Relationship between pH and medium dissolved solids in terms of growth and metabolism of lactobacilli and Saccharomyces cerevisiae during ethanol production

The specific growth rates of four species of lactobacilli decreased linearly with increases in the concentration of dissolved solids (sugars) in liquid growth medium. This was most likely due to the osmotic stress exerted by the sugars on the bacteria. The reduction in growth rates corresponded to decreased lactic acid production. Medium pH was another factor studied. As the medium pH decreased from 5.5 to 4.0, there was a reduction in the specific growth rate of lactobacilli and a corresponding decrease in the lactic acid produced. In contrast, medium pH did not have any significant effect on the specific growth rate of yeast at any particular concentration of dissolved solids in the medium. However, medium pH had a significant (P < 0.001) effect on ethanol production. A medium pH of 5.5 resulted in maximal ethanol production in all media with different concentrations of dissolved solids. When the data were analyzed as a 4 (pH levels) by 4 (concentrations of dissolved solids) factorial experiment, there was no synergistic effect (P > 0.2923) observed between pH of the medium and concentration of dissolved solids of the medium in reducing bacterial growth and metabolism. The data suggest that reduction of initial medium pH to 4.0 for the control of lactobacilli during ethanol production is not a good practice as there is a reduction (P < 0.001) in the ethanol produced by the yeast at pH 4.0. Setting the mash (medium) with > or =30% (wt/vol) dissolved solids at a pH of 5.0 to 5.5 will minimize the effects of bacterial contamination and maximize ethanol production by yeast.     

Effect of pH and lactic or acetic acid on ethanol productivity by Saccharomyces cerevisiae in corn mash

The effects of lactic and acetic acids on ethanol production by Saccharomyces cerevisiae in corn mash, as influenced by pH and dissolved solids concentration, were examined. The lactic and acetic acid concentrations utilized were 0, 0.5, 1.0, 2.0, 3.0 and 4.0% w/v, and 0, 0.1, 0.2, 0.4, 0.8 and 1.6% w/v, respectively. Corn mashes (20, 25 and 30% dry solids) were adjusted to the following pH levels after lactic or acetic acid addition: 4.0, 4.5, 5.0 or 5.5 prior to yeast inoculation. Lactic acid did not completely inhibit ethanol production by the yeast. However, lactic acid at 4% w/v decreased (P<0.05) final ethanol concentration in all mashes at all pH levels. In 30% solids mash set at pH ≤5, lactic acid at 3% w/v reduced (P<0.05) ethanol production. In contrast, inhibition by acetic acid increased as the concentration of solids in the mash increased and the pH of the medium declined. Ethanol production was completely inhibited in all mashes set at pH 4 in the presence of acetic acid at concentrations ≥0.8% w/v. In 30% solids mash set at pH 4, final ethanol levels decreased (P<0.01) with only 0.1% w/v acetic acid. These results suggest that the inhibitory effects of lactic acid and acetic acid on ethanol production in corn mash fermentation when set at a pH of 5.0–5.5 are not as great as that reported thus far using laboratory media.     

Effect of pH on growth and ethanol production byZymomonas

Summary In a mineral salts medium containing yeast extract, NH₄Cl and glucose (50g/L), the pH range producing the fastest growth ofZ. mobilis was 5.5–6.5 with an apparent optimum at 6.5. At constant growth rate of 0.15hr^–1, the specific rates of glucose utilization (q_s) and ethanol production (q_p) were relatively unaffected by pH over the range 7.0–5.5 but increased sharply as the pH was further decreased below 5.5 to 4.0. Under these conditions the ethanol yield was unaffected by pH over the range 4.0–6.5 but decreased markedly at pH of 7.     

Fermentation of starch hydrolysates byLactobacillus plantarum

Summary Lactic acid production by an isolated ofLactobacillus plantarum was standardised on enzyme-hydrolysed tapioca (Manihot esculenta) flour, tapioca starch and soluble starch. Calculated yields of lactic acid (g from 100 g reducing sugars used) in nutrient media containing the abovementioned hydrolysates (10% reducing sugars) were 21.8%, 16.2% and 16.2%, respectively. Higher yields (29–34%) were obtained in media containing 5% reducing sugars. A conversion efficiency of 80–99% was achieved when the acid produced in the broth was neutralised periodically. One hundred milliliters of the medium (5% sugars) yielded 4.0–4.5 g of calcium lactate. These results indicate that unrefined starchy material can be successfully employed for the economic production of lactic acid. The same substrate can also be utilised for biomass production, as viable lactobacilli are being used for therapy in medicine.     

