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)).     

Inhibition of Listeria monocytogenes by piscicolin 126 in milk and Camembert cheese manufactured with a thermophilic starter

The effect of bacteriocin, piscicolin 126, on the growth of Listeria monocytogenes and cheese starter bacteria was investigated in milk and in Camembert cheese manufactured from milk challenged with 10(2) cfu ml(-1) L. monocytogenes. In milk incubated at 30 degrees C, piscicolin 126 added in the range of 512-2,048 AU ml(-1) effectively inhibited growth of L. monocytogenes for more than 20 d when challenged with approximately 10(2) cfu ml(-1) L. monocytogenes. At higher challenge levels (10(4) and 10(6) cfu ml(-1)), piscicolin 126 reduced the viable count of L. monocytogenes by 4-5 log units immediately after addition of the bacteriocin; however, growth of Listeria occurred within 24 h. The minimum inhibitory concentration (MIC) of piscicolin 126 against lactic acid cheese starter bacteria was generally greater than 204,800 AU ml(-1) , and the viable count and acid production of these starter cultures in milk were not affected by the addition of 2,048 AU ml(-1) piscicolin 126. Camembert cheeses made from milk challenged with L. monocytogenes and with added piscicolin 126 showed a viable count of L. monocytogenes 3-4 log units lower than those without piscicolin 126. Inactivation of piscicolin 126 by proteolytic enzymes from cheese starter bacteria and mould together with the emergence of piscicolin 126-resistant isolates was responsible for the recovery of L. monocytogenes in the cheeses during ripening.     

Enhanced production of cellulase usingAcidothermus cellulyticus in fed-batch culture

Summary Fed-batch fermentations of Acidothermus cellulolyticus utilizing mixtures of cellulose and sugars were investigated for potential improvements in cellulase enzyme production. In these fermentations, we combined cellulose from several sources with various simple sugars at selected concentrations. The best source of cellulose for cellulase production was found to be ball-milled Solka Floc at 15 g/l. Fed-batch fermentations with cellobiose and Solka Floc increased cell mass only slightly, but succeeded in significantly enhancing cellulase synthesis compared to batch conditions. Maximum cellulase activities obtained from fermentations initiated with 2.5 g cellobiose/l and 15 g Solka Floc/l were 0.187 units (U)/ml, achieved by continuous feeding to maintain <0.1 g cellobiose/l, and 0.215 U/ml using the same initial medium when 2.5 g cellobiose/l was step-fed after the sugar was nearly consumed. In batch, dual-substrate systems consisting of simple sugars with Solka Floc, substrate inhibition was evident in terms of specific growth rates, specific productivity values, and maximum enzyme yields. Limiting concentrations of glucose or sucrose at 5 g/l, and cellobiose at 2.5 g/l, in the presence of Solka Floc, yielded cellulase activities of 0.134, 0.159, and 0.164 U/ml, respectively. Offprint requests to: M. E. Himmel     

Effects of lactobacilli on yeast-catalyzed ethanol fermentations.

Normal-gravity (22 to 24 degrees Plato) wheat mashes were inoculated with five industrially important strains of lactobacilli at approximately 10(5), approximately 10(6), approximately 10(7), approximately 10(8), and approximately 10(9) CFU/ml in order to study the effects of the lactobacilli on yeast growth and ethanol productivity. Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus #3, Lactobacillus rhamnosus, and Lactobacillus fermentum were used. Controls with yeast cells but no bacterial inoculation and additional treatments with bacteria alone inoculated at approximately 10(7) CFU/ml of mash were included. Decreased ethanol yields were due to the diversion of carbohydrates for bacterial growth and the production of lactic acid. As higher numbers of the bacteria were produced (depending on the strain), 1 to 1.5% (wt/vol) lactic acid resulted in the case of homofermentative organisms. L. fermentum, a heterofermentative organism, produced only 0.5% (wt/vol) lactic acid. When L. plantarum, L. rhamnosus, and L. fermentum were inoculated at approximately 10(6) CFU/ml, an approximately 2% decrease in the final ethanol concentration was observed. Smaller initial numbers (only 10(5) CFU/ml) of L. paracasei or Lactobacillus #3 were sufficient to cause more than 2% decreases in the final ethanol concentrations measured compared to the control. Such effects after an inoculation of only 10(5) CFU/ml may have been due to the higher tolerance to ethanol of the latter two bacteria, to the more rapid adaptation (shorter lag phase) of these two industrial organisms to fermentation conditions, and/or to their more rapid growth and metabolism. When up to 10(9) CFU of bacteria/ml was present in mash, approximately 3.8 to 7.6% reductions in ethanol concentration occurred depending on the strain. Production of lactic acid and a suspected competition with yeast cells for essential growth factors in the fermenting medium were the major reasons for reductions in yeast growth and final ethanol yield when lactic acid bacteria were present.     

