Lactic Acid Production in a Mixed-Culture Biofilm Reactor

Novel solid supports, consisting of polypropylene blended with various agricultural materials (pp composite), were evaluated as supports for pure- and mixed-culture continuous lactic acid fermentations in biofilm reactors. Streptomyces viridosporus T7A (ATCC 39115) was used to form a biofilm, and Lactobacillus casei subsp. rhamnosus (ATCC 11443) was used for lactic acid production. For mixed-culture fermentations, a 15-day continuous fermentation of S. viridosporus was performed initially to establish the biofilm. The culture medium was then inoculated with L. casei subsp. rhamnosus. For pure-culture fermentation, L. casei subsp. rhamnosus was inoculated directly into the reactors containing sterile pp composite chips. The biofilm reactors containing various pp composite chips were compared with a biofilm reactor containing pure polypropylene chips and with a reactor containing a suspension culture. Continuous fermentation was started, and each flow rate (0.06 to 1.92 ml/min) was held constant for 24 h; steady state was achieved after 10 h. Lactic acid production was determined throughout the 24-h period by high-performance liquid chromatography. Production rates that were two to five times faster than those of the suspension culture (control) were observed for the pure- and mixed-culture bioreactors. Both lactic acid production rates and lactic acid concentrations in the culture medium were consistently higher in mixed-culture than in pure-culture fermentations. Biofilm formation on the chips was detected at harvest by chip clumping and Gram staining.     

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     

Evaluation of plastic composite-supports for enhanced ethanol production in biofilm reactors

Biofilms are a natural form of cell immobilization that result from microbial attachment to solid supports. Biofilm reactors with polypropylene composite-supports containing up to 25% (w/w) of various agricultural materials (corn hulls, cellulose, oat hulls, soybean hulls or starch) and nutrients (soybean flour or zein) were used for ethanol production. Pure cultures ofZymomonas mobilis, ATCC 31821 orSaccharomyces cerevisiae ATCC 24859 and mixed cultures with either of these ethanol-producing microorganisms and the biofilm-formingStreptomyces viridosporus T7A ATCC 39115 were evaluated. An ethanol productivity of 374g L^–1 h^–1 (44% yield) was obtained on polypropylene composite-supports of soybean hull-zein-polypropylene by usingZ. mobilis, whereas mixed-culture fermentations withS. viridosporus resulted in ethanol productivity of 147.5 g L^–1 h^–1 when polypropylene composite-supports of corn starch-soybean flour were used. WithS. cerevisiae, maximum productivity of 40 g L^–1 h^–1 (47% yield) was obtained on polypropylene composite-supports of soybean hull-soybean flour, whereas mixed-culture fermentation withS. viridosporus resulted in ethanol productivity of 190g L^–1 h^–1 (35% yield) when polypropylene composite-supports of oat hull-polypropylene were used. The maximum productivities obtained without supports (suspension culture) were 124 g L^–1 h^–1 and 5 g L^–1 h^–1 withZ. mobilis andS. cerevisiae, respectively. Therefore, forZ. mobilis andS. cerevisiae, ethanol productivities in biofilm fermentations were three- and eight-fold higher than suspension culture fermentations, respectively. Biofilm formation on the chips was detected by weight change and Gram staining of the support material at the end of the fermentation. The ethanol production rate and concentrations were consistently greater in biofilm reactors than in suspension cultures.This is Journal Paper No. J-16356 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 3253     

Effect of lactic acid on diacetyl and acetoin production byLactobacillus casei subsp.rhamnosus ATCC 7469

Lactic acid or its acidity apparently play an important role in the regulation of the biosynthesis of flavor compounds inLactobacillus casei subsp.rhamnosus ATCC 7469. In pyruvate-containing media,L. casei produces lactic acid, acetoin, and diacetyl. A specific pH-dependent system is necessary for both the use of pyruvate and the induction of acetoin and diacetyl production. In cell extracts ofL. casei, lactic acid inhibits the enzymatic activity of acetolactate decarboxylase (ALD) and acetolactate synthetase (ALS); this effect does not occur in whole cells under standard physiological conditions. Lactic acid prevents the use of pyruvate, and the induction of acetoin and diacetyl production. When pyruvate-containing media are used, the pH must be kept close to 6.0 in order to obtain the best production of acetoin and diacetyl.     