Effect of yeast inoculation rate on the metabolism of contaminating lactobacilli during fermentation of corn mash

Two separate 4 (bacterial concentrations)×6 (yeast concentrations) full factorial experiments were conducted in an attempt to identify a novel approach to minimize the effects caused by bacterial contamination during industrial production of ethanol from corn. Lactobacillus plantarum and Lactobacillus paracasei, commonly occurring bacterial contaminants in ethanol plants, were used in separate fermentation experiments conducted in duplicate using an industrial strain of Saccharomyces cerevisiae, Allyeast Superstart. Bacterial concentrations were 0, 1×10⁶, 1×10⁷ and 1×10⁸ cells/ml mash. Yeast concentrations were 0, 1×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, and 4×10⁷ cells/ml mash. An increased yeast inoculation rate of 3×10⁷ cells/ml resulted in a greater than 80% decrease (P<0.001) and a greater than 55% decrease (P<0.001) in lactic acid production by L. plantarum and L. paracasei, respectively, when mash was infected with 1×10⁸ lactobacilli/ml. No differences (P>0.25) were observed in the final ethanol concentration produced by yeast at any of the inoculation rates studied, in the absence of lactobacilli. However, when the mash was infected with 1×10⁷ or 1×10⁸ lactobacilli/ml, a reduction of 0.7–0.9% v/v (P<0.005) and a reduction of 0.4–0.6% v/v (P<0.005) in the final ethanol produced was observed in mashes inoculated with 1×10⁶ and 1×10⁷ yeast cells/ml, respectively. At higher yeast inoculation rates of 3×10⁷ or 4×10⁷ cells/ml, no differences (P>0.35) were observed in the final ethanol produced even when the mash was infected with 1×10⁸ lactobacilli/ml. The increase in ethanol corresponded to the reduction in lactic acid production by lactobacilli. This suggests that using an inoculation rate of 3×10⁷ yeast cells/ml reduces the growth and metabolism of contaminating lactic bacteria significantly, which results in reduced lactic acid production and a concomitant increase in ethanol production by yeast.     

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.     

Selection and Optimization of Culture Medium for Exopolysaccharide Production by Coriolus (Trametes) Versicolor

Summary Coriolus versicolor is a medicinal fungus producing exopolysaccharides (EPS). Five well-defined culture media were studied to select the medium that maximizes production of EPS by C. versicolor. Biomass, reducing sugars and EPS concentrations along with the rheological behaviour of the broth were followed during fermentations lasting 9 days. The yeast malt extract medium (YM) was shown to yield the highest production of EPS. Fermentation conditions with YM medium were further investigated to optimize EPS production by C. versicolor. An experimental design to do this was adopted, in which the effects of pH and initial substrate concentration were considered. The effects of initial glucose concentration (5, 15 and 25 g l⁻¹) and pH (4.0, 5.5 and 7.0) were evaluated. The initial glucose concentration was found to be the most important factor in EPS production and also cell growth.     

Fungal Invertase as an Aid for Fermentation of Cane Molasses into Ethanol

Comparative studies of the fermentation of cane molasses into ethanol by Saccharomyces cerevisiae in the presence or absence of fungal invertase were performed. When cane molasses was fermented by the yeast at 30°C and pH 5.0, the presence of the enzyme had no effect on ethanol production. At pH 3.5, ethanol production was increased by the addition of invertase. At 40°C, the addition of invertase increased ethanol production by 5.5% at pH 5.0 and by 20.9% at pH 3.5.     