Probiotic properties of Lactobacillus strains isolated from the feces of breast-fed infants and Taiwanese pickled cabbage

This study assessed potential probiotic Lactobacillus strains isolated from the feces of breast-fed infants and from Taiwanese pickled cabbage for their possible use in probiotic fermented foods by evaluating their (i) in vitro adhesive ability, resistance to biotic stress, resistance to pathogenic bacteria, and production of β-galactosidase; (ii) milk technological properties; and (iii) in vivo adhesive ability, intestinal survival and microbial changes during and after treatment. Five Lactobacillus isolates identified as Lactobacillus reuteri F03, Lactobacillus paracasei F08, Lactobacillus rhamnosus F14, Lactobacillus plantarum C06, and Lactobacillus acidophilus C11 that showed resistance to gastric juice and bile salts were selected for further evaluation of their probiotic properties. All the strains demonstrated the ability to adhere to Caco-2 cells, particularly, strain L. plantarum C06 and L. reuteri F03 showed satisfactory abilities, which were similar to that of the reference strain L. rhamnosus GG. The strains L. paracasei F08 and L. acidophilus C11 had the highest β-galactosidase activity. Most of the strains were resistant to aminoglycosides and vancomycin but sensitive to ampicillin, erythromycin, and penicillin. All the 5 strains elicited antibacterial activity against both Gram-positive (Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus) and -negative (Escherichia coli and Salmonella enterica) pathogens. Moreover, the strains L. reuteri F03, L. paracasei F08, and L. plantarum C06 could grow rapidly in milk without nutrient supplementation and reached 10? cfu/mL after 24 h of fermentation at 37 °C. The viable cell counts of the 3 strains remained above 10? cfu/mL after 21 d of storage at 4 °C. In the animal feeding trial, the number of intestinal lactobacilli increased significantly after administration of milk fermented with the 3 strains, and the counts of fecal coliforms and Clostridium perfringens were markedly reduced. Lactobacillus strains could also survive in the ileal intestinal tissue of the treated rats. Technologically interesting Lactobacillus isolates may be used in the future as probiotic starter cultures for manufacturing novel fermented foods.     

Biosynthesis of protease from Lactobacillus paracasei: kinetic analysis of fermentation parameters

Fifteen strains of Lactobacillus species, isolated from different samples of curd were screened for their ability to produce more extracellular protease. The proteolytic activities of these strains based on casein hydrolysis showed a variation of 1.26-5.80 U ml(-l), with Lactobacillus IH8 showing the maximum activity and was identified as L. paracasei. Different cultural conditions for enhanced production of protease by L. paracasei were optimized. The optimal conditions for production of the enzyme were an incubation temperature of 35 degrees C and a medium pH of 6.0. The maximum proteolytic activity of L. paracasei (7.28 Uml(-1)) was achieved after 48 h of cultivation. The kinetic parameters such as product yield (Yp/x,), growth yield (Yx/s), specific product yield (qp) and specific growth yield (qs) coefficients also revealed that the values of experimental results were kinetically significant.     

Growth and morphology of thermophilic dairy starters in alginate beads

The aim of this research was to produce concentrated biomasses of thermophilic lactic starters using immobilized cell technology (ICT). Fermentations were carried out in milk using pH control with cells microentrapped in alginate beads. In the ICT fermentations, beads represented 17% of the weight. Some assays were carried out with free cells without pH control, in order to compare the ICT populations with those of classical starters. With Streptococcus thermophilus, overall populations in the fermentor were similar, but maximum bead population for (8.2 x 10(9) cfu/g beads) was 13 times higher than that obtained in a traditional starter (4.9 x 10(8) cfu/ml). For both Lactobacillus helveticus strains studied, immobilized-cell populations were about 3 x 10(9) cfu/g beads. Production of immobilized Lb. bulgaricus 210R strain was not possible, since no increases in viable counts occurred in beads. Therefore, production of concentrated cell suspension in alginate beads was more effective for S. thermophilus. Photomicrographs of cells in alginate beads demonstrated that, while the morphology of S. thermophilus remained unchanged during the ICT fermentation, immobilized cells of Lb. helveticus appeared wider. In addition, cells of Lb. bulgaricus were curved and elongated. These morphological changes would also impair the growth of immobilized lactobacilli.     