Mannitol production by lactic acid bacteria grown in supplemented carob syrup

Detailed kinetic and physiological characterisation of eight mannitol-producing lactic acid bacteria, Leuconostoc citreum ATCC 49370, L. mesenteroides subsp. cremoris ATCC19254, L. mesenteroides subsp. dextranicum ATCC 19255, L. ficulneum NRRL B-23447, L. fructosum NRRL B-2041, L. lactis ATCC 19256, Lactobacillus intermedius NRRL 3692 and Lb. reuteri DSM 20016, was performed using a carob-based culture medium, to evaluate their different metabolic capabilities. Cultures were thoroughly followed for 30 h to evaluate consumption of sugars, as well as production of biomass and metabolites. All strains produced mannitol at high yields (>0.70 g mannitol/g fructose) and volumetric productivities (>1.31 g/l h), and consumed fructose and glucose simultaneously, but fructose assimilation rate was always higher. The results obtained enable the studied strains to be divided mainly into two groups: one for which glucose assimilation rates were below 0.78 g/l h (strains ATCC 49370, ATCC 19256 and ATCC 19254) and the other for which they ranged between 1.41 and 1.89 g/l h (strains NRRL B-3692, NRRL B-2041, NRRL B-23447 and DSM 20016). These groups also exhibited different mannitol production rates and yields, being higher for the strains with faster glucose assimilation. Besides mannitol, all strains also produced lactic acid and acetic acid. The best performance was obtained for L. fructosum NRRL B-2041, with maximum volumetric productivity of 2.36 g/l h and the highest yield, stoichiometric conversion of fructose to mannitol.     

Lactic acid production by mixed cultures of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus helveticus

Lactic acid production using Kluyveromyces marxianus (IFO 288), Lactobacillus delbrueckii ssp. bulgaricus (ATCC 11842) and Lactobacillus helveticus (ATCC 15009) individually or as mixed culture on cheese whey in stirred or static fermentation conditions was evaluated. Lactic acid production, residual sugar and cell biomass were the main features examined. Increased lactic acid production was observed, when mixed cultures were used in comparison to individual ones. The highest lactic acid concentrations were achieved when K. marxianus yeast was combined with L. delbrueckii ssp. bulgaricus, and when all the strains were used revealing possible synergistic effects between the yeast and the two lactic acid bacteria. The same synergistic effects were further observed and verified when the mixed cultures were applied in sourdough fermentations, proving that the above microbiological system could be applied in the food fermentations where high lactic acid production is sought.     

Effect of temperature and various nitrogen sources on L(+)-lactic acid production by Lactobacillus casei

 Two homofermentative strains, Lactobacillus casei NRRL B-441 and Lactobacillus casei subsp. rhamnosus NRRL B-445 were selected for further study from 17 lactic acid bacterial strains screened for lactic acid production. The effect of temperature on lactic acid production with the selected strains was investigated by adapting both strains to four different temperatures. The production of L(+)-lactic acid by both strains was most efficient at 37°C, although with L. casei the highest lactic acid concentration was obtained at 41°C. The maximal volumetric productivity with L. casei was 4.1 g l^-1 h^-1 and with L. casei subsp. rhamnosus 3.5 g l^-1 h^-1. The composition of the medium was studied in order to replace the costly yeast extract with less expensive sources of nitrogen and amino acids. From 11 different nitrogen sources investigated at 37°C, barley malt sprouts (88 g l^-1 lactic acid in 66 h) and grass extract (74 g l^-1 lactic acid in 73 h) were the best economic alternatives. The effect of different combinations of yeast extract, peptone and malt sprouts was further studied by using statistical experimental design, and an empirical second-order polynomial model was constructed on the basis of the results. With the right combination most of the yeast extract could be substituted by barley malt sprouts for efficient lactic acid production. A method for extraction of nutrients and growth factors from malt sprouts is also described. Received: 25 September 1995/Accepted: 24 October 1995     

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.     