Influence of process parameters on growth of Clostridium ljungdahlii and Clostridium autoethanogenum on synthesis gas

Effects of initial medium pH and gas flow rate on Clostridium ljungdahlii and Clostridium autoethanogenum in liquid batch, continuous gas fermentations were investigated. Synthesis gas components were supplied at varying flow rates (5, 7.5 and 10 mL/min) for C. ljungdahlii (pH 6.8 and 5.5) and C. autoethanogenum (pH 6.0). Growth on synthesis gas was slower than growth on sugars. For C. ljungdahlii, higher cell densities were achieved at pH 6.8 (579 mg/L) compared to pH 5.5 (378 mg/L). The ethanol concentration at pH 6.8 was also 110% greater than that at pH 5.5. The interaction of flow rate and pH was statistically significant with the greatest acetate production in the 10 mL/min, pH 6.8 treatment. The ethanol to acetate ratios were smaller at lower pH levels and higher flow rates. In C. autoethanogenum fermentations, higher flow rates resulted in greater end product formation with no significant effect on product ratios.     

Production of amylase(s) by Schwanniomyces castellii and Endomycopsis fibuligera

Schwanniomyces castellii and Endomycopsis fibuligera Produced extracellular amylase(s) when grown on various carbon sources and at different pH values. Both yeast species showed significant amylase synthesis in the presence of either maltose or soluble starch. On the other substrates tested (glucose, cellobiose, sucrose, trehalose, melezitose, raffinose, ethanol, glycerol) differences were found regarding growth and amylase production. Free glucose in the culture medium apparently inhibited enzyme synthesis. The pH range allowing maximal growth and amylase production was 4.5–6.0 for E. fibuligera and 5.5–7.0 for S. castellii.     

Modeling of Growth of Lactobacillus sanfranciscensis and Candida milleri in Response to Process Parameters of Sourdough Fermentation

We investigated the effect of the ecological factors pH, temperature, ionic strength, and lactate, acetate, and ethanol levels on Candida milleri and two strains of Lactobacillus sanfranciscensis, organisms representative of the microflora of sourdough. A mathematical model describing the single and combined effects of these factors on the growth of these organisms was established in accordance with the following criteria: quality of fit, biological significance of the parameters, and applicability of the in vitro data to in situ processes. The growth rates of L. sanfranciscensis LTH1729 and LTH2581 were virtually identical under all conditions tested. These organisms tolerated >160 mmol of undissociated acetic acid per liter. Growth occurred in the pH range of 3.9 to 6.7 and was completely inhibited by 4% NaCl. C. milleri had a lower optimum temperature for growth (27°C) than the lactobacilli. The growth of the yeast was not affected by pH in the range of 3.5 to 7, and up to 8% NaCl was tolerated. Complete inhibition of growth occurred at 150 mmol of undissociated acetic acid per liter, but acetate at concentrations of up to 250 mmol/liter exerted virtually no effect. The model provides insight into factors contributing to the stability of the sourdough microflora and can facilitate the design of novel sourdough processes.     

Development of Bacterial Contamination during Production of Yeast Extracts

Baker’s yeast suspensions having bacterial populations of 10⁶ and 10⁸ CFU/ml were subjected to autolysis processes designed to obtain yeast extracts (YE). The bacterial contaminants added to the yeast cell suspensions were produced with spent broths obtained from a commercial yeast production plant and contained 59% cocci (Leuconostoc, Aerococcus, Lactococcus) as well as 41% bacilli (Bacillus). Autolyses were conducted at four different pH levels (4.0, 5.5, 7.0, and 8.5) and with two autolysis-promoting agents (ethyl acetate and chitosan). Processing parameters were more important than the initial bacterial population in the development of contaminating bacteria during manufacture of YE. Drops in the viable bacterial population after a 24-h autolysis were observed when pH was adjusted to 4.0 or when ethyl acetate was added. A significant interaction was found between the effects of pH and autolysis promoters on the bacterial population in YE, indicating that the activity of ethyl acetate, as opposed to that of chitosan, was not influenced by pH.     