Effects of live Lactobacillus paracasei on plasma lipid concentration in rats fed an ethanol-containing diet

The protective effects of live Lactobacillus paracasei NFRI 7415 on alcoholic liver disease were investigated. Male Fischer 344 rats were fed a control diet (CD), an ethanol diet (ED) (35.8% of total energy from ethanol), or an ethanol diet containing 20% live Lb. paracasei NFRI 7415 (10(7) cfu/g) (LD) for 10 weeks. The results indicated that live Lb. paracasei NFRI 7415 reduced the total cholesterol concentration of the plasma and liver in the rats fed the LD. The level of docosahexaenoic acid (DHA; 22:6n-3) in the plasma and liver of the LD group was higher than in the ED group. Chronic alcohol consumption decreased the level of n-3 fatty acid in the plasma and liver of the ED group. These results indicated that live Lb. paracasei NFRI 7415 can adjust the fatty acid composition of the plasma and liver, and that it is possible to decrease liver damage due to chronic alcohol intake.     

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.     

Electrotransformation of intact cells ofBrevibacterium flavum MJ-233

Summary Electroporation allowed transformation of intact cells ofBrevibacterium flavum MJ-233. The two plasmids used for electroporation were pCRY2 (6.3 kilobases) and pCRY3 (8.2 kilobases). Both plasmids contain the chloramphenicol-resistance gene and the autonomous replication origin inB. flavum MJ-233. The efficiency of electrotransformation was optimal with cells harvested at the middle log phase of growth, and was imporved by the addition of 1.0U/ml of penicillin G to the culture medium. The optimum yield of transformants per g DNA was 5×10⁴ when the cell suspension was pulsed at a cell density of 1×10¹⁰/ml and at a DNA amount of 1.0g.     

Functional and safety characterization of autochthonous Lactobacillus paracasei FS103 isolated from sheep cheese and its survival in sheep and cow fermented milks during cold storage

The main objective of this study was to evaluate some probiotic characteristics of Lactobacillus spp. isolated from traditional sheep cheese, and to investigate the fermentative ability and viability in sheep and cow milks of a selected potential probiotic Lactobacillus (L.) strain, i.e., L. paracasei FS103. A total of 54 autochthonous Lactobacillus isolates were characterized for (i) acidity and bile salt resistance, (ii) tolerance to gastric and intestinal juice models, and (iii) antagonistic activity against pathogens and antibiotic resistance. Potential probiotic Lactobacillus has been used in sheep and cow milks for the manufacturing of experimental fermented milks. In these latter, pH value, microbial count, and sensory analysis were carried out. Lactobacillus FS103 classified as L. paracasei subsp. paracasei had a good survival in gastric and intestinal juice models, inhibited the growth of undesirable bacteria, and was susceptible to chloramphenicol, clindamycin, penicillin, amoxicillin, erythromycin, tetracycline, and ampicillin. Moreover, when used to produce experimental sheep and cow fermented milks, L. paracasei FS103 was able to acidify both milk types leading to a continuous pH decrease during all fermentation time (24 h). FS103 population remains viable at a level > 108 CFU mL−1 after 21 days of cold (4 °C) storage. The results of sensory analysis showed that scores related to consistency, taste, and astringent were significantly higher in sheep fermented milk while animal-like was less acceptable compared to cow fermented milk. Lactobacillus paracasei FS103 isolated from sheep cheese exhibited potential probiotic properties and suitable features for sheep and cow fermented milks maintaining high vitality during cold storage.     

Lactococcus lactis release from calcium alginate beads.

Cell release during milk fermentation by Lactococcus lactis immobilized in calcium alginate beads was examined. Numbers of free cells in the milk gradually increased from 1 x 10(6) to 3 x 10(7) CFU/ml upon successive reutilization of the beads. Rinsing the beads between fermentations did not influence the numbers of free cells in the milk. Cell release was not affected by initial cell density within the beads or by alginate concentration, although higher acidification rates were achieved with increased cell loading. Coating alginate beads with poly-L-lysine (PLL) did not significantly reduce the release of cells during five consecutive fermentations. A double coating of PLL and alginate reduced cell release by a factor of approximately 50. However, acidification of milk with beads having the PLL-alginate coating was slower than that with uncoated beads. Immersing the beads in ethanol to kill cells on the periphery reduced cell release, but acidification activity was maintained. Dipping the beads in aluminum nitrate or a hot CaCl2 solution was not as effective as dipping them in ethanol. Ethanol treatment or heating of the beads appears to be a promising method for maintaining acidification activity while minimizing viable cell release due to loosely entrapped cells near the surface of the alginate beads.     

Lactococcus lactis release from calcium alginate beads.