Inhibition of beet molasses alcoholic fermentation by lactobacilli

Summary Alcohol production rate decreases as the concentration of bacterial contaminants increases. In complex medium, such as beet molasses, an alternative mechanism can be used by homofermentative lactic bacteria (Lactobacillus casei). Lactic acid and associated products, especially acetic acid, are liberated into the medium. The inhibition induced by these metabolites was reinforced by the presence of viable lactobacilli. Offprint requests to: P. Villa     

Influence of polysorbate 80 and cyclopropane fatty acid synthase activity on lactic acid production by Lactobacillus casei ATCC 334 at low pH

Lactic acid is an important industrial chemical commonly produced through microbial fermentation. The efficiency of acid extraction is increased at or below the acid’s pKa (pH 3.86), so there is interest in factors that allow for a reduced fermentation pH. We explored the role of cyclopropane synthase (Cfa) and polysorbate (Tween) 80 on acid production and membrane lipid composition in Lactobacillus casei ATCC 334 at low pH. Cells from wild-type and an ATCC 334 cfa knockout mutant were incubated in APT broth medium containing 3 % glucose plus 0.02 or 0.2 % Tween 80. The cultures were allowed to acidify the medium until it reached a target pH (4.5, 4.0, or 3.8), and then the pH was maintained by automatic addition of NH₄OH. Cells were collected at the midpoint of the fermentation for membrane lipid analysis, and media samples were analyzed for lactic and acetic acids when acid production had ceased. There were no significant differences in the quantity of lactic acid produced at different pH values by wild-type or mutant cells grown in APT, but the rate of acid production was reduced as pH declined. APT supplementation with 0.2 % Tween 80 significantly increased the amount of lactic acid produced by wild-type cells at pH 3.8, and the rate of acid production was modestly improved. This effect was not observed with the cfa mutant, which indicated Cfa activity and Tween 80 supplementation were each involved in the significant increase in lactic acid yield observed with wild-type L. casei at pH 3.8.     

Molecular Discrimination of Lactobacilli Used as Starter and Probiotic Cultures by Amplified Ribosomal DNA Restriction Analysis

Lactic acid bacteria such as Lactobacillus helveticus, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, L. acidophilus, and L. casei related taxa which are widely used as starter or probiotic cultures can be identified by amplified ribosomal DNA restriction analysis (ARDRA). The genetic discrimination of the related species belonging to these groups was first obtained by PCR amplifications by using group-specific or species-specific 16S rDNA primers. The numerical analysis of the ARDRA patterns obtained by using CfoI, HinfI, Tru9I, and ScrFI was an efficient typing tool for identification of species of the L. acidophilus and L. casei complex. ARDRA by using CfoI was a reliable method for differentiation of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. Finally, strains ATCC 393 and ATCC 15820 exhibited unique ARDRA patterns with CfoI and Tru9I restriction enzymes as compared with the other strains of L. casei, L. paracasei, and L. rhamnosus. Received: 30 August 2000 / Accepted: 2 October 2000     

Determining the Suitability of Lactobacilli Antifungal Metabolites for Inhibiting Mould Growth

Summary In recent years, public concern about indoor mould growth has increased dramatically in the United States. In this study, lactic acid bacteria (LAB), which are known to produce antimicrobial compounds important in the biopreservation of food, were evaluated to determine if the same antimicrobial properties can be used to inhibit mould fungi that typically colonize wood. Based on biomass measurement, cell-free supernatants from Lactobacillus casei subsp. rhamnosus and Lactobacillus acidophilus grown in deMan Rogosa Sharpe (MRS) broth inhibited 95–100% growth of three mould fungi and one stain fungus associated with wood-based building materials. Lactic acid and four unknown compounds ⩽ kDa molecular weight were fractionated from the culture supernatant by thin layer chromatography and high-performance liquid chromatography. Antifungal activity, which was attributed to one or more unknown metabolites, was retained during heating and neutralization. A 1:2 dilution of L. casei supernatant inhibited 100% growth of all test fungi.     

Nutrient Leaching and End Product Accumulation in Plastic Composite Supports for L-(+)-Lactic Acid Biofilm Fermentation

