production rate) and the variables was formulated. The maximum acid production rate was found when the values of pH and temperature were 5.4 and 38°C, respectively. All conclusions are restricted to the experimental range studied.     

Kinetic model-based feed-forward controlled fed-batch fermentation of Lactobacillus rhamnosus for the production of lactic acid from Arabic date juice

Arabic date is overproduced in Arabic countries such as Saudi Arabia and Iraq and is mostly composed of sugars (70–80 wt%). Here we developed a fed-batch fermentation process by using a kinetic model for the efficient production of lactic acid to a high concentration from Arabic date juice. First, a kinetic model of Lactobacillus rhamnosus grown on date juice in batch fermentation was constructed in EXCEL so that the estimation of parameters and simulation of the model can be easily performed. Then, several fed-batch fermentations were conducted by employing different feeding strategies including pulsed feeding, exponential feeding, and modified exponential feeding. Based on the results of fed-batch fermentations, the kinetic model for fed-batch fermentation was also developed. This new model was used to perform feed-forward controlled fed-batch fermentation, which resulted in the production of 171.79 g l^?1 of lactic acid with the productivity and yield of 1.58 and 0.87 g l^?1 h^?1, respectively.     

Batch fermentation of whey ultrafiltrate by Lactobacillus helveticus for lactic acid production

Summary Cheese whey ultrafiltrate (WU) was used as the carbon source for the production of lactic acid by batch fermentation with Lactobacillus helveticus strain milano. The fermentation was conducted in a 400 ml fermentor at an agitation rate of 200 rpm and under conditions of controlled temperature (42° C) and pH. In the whey ultrafiltrate-corn steep liquor (WU-CSL) medium, the optimal pH for fermentation was 5.9. Inoculum propagated in skim milk (SM) medium or in lactose synthetic (LS) medium resulted in the best performance in fermentation (in terms of growth, lactic acid production, lactic acid yield and maximum productivity of lactic acid), as compared to that propagated in glucose synthetic (GS) medium. The yeast extract ultrafiltrate (YEU) used as the nitrogen/growth factor source in the WU medium at 1.5% (w/v) gave the highest maximum productivity of lactic acid of 2.70 g/l-h, as compared to the CSL and the tryptone ultrafiltrate (TU). L. helveticus is more advantageous than Streptococcus thermophilus and Lactobacillus delbrueckii for the production of lactic acid from WU. The L. helveticus process will provide an alternative solution to the phage contamination in dairy industries using Lactobacillus bulgaricus.     

Lactic acid fermentation in a recycle batch reactor using immobilized Lactobacillus casei

Lactic acid production by recycle batch fermentation using immobilized cells of Lactobacillus casei subsp. rhamnosus was studied. The culture medium was composed of whey treated with an endoprotease, and supplemented with 2.5 g/L of yeast extract and 0.18 mM Mn(2+) ions. The fermentation set-up comprised of a column packed with polyethyleneimine-coated foam glass particles, Pora-bact A, and connected with recirculation to a stirred tank reactor vessel for pH control. The immobilization of L. casei was performed simply by circulating the culture medium inoculated with the organism over the beads. At this stage, a long lag period preceded the cell growth and lactic acid production. Subsequently, for recycle batch fermentations using the immobilized cells, the reducing sugar concentration of the medium was increased to 100 g/L by addition of glucose. The lactic acid production started immediately after onset of fermentation and the average reactor productivity during repeated cycles was about 4.3 to 4.6 g/L . h, with complete substrate utilization and more than 90% product yield. Sugar consumption and lactate yield were maintained at the same level with increase in medium volume up to at least 10 times that of the immobilized biocatalyst. The liberation of significant amounts of cells into the medium limited the number of fermentation cycles possible in a recycle batch mode. Use of lower yeast extract concentration reduced the amount of suspended biomass without significant change in productivity, thereby also increasing the number of fermentation cycles, and even maintained the D-lactate amount at low levels. The product was recovered from the clarified and decolorized broth by ion-exchange adsorption. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:841-853, 1997.     

Lactic acid fermentation by Lactobacillus casei in free cell form and immobilised on gluten pellets

A comparative study of the fermentation of a range of carbohydrate substrates, at various temperatures, was carried out using a commercial Lactobacillus casei strain in a free cell form and immobilised on gluten pellets. This strain required yeast extract, l-cysteine HCl and Mn²⁺ at 5, 0.5 and 0.1 g l^–1, respectively, for maximum growth and lactic acid production. Sugar fermentation using free cells showed preference in the order glucose, sucrose, fructose while lactose was poorly utilised. Optimum temperature for growth and lactic acid production over (18–30 h) was 43 °C. L. casei was successfully immobilised on gluten pellets and fermented glucose and sucrose in a shorter time (18 h) with increased lactic acid production (42 and 41 g l^–1 on glucose and sucrose, respectively).     