Bioconversion of hemicelluloses into fungal protein

Summary The utilization of cellulose from one ton of lignocellulose for ethanol production would yield 150–250 kg of hemicelluloses. The total soluble solids in the hemicellulose fraction (HF) obtained with the Université de Sherbrooke (UdeS) process contained about 56% carbohydrates. These carbohydrates were present in the form of oligomers of various sugars, predominantly xylose. All the test fungi,Chaetomium cellulolyticum, C. cellulolyticum (asporogenous mutant) andPleurotus sajor-caju, were capable of utilizing all the carbohydrates present in HF.C. cellulolyticum gave the highest amount of protein (7 g/l) from 19 g carbohydrates/l. The yield of protein was higher than expected, indicating that carbon compounds other than reducing sugars present in HF might have been consumed for fungal growth. The inhibitory effect of toxic compounds on protein production increased with an increase in concentration of soluble solids in HF. The inhibitory effect was overcome by increasing the pH of the medium to 6.0 or 7.0. Fungal protein production from hemicelluloses will give extra revenue in our integrated approach for ethanol production from lignocelluloses.NRCC publication No. 26277.     

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.     

Production,purification and properties of a Pichia kluyveri killer toxin

Production of the killer toxin of Pichia kluyveri 1002 was stimulated in the presence of yeast extract. In a minimal medium production was optimal at pH 3.8–4.0 and 22–25°C. Addition of gelatin and nonionic detergents, like Brij-58 (polyoxyethylene 20 cetyl ether) and Triton-X-100, to this medium enhanced production significantly.The killer toxin was purified 140-fold by use of a stepwise ethanol precipitation and butyl Sepharose column chromatography. The purified killer toxin, which still contained some carbohydrates, appeared to be glycoprotein with a mol wt of about 19000 and an isoelectric point of 4.3. It was stable between pH 2.5 and 4.7 and up to 40°C.     

Indonesian Tapé Ketan Fermentation

Indonesian tapé ketan is a fermentation in which a mold, Amylomyces rouxii Calmette (Chlamydomucor oryzae Went and Prinsen Geerligs), in combination with one or more yeasts such as Endomycopsis burtonii converts steamed rice to a sweet-sour, slightly alcoholic paste. A study was made to determine the biochemical changes that occur in the substrate during fermentation. It was found that the product was ready for consumption after fermentation at 30°C for 36 to 48 h. A. rouxii used about 30% of the total rice solids, resulting in a crude protein of 12% in 96 h, whereas the combination of the mold with E. burtonii reduced total solids by 50% in 192 h, causing crude protein to increase to 16.5%. Soluble solids increased from 5 to about 67% in 36 h and decreased to 12% at 192 h with A. rouxii alone, whereas soluble solids fell to about 8% at 192 h in the fermentation with both the mold and the yeast. The mold, by itself, reduced the starch content of the rice from 78 to 10% in 48 h and to less than 2% in 144 h. The mold plus yeast reduced the starch content to about 18% in 48 h; however the “starch” content did not fall below 6% even at 192 h, presumably because the yeast was producing glycogen, which was determined along with the residual starch. With both the mold and the mold plus yeast fermentations, reducing sugars increased from less than 1% to approximately 5% in 24 h and reached maximum concentration, 16 to 17%, between 36 and 48 h. A. rouxii by itself produced a maximum of about 5.6% (vol/vol) ethanol at 96 h. The highest concentration of ethanol (8%, vol/vol) was produced by the mold plus E. burtonii at 144 h. The mold by itself reduced the starting pH from 6.3 to about 4.0 in 48 h. The combination of the mold and yeast reduced the pH to 4.1 in 144 h. The mold increased total acidity to approximately 6.2 meq of H⁺ per 100 ml, and the combination of the mold and yeast increased the total acidity to 7.8 meq of H⁺ per 100 ml in 192 h. At 48 h there was practically no difference in the volatile acidity (0.20) for the combined fermentation compared with 0.26 meq of H⁺ per 100 ml for the mold fermentation. The mold and at least one species of yeast were required to develop the rich aroma and flavor of typical Indonesian tapé.     