Investigations on the leachate bioavailability, leaching rate, and lactic acid accumulation properties of plastic composite supports (PCS) were essential for large-scale or long-term lactic acid fermentation. Leachates from PCS and polypropylene discs (controls) were analyzed by the micro-Kjeldahl method; by absorbances at 260, 275, and 280 nm; and by bioassays with Lactobacillus casei subsp. rhamnosus (ATCC 11443). The amount of leached nitrogen in a 20-ml initial soaking solution had a high correlation with the soaking solution's cell density (r = 0.87) and absorbance at 260 nm (r = 0.95). Leaching rates of various PCS were evaluated by 20 20-ml simulated repeated-batch fermentations (RBF). PCS with only yeast extract as the minor agricultural ingredient had a high leaching rate and leached out 51 to 60% of the total nitrogen during the first RBF. PCS blended with dried bovine albumin, dried bovine erythrocytes, and/or soybean flour had slowed nutrient leaching (20 to 30% of the initial leached nitrogen). Hence, they could still maintain 1 g of lactic acid per liter and measurable cell density (absorbance at 620 nm, 0.4 to 0.6) at the 20th 20-ml RBF. Lactic acid accumulation properties of PCS were evaluated by soaking the supports in a 30% lactic acid solution for 72 h at 45(deg)C. The lactic acid-soaked supports were rinsed three times and then heat treated (121(deg)C, 15 min) in 15 ml of deionized water. The results showed that lactic acid accumulation in PCS was mainly due to absorption and had no correlation with lactic acid production or biofilm formation.     

Evaluation of plastic-composite supports in repeated fed-batch biofilm lactic acid fermentation by Lactobacillus casei

A customized stirred-tank biofilm reactor was designed for plastic-composite supports (PCS). In repeated-batch studies, the PCS-biofilm reactors outperformed the suspended-cell reactors by demonstrating higher lactic acid productivities (2.45 g l(-1) h(-1) vs 1.75 g l(-1) h(-1)) and greater glucose consumption rates (3.27 g l(-1) h(-1) vs 2.09 g l(-1) h(-1)). In the repeated fed-batch studies, reactors were spiked periodically with concentrated glucose (75%) to maintain a concentration of approximately 80 g of glucose l(-1) in the bioreactor. In suspended-cell fermentations with 10 g of yeast extract (YE) l(-1) and zero, one, two, and three glucose spikes, the lactic acid productivities were 2.64, 1.58, 0.80, and 0.62 g l(-1) h(-1), respectively. In comparison, biofilm reactors with 7 g of YE l(-1) and zero, one, two, and three glucose spikes achieved lactic acid productivities of 4.20, 2.78, 0.66, and 0.94 g l(-1) h(-1), respectively. The use of nystatin (30 U ml(-1)) subdued the contaminating yeast population with no effect on the lactic acid productivity of the biofilm reactors, but it did affect productivity in the suspended-cell bioreactor. Overall, in repeated fed-batch fermentations, the biofilm reactors consistently outperformed the suspended-cell bioreactors, required less YE, and produced up to 146 g of lactic acid l(-1) with 7 g of YE l(-1), whereas the suspended-cell reactor produced 132 g l(-1) with 10 g of YE l(-1).     

Inhibition of yeast by lactic acid bacteria in continuous culture: nutrient depletion and/or acid toxicity?

Lactic acid was added to batch very high gravity (VHG) fermentations and to continuous VHG fermentations equilibrated to steady state with Saccharomyces cerevisiae. A 53% reduction in colony-forming units (CFU) ml^–1 of S. cerevisiae was observed in continuous fermentation at an undissociated lactic acid concentration of 3.44% w/v; and greater than 99.9% reduction was evident at 5.35% w/v lactic acid. The differences in yeast cell number in these fermentations were not due to pH, since batch fermentations over a pH range of 2.5–5.0 did not lead to changes in growth rate. Similar fermentations performed in batch showed that growth inhibition with added lactic acid was nearly identical. This indicates that the apparent high resistance of S. cerevisiae to lactic acid in continuous VHG fermentations is not a function of culture mode. Although the total amount of ethanol decreased from 48.7 g l^–1 to 14.5 g l^–1 when 4.74% w/v undissociated lactic acid was added, the specific ethanol productivity increased ca. 3.2-fold (from 7.42×10^–7 g to 24.0×10^–7 g ethanol CFU^–1 h^–1), which indicated that lactic acid stress improved the ethanol production of each surviving cell. In multistage continuous fermentations, lactic acid was not responsible for the 83% (CFU ml^–1) reduction in viable S. cerevisiae yeasts when Lactobacillus paracasei was introduced to the system at a controlled pH of 6.0. The competition for trace nutrients in those fermentations and not lactic acid produced by L. paracasei likely caused the yeast inhibition.     