Cell release during milk fermentation by Lactococcus lactis immobilized in calcium alginate beads was examined. Numbers of free cells in the milk gradually increased from 1 x 10(6) to 3 x 10(7) CFU/ml upon successive reutilization of the beads. Rinsing the beads between fermentations did not influence the numbers of free cells in the milk. Cell release was not affected by initial cell density within the beads or by alginate concentration, although higher acidification rates were achieved with increased cell loading. Coating alginate beads with poly-L-lysine (PLL) did not significantly reduce the release of cells during five consecutive fermentations. A double coating of PLL and alginate reduced cell release by a factor of approximately 50. However, acidification of milk with beads having the PLL-alginate coating was slower than that with uncoated beads. Immersing the beads in ethanol to kill cells on the periphery reduced cell release, but acidification activity was maintained. Dipping the beads in aluminum nitrate or a hot CaCl2 solution was not as effective as dipping them in ethanol. Ethanol treatment or heating of the beads appears to be a promising method for maintaining acidification activity while minimizing viable cell release due to loosely entrapped cells near the surface of the alginate beads.     

Production and application of methylotrophic yeast Pichia pastoris

Pichia pastoris is a methylotrophic yeast that makes use bf the enzyme alcohol oxidase to catalyze the first step of the dissimilatory pathway that enables it to grow on methanol. Because of its stability and low substrate specificity, alcohol oxidase is of considerable interest for a range of biotechnological processes. Various feeding regimes were evaluated in an effort to increase the biomass concentration and productivity that could be achieved from fermentations using this organism. Through continuous or semicontinuous feeding, biomass concentrations were increased 10-fold over those achieved in batch fermentations. In subsequent trials, nongrowing whole cells were applied successfully to convert ethanol to acetaldehyde. Quantitative conversions of 20-g/L solutions of ethanol have been achieved in 2 h, and acetaldehyde concentrations of up to 35 g/L have been achieved using extended reaction times of 5 h. The conversion reaction was limited by end product inhibition and by acetaldehyde holdup within the yeast cells.     

Fermentation of paddy malt mash to ethanol by mixed cultures of Saccharomyces cerevisiae and Zymomonas mobilis ZM4 with penicillin G

Traditional fermentation of paddy malt mash (containing 18.1% w/v dextrose equivalent) to paddy arrack using paddy husk as source of inoculum yielded very low level of ethanol (4.25% v/v). Use of yeast isolates obtained from paddy husk as well as a potent ethanol producer like Zymomonas mobilis ZM4 and their combinations in the fermentation revealed that a combination of an yeast isolate PH 03 (Saccharomyces cerevisiae) and Z. mobilis ZM4 produced synergistically and statistically more ethanol (10.1% v/v) than the individual and other combination of cultures. In this process, addition of penicillin G at a concentration of 20 U/ml rather than heat sterilization, helped retention of the limited amylase activity in the mash for simultaneous saccharification and fermentation over 7 d at 30°C. About 98.5% of the carbohydrate was accountable in the fermentation which yielded 86.7% of the theoretical yield of ethanol, apart from biomass and acids.     

High concentration cultivation of Bifidobacterium bifidum in a submerged membrane bioreactor

In batch cultures, after 25 h, the maximum cell mass of Bifidobacterium bifidum BGN4 was 4.5 g/L, and the maximum cell count was 3.0 x 10(9) cfu/mL at pH 6.0 and 50 g/L sucrose. To increase the viable counts of bifidobacteria, cell retentive culture was applied using a submerged membrane bioreactor with suction and gas sparging. The maximum mass, count, and productivity of the cells after 36 h were 12.0 g/L, 2.2 x 10(10) cfu/mL, and 6.1 x 10(8) cfu/mL x h, respectively, at the feeding (dilution) rate of 120 mL/h (0.06 h-1) in the feeding medium. The accumulated levels of organic acids and ammonium ions at the end of the cultivation were 1.5 and 1.0 g/L, respectively. The viable counts and volumetric productivity of the cells after the cell retentive culture were 7.3- and 5.1-fold higher, respectively, than the values obtained during batch culture. These high viable counts and volumetric productivities were obtained by maintaining lower concentrations of organic acids and ammonium ions so that the growth of B. bifidum BGN4 was not inhibited. The submerged membrane bioreactor produced the highest viable counts of B. bifidum without membrane fouling and cell damage.     