Semi-continuous lactic fermentation of whey by Lactobacillus bulgaricus

Semi-continuous tests of lactic fermentation of whey by Lactobacillus buigaricus were carried out using a mixture of hydrolysed milk and vitamins. The volume of the Inoculum varied from 20% to 50% of the reactor working volume. A Monod-like equation correlates the lactic acid productivity and the volume fraction of inoculum.The authors are with the Centro de Desenvolvimento Biotecnológico de Santa Catarina (Biotechnological Development Centre of Santa Catarina), R. Princesa isabel, 114, 89200, Joinville, SC, Brazil. CDEB. L.A. Kulay was an undergraduate student, in 1989, of the Escola de Engenharia Mauá (Mauá Engineering School), São Caetano do Sul, SP, Brazil. W. Borzani is the corresponding author.     

Production of lactic acid by direct fermentation of starchy wastes by an amylase-producing Lactobacillus

Lactobacillus cellobiosus, isolated from city wastes, produced an extracellular alpha-amylase and produced lactic acid by direct fermentation of waste potato mash. Using a 5% (w/v) potato mash with 3% (w/v) CaCO to neutralise the lactic acid produced, 50% conversion of starch to lactic acid occurred in 48 h without any other media supplement.     

Long-term repeated fed-batch ethanol fermentation in aerated condition

In this study, we attempted to assess the process stability of long-term fed-batch ethanol fermentation in the absence and presence of aeration (0.33 vvm). To examine the effect of aeration, a long-term repeated fed-batch operation was conducted for 396 h to mimic a long-term industrial bioethanol production process. In this long-term repeated fed-batch ethanol fermentation experiments, withdrawal-fill operation were conducted every 36 h for 10 repeat cycles. The whole operation was stably sustained in a quasi-steady state. The average maximal cell concentration and the average maximal ethanol production during operation were increased by 81.63 and 12.12%, respectively, when aeration was used. In addition, since aeration was carried out, the average ethanol yield slightly decreased by 4.03% and the average specific ethanol production rate decreased by 46.75% during operation. However, the average ethanol productivity increased by 17.53% when aeration was carried out. After 396 h of long-term repeated fed-batch ethanol fermentation, 1,908.9 g of ethanol was cumulatively produced when aeration was used, which was 12.47%, higher than when aeration was not used (1,697.2 g). Meanwhile, glycerol production was greatly decreased during long-term repeated fed-batch ethanol fermentation, in which the glycerol concentration in the culture broth decreased from about 34∼15 g/L. Thus, we can conclude that cell growth was greatly improved by overcoming ethanol inhibition and glycerol production was remarkably decreased when aeration was carried out, although aeration in ethanol fermentation decreased the specific ethanol production rate and ethanol yield.     

L-lactic acid production by Lactobacillus casei fermentation with corn steep liquor-supplemented acid-hydrolysate of soybean meal

Batch and fed-batch fermentation studies were performed to evaluate the potential of corn steep liquor (CSL)-supplemented acid-hydrolysate of soybean meal (AHSM) as an alternative to yeast extract (YE) for the production of L-lactic acid by Lactobacillus casei LA-04-1. The CSL-supplemented AHSM gave an outstanding result in supporting L-lactic acid production from glucose. In the exponential fed-batch fermentation, the concentration, yield and productivity of L-lactic acid were 162.5 g/L, 89.7% and 1.69 g/L per h, respectively, which were lower than those with 20 g/L YE (180 g/L, 90.3%, 2.14 g/L per h) after 96 h of fermentation. However, the raw material cost of the nitrogen resource was estimated as only 25% of that using the YE.     