Production of lactic acid from date juice extract with free cells of single and mixed cultures of Lactobacillus casei and Lactococcus lactis

The production of lactic acid from date juice by single and mixed cultures of Lactobacillus casei and Lactococcus lactis was investigated. In the present conditions, the highest concentration of lactic acid (60.3 g l⁻¹) was obtained in the mixed culture system while in single culture fermentations of Lactobacillus casei or Lactococcus lactis, the maximum concentration of lactic acid was 53 and 46 g l⁻¹, respectively. In the case of single Lactobacillus casei or Lactococcus lactis, the total percentage of glucose and fructose utilized were 82.2; 94.4% and 93.8; 60.3%, respectively, whereas in the case of mixed culture, the total percentage of glucose and fructose were 96 and 100%, respectively. These results showed that the mixed culture system gave better results than single cultures regarding lactic acid concentration, and sugar consumption.     

<Emphasis Type="Italic">Lactobacillus casei</Emphasis> as a biocatalyst for biofuel production

Microbial fermentation of sugars from plant biomass to alcohols represents an alternative to petroleum-based fuels. The optimal biocatalyst for such fermentations needs to overcome hurdles such as high concentrations of alcohols and toxic compounds. Lactic acid bacteria, especially lactobacilli, have high innate alcohol tolerance and are remarkably adaptive to harsh environments. This study assessed the potential of five Lactobacillus casei strains as biocatalysts for alcohol production. L. casei 12A was selected based upon its innate alcohol tolerance, high transformation efficiency and ability to utilize plant-derived carbohydrates. A 12A derivative engineered to produce ethanol (L. casei E1) was compared to two other bacterial biocatalysts. Maximal growth rate, maximal optical density and ethanol production were determined under conditions similar to those present during alcohol production from lignocellulosic feedstocks. L. casei E1 exhibited higher innate alcohol tolerance, better growth in the presence of corn stover hydrolysate stressors, and resulted in higher ethanol yields.     

Lactate from cultures of Lactobacillus casei recovered in a fluidized bed column using ion exchange resin

Summary Lactic acid produced by continuous culture of L.casei in an upflow packed bed reactor, was recovered with Amberlite IRA 400 in a fluidized bed column. Bed expansions of 1.25 and 2.25 were applied. Reutilization did not alter the capability of net recovery of 0.048 ± 0.01 g lactic acid/g resin. When 2200 cm/h of ascensional velocity was used, (bed expansion of 2.25), the resin adsorbed 39.3% of the initial lactic acid and 63.5% was eluted. This resin supported the highest exchange capacity of 0.126 g lactic acid/g resin. Applying high flow rates, the process has potential industrial applications due to the short time employed.     

Characteristics of aerobic,solid substrate fermentation of swine waste-corn mixtures

Summary Aerobic fermentation of swine waste combined with corn produced differences in microbial and biochemical patterns dependent on use of fresh or stored excrement. Lactic acid fermentation and odor control resulted with either waste. Homofermentative lactic acid bacteria were present initially at 10⁷ organisms/dry g with stored waste-corn cultures and total microflora amounted to 10⁸ organisms/dry g. Fresh waste-corn fermentations initially yielded heterofermentative lactic acid bacteria at 10⁷ organisms/dry g and total viable population was 10⁹ organisms/dry g. These respective groups of lactic acid bacteria dominated from 12 through 144 h in cultures with either waste, and acid production (0.2 meq/dry g) decreased pH by 2 units to 4.5. The major acid component with stored waste-corn was lactic acid, whereas fresh waste-corn fermentation produced both lactic and homologous fatty acids from acetic through valeric acid. Coliform bacteria present initially at 10⁵ organisms/dry g in stored waste-corn cultures were not detected after 36 h; coliform bacteria in fresh waste-corn fermentations persisted at 10⁶ organisms/dry g. A silage-like fermentation product resulted which may have use in animal feed formulations.     

Lactic acid fermentation on barley flour without additional nutrients

Summary An amylolytic lactic acid producing Lactobacillus amylovorus produced 36 g/l of lactic acid in mixed cultures with L. casei without additional nutrients at 37 °C in 48 h, when barley flour concentration was 180 g/l (appr. 108 g/l starch) and barley malt quantity 0.8% of flour weight. This represented an improvement of up to 20% in comparison to the fermentation with L. amylovorus or L. casei alone. By simultaneous glucoamylase addition lactic acid production yield was about doubled. With L. casei the lactic acid yield was from 580 g in 72 h to 667 g in 144 h per kg barley flour.