Fermentation kinetics of Zymomonas mobilis near zero growth rate

The fermentation kinetics Zymomonas mobilis were studied near zero growth rate in fed-batch cultures and continuous cultures with complete cell recycle. The results show the ethanol enhances that specific substrate conversion rate under these conditions. The maximum achievable ethanol concentration in continuous cultures with cell recycle (66 g/L) was significantly lower than in fed-batch cultures (100 g/L). The results indicate that growth-rate-independent metabolism is not instantaneous and can lag behind steadily increasing ethanol concentrations in fed-batch fermentations. A model is proposed to account for this slow adaptation.     

Effect of Dissolved Oxygen, Temperature, Initial Cell Count, and Sugar Concentration on the Viability of Saccharomyces cerevisiae in Rapid Fermentations

By using 7 x 10(8) cells of Saccharomyces cerevisiae per ml with which 25 degrees Brix honey solutions were fermented to 9.5% (wt/vol; 12% vol/vol) ethanol in 2.5 to 3 h at 30 C, i.e., rapid fermentation, the death rate was found to be high, with only 2.1% of the yeast cells surviving at the end of 3 h under anaerobic conditions. As the dissolved oxygen in the medium was increased from 0 to 13 to 20 to 100% in rapid fermentations at 30 C, there was a progressive increase in the percentage of cells surviving. The ethanol production rate and total were not seriously affected by a dissolved oxygen concentration of 13%, but fermentation was retarded by 20% dissolved oxygen and still further decreased as the dissolved oxygen content reached 100%. When the fermentation temperature was decreased to 15 C (at 13% dissolved oxygen), the rate of fermentation decreased, and the fermentation time to 9.5% ethanol (wt/vol) increased to 6 h. It was found that the higher the temperature between 15 and 30 C, the greater the rate of death as initial cell counts were increased from 1.1 x 10(7) to 7.8 x 10(8) cells per ml. At the lowest level of inoculum, 1.1 x 10(7) cells per ml, there was actual multiplication, even at 30 C; however, the fermentation was no longer rapid. The addition of 15% sugar, initially followed after an hour by the remaining 10%, or addition of the sugar in increments of 2.5 or 5% yielded a better survival rate of yeast cells than when the fermentation was initiated with 25% sugar.     

Changes in steady state on introduction of a Lactobacillus contaminant to a continuous culture ethanol fermentation

Lactobacillus paracasei was introduced as a contaminant into a multistage continuous culture ethanol fermentation system at ratios of 1:100, 1:1, and 70:1 with Saccharomyces cerevisiae, but failed to overtake the yeast. None of the inoculation ratios allowed L. paracasei to affect S. cerevisiae in the first fermentor in the multistage system. S. cerevisiae remained constant at ∼3×10⁷ CFU/ml regardless of the bacterial inoculation level, and even at the 70:1 inoculation ratio, glucose, ethanol, and lactic acid concentrations did not change from the steady-state concentrations seen before bacterial inoculation. However, L. paracasei decreased steadily from its initial inoculation level of ∼2.2×10⁹ CFU/ml and stabilized at 3.7×10⁵ CFU/ml after 10 days of steady-state operation. Both organisms then persisted in the multistage system at an approximate L. paracasei/S. cerevisiae ratio of 1:100 which confirms that, in continuous fuel ethanol production, it would be difficult to eliminate this bacterium. Only when the pH was controlled at 6.0 in fermentor 1 (F1) were changes seen which would affect the multistage system. Ethanol concentration then decreased by 44% after 4 days of pH-controlled operation. This coincided with an increase in L. paracasei to >10¹⁰ CFU/ml, and a 4× increase in lactic acid concentration to 20 g/l. When the clarified contents from other fermentors (F2–F5) in the multistage system were used as growth media, L. paracasei was not able to grow in batch culture. This indicated that the first fermentor in the multistage system was the only fermentor capable of supporting the growth of L. paracasei under the described conditions. Journal of Industrial Microbiology & Biotechnology (2001) 27, 39–45. Received 26 February 2001/ Accepted in revised form 29 May 2001     

Control of feed rate to a fed-batch culture using a heat-flux sensor

Summary Substrate inhibition in batch fermentations can be avoided by employing the fed-batch technique in which substrate concentration is kept at low levels by a programmed feed rate. This research demonstrates the use of a heat-flux sensor to control substrate addition by continuously monitoring evolving heat which is proportional to fermentation rate. Batch fermentation with 240 g/L glucose in the medium was compared with a fed-batch starting with 20 g/L glucose in the medium and increased, with 500 g/L glucose, to a final equivalent glucose concentration of 240 g/L. The batch fermentation produced 106 g/L ethanol in 39 hr at 2.72 g/L/h, while the best fed-batch produced 114 g/L ethanol in 34 hr at 3.35 g/L/h with the same nutrients.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.