Model aided design of repeated fed-batch penicillin fermentation

Structured models of antibiotic fermentation that quantify maturation and aging of product forming biomass are fitted to experimental data. Conditions of superiority of repeated fed batch cultivation are characterized on the basis of a performance criterion that includes penicillin productivity and costs of operation. Emphasis is placed on the relevance of such research to the model aided design of optimal cyclic operation.List of Symbols c IU/mg cost factor - D s^–1 dilution rate - J IU · cm^–3 · h^–1 net productivity - k _p IU · mg^–11 · h^–1 specific product formation rate - k _pm IU · mg^–1 · h^–1 maximum specific product formation rate - p IU/cm³ concentration of penicillin - T s final time of fermentation - t s fermentation time - X kg/m³ concentration of biomass dry weight - X ₁kg/m³ concentration of young, immature biomass - X ₂ kg/m³ concentration of mature product forming biomass - X _c kg/m³ biomass concentration of the end of growth phase - X _mkg/m³ maximum biomass concentration Greek Letters s^–1 specific maturation rate - s^–1 specific aging rate - s^–1 specific growth rate - _m s^–1 maximum specific growth rate - _p s^–1 specific growth rate during the product formation phase - s cycle time - % volume fraction of draw-off Abbreviations CC chemostat culture - RFBC repeated fed batch culture - RBC repeated batch culture     

Continuous lactic acid fermentation using a plastic composite support biofilm reactor

An immobilized-cell biofilm reactor was used for the continuous production of lactic acid by Lactobacillus casei subsp. rhamnosus (ATCC 11443). At Iowa State University, a unique plastic composite support (PCS) that stimulates biofilm formation has been developed. The optimized PCS blend for Lactobacillus contains 50% (wt/wt) agricultural products [35% (wt/wt) ground soy hulls, 5% (wt/wt) soy flour, 5% (wt/wt) yeast extract, 5% (wt/wt) dried bovine albumin, and mineral salts] and 50% (wt/wt) polypropylene (PP) produced by high-temperature extrusion. The PCS tubes have a wall thickness of 3.5 mm, outer diameter of 10.5 mm, and were cut into 10-cm lengths. Six PCS tubes, three rows of two parallel tubes, were bound in a grid fashion to the agitator shaft of a 1.2-1 vessel for a New Brunswick Bioflo 3000 fermentor. PCS stimulates biofilm formation, supplies nutrients to attached and suspended cells, and increases lactic acid production. Biofilm thickness on the PCS tubes was controlled by the agitation speed. The PCS biofilm reactor and PP control reactor achieved optimal average production rates of 9.0 and 5.8 g l(-1) h(-1), respectively, at 0.4 h(-1) dilution rate and 125-rpm agitation with yields of approximately 70%.     

Production of mannitol and lactic acid by fermentation with Lactobacillus intermedius NRRL B-3693

Lactobacillus intermedius B-3693 was selected as a good producer of mannitol from fructose after screening 72 bacterial strains. The bacterium produced mannitol, lactic acid, and acetic acid from fructose in pH-controlled batch fermentation. Typical yields of mannitol, lactic acid, and acetic acid from 250 g/L fructose were 0.70, 0.16, and 0.12 g, respectively per g of fructose. The fermentation time was greatly dependent on fructose concentration but the product yields were not dependent on fructose level. Fed-batch fermentation decreased the time of maximum mannitol production from fructose (300 g/L) from 136 to 92 h. One-third of fructose could be replaced with glucose, maltose, galactose, mannose, raffinose, or starch with glucoamylase (simultaneous saccharification and fermentation), and two-thirds of fructose could be replaced with sucrose. L. intermedius B-3693 did not co-utilize lactose, cellobiose, glycerol, or xylose with fructose. It produced lactic acid and ethanol but no acetic acid from glucose. The bacterium produced 21.3 +/- 0.6 g lactic acid, 10.5 +/- 0.3 g acetic acid, and 4.7 +/- 0.0 g ethanol per L of fermentation broth from dilute acid (15% solids, 0.5% H(2)SO(4), 121 degrees C, 1 h) pretreated enzyme (cellulase, beta-glucosidase) saccharified corn fiber hydrolyzate.     

Evaluation of lactic acid bacteria autolysate for the supplementation of lactic acid bacteria fermentation

At the end of culture in a carbon-limited medium, i.e. the best conditions for subsequent autolysis, lactic acid bacteria were harvested and autolysed at 50 °C for 24 h. The resulting supernatant was then successfully tested as a substitute for industrial yeast extract for the supplementation of whey permeate and its conversion into lactic acid: for almost equivalent total nitrogen amounts of both supplements, the same growth and production rates were recorded.     

Behaviour of different Lactobacillus species in the production of lactic acid by the fermentation of whey

The behaviour of different Lactobacillus casei and Lactobacillus plantarum species in the fermentation of Manchego whey was experimentally studied and the results were statistically analyzed using a hypothesis contrast method. The steadiness of the velocity of the production of lactic acid during the fermentation process allowed the use of this variable to compare the different microorganisms. From this comparison it was inferred that the individuals of the same population behave alike and that the L. casei population produces lactic acid at a higher rate than the L. plantarum population. A competitive effect among the members of the L. casei population was also observed.