Characterization of ethanol fermentation waste and its application to lactic acid production by Lactobacillus paracasei

In this study, an ethanol fermentation waste (EFW) was characterized for use as an alternative to yeast extract for bulk fermentation processes. EFW generated from a commercial plant in which ethanol is produced from cassava/rice/wheat/barley starch mixtures using Saccharomyces cerevisiae was used for lactic acid production by Lactobacillus paracasei. The effects of temperature, pH, and duration on the autolysis of an ethanol fermentation broth (EFB) were also investigated. The distilled EFW (DEFW) contained significant amounts of soluble proteins (2.91 g/l), nitrogen (0.47 g/l), and amino acids (24.1 mg/l). The autolysis of the EFB under optimum conditions released twice as much amino acids than in the DEFW. Batch fermentation in the DEFW increased the final lactic acid concentration, overall lactic acid productivity, and lactic acid yield on glucose by 17, 41, and 14 %, respectively, in comparison with those from comparable fermentation in a lactobacillus growth medium (LGM) that contained 2 g/l yeast extract. Furthermore, the overall lactic acid productivity in the autolyzed then distilled EFW (ADEFW) was 80 and 27 % higher than in the LGM and DEFW, respectively.     

Effects of Acid on Plant Litter Decomposition in an Arctic Lake

The effects of acid on the microbial decomposition of the dominant aquatic macrophyte (Carex sp.) in Toolik Lake, Alaska were studied in microcosms during the ice-free season of 1980. Toolik Lake is slightly buffered, deep, and very oligotrophic. Microbial activities, as determined by ¹⁴C-acetate incorporation into extractable lipids, associated with Carex litter were significantly (P < 0.01) reduced within 2 days at pHs of 3.0 and 4.0, but not 5.0, 5.5, or 6.0, as compared with ambient controls (pH 7.4). ATP levels were significantly reduced at pH 3.0, but not at the other pHs tested. After 18 days, microbial activity significantly correlated with weight loss (P < 0.05), nitrogen content (P < 0.01), and C/N ratios (P < 0.01) of the litter, but did not correlate with ATP levels. Scanning electron microscopy of the litter surface revealed that the fungi present at ambient pH did not become dominant at pHs below 5.5, diatoms were absent below pH 4.0, and bacterial numbers and extracellular slime were greatly reduced at pH 4.0 and below. Mineralization of Carex¹⁴C-lignin-labeled or ¹⁴C-cellulose-labeled lignocellulose was reduced at pH 2.0, but not at pH 4.0, 5.0, or 6.0, compared with controls (pH 7). We concluded that if the pH of the water from this slightly buffered lake was sufficiently reduced, rates of litter decomposition would be significantly reduced.     

Ethanol Inhibition Kinetics of Kluyveromyces marxianus Grown on Jerusalem Artichoke Juice

The kinetics of ethanol inhibition on cell growth and ethanol production by Kluyveromyces marxianus UCD (FST) 55-82 were studied during batch growth. The liquid medium contained 10% (wt/vol) inulin-type sugars derived from an extract of Jerusalem artichoke (Helianthus tuberosus) tubers, supplemented with small amounts of Tween 80, oleic acid, and corn steep liquor. Initial ethanol concentrations ranging from 0 to 80 g/liter in the liquid medium were used to study the inhibitory effect of ethanol on the following parameters: maximum specific growth rate (μ_max), cell and ethanol yields, and sugar utilization. It was found that as the initial ethanol concentration increased from 0 to 80 g/liter, and maximum specific growth rate of K. marxianus cells decreased from 0.42 to 0.09 h⁻¹, whereas the ethanol and cell yields and sugar utilization remained almost constant. A simple kinetic model was used to correlate the μ_max results and the rates of cell and ethanol production, and the appropriate constants were evaluated.     

Promotive action of ceramics on yeast ethanol production,and its relationship to pH,glycerol and alcohol dehydrogenase activity

Summary A yeast strain, Saccharomyces cerevisiae KPY32 isolated from pito, a traditional West-African alcoholic beverage, was immobilized in porous ceramic beads as a means of improving its ethanol production. Stationary fermentation cultures at different temperatures were made using semi-synthetic medium and fermentation parameters including ethanol production, sugar consumption, cell growth and pH were monitored. Glycerol production, and the activity of alcohol dehydrogenase (ADH) of the various systems were monitored. It was found that immobilization of the yeast resulted in improved ethanol production, at conversion rates above 93% of the theoretical value. The pH of the immobilized systems was also stabilized at around 4.0, glycerol production was higher, and the ADH activities were higher than those of free-cell systems. Ethanol production at the high temperature of 37° C was also improved by immobilization. The promotive action was found to be related to the pH, presence of glycerol and the enhancement of ADH activity.Offprint requests to: B. Demuyakor