Inhibited Ethanol Production Research Articles

Mannitol and erythritol reduce the ethanol yield during Chinese Baijiu production

Chinese Baijiu is prepared using multiple microbial strains and complex metabolites by simultaneous saccharification and fermentation (SSF). Yeasts are challenged by various endogenous and exogenous factors, detrimentally affecting the ethanol yield. It is imperative to identify and control inhibitory factors. In the present study, microbial taxa and metabolites during Baijiu fermentation were evaluated to identify inhibitors of ethanol production. We found that filamentous fungi and Bacillus, contributing to saccharification, were negatively related to the ethanol content (Spearman's |ρ| > 0.5, P < 0.05). To explore how they affect ethanol production, ten filamentous fungi and three Bacillus strains were isolated. In addition to glucose and maltose, polyols were simultaneously generated by filamentous fungi and Bacillus via the hydrolysis of starch, among which mannitol and erythritol had the highest contents of up to 41.56 ± 2.01 g/kg and 16.16 ± 1.13 g/kg, respectively. The presence of mannitol and erythritol inhibited ethanol production by the functional yeasts Saccharomyces cerevisiae and Pichia kudriavzevii. The presence of 10.0 g/L mannitol significantly (P < 0.01) decreased the ethanol yield of S. cerevisiae by 12.67% (from 39.34 ± 0.02% to 32.71 ± 0.49%). These results revealed that polyols may inhibit the production of Baijiu and other fermented foods, suggesting that the origin and influence of polyols should be a focus of future research.

Homoacetogenesis and solventogenesis from H2/CO2 by granular sludge at 25, 37 and 55 °C

CO2 fermentation is a promising process to produce biofuels like ethanol. It can be integrated in third generation biofuel production processes to substitute traditional sugar fermentation when supplied with cheap electron donors, e.g. hydrogen derived from wind energy or as surplus gas in electrolysis. In this study, granular sludge from an industrial wastewater treatment plant was tested as inoculum for ethanol production from H2/CO2 via non-phototropic fermentation at submesophilic (25 °C), mesophilic (37 °C) and thermophilic (55 °C) conditions. The highest ethanol concentration (17.11 mM) was obtained at 25 °C and was 5-fold higher than at 37 °C (3.36 mM), which was attributed to the fact that the undissociated acid (non-ionized acetic acid) accumulation rate constant (0.145 h−1) was 1.39 fold higher than at 25 °C (0.104 h−1). Methane was mainly produced at 55 °C, while neither acetic acid nor ethanol were formed. Ethanol production was linked to acetic acid production with the highest ethanol to acetic acid ratio of 0.514 at 25 °C. The carbon recovery was 115.7%, 131.2% and 117.1%, while the electron balance was almost closed (97.1%, 110.1% and 109.1%) at 25 °C, 37 °C and 55 °C, respectively. The addition of bicarbonate inhibited ethanol production both at 25 °C and 37 °C. Clostridium sp. were the prevalent species at both 25 and 37 °C at the end of the incubation, which possibly contributed to the ethanol production.

Biofilm formation and ethanol inhibition by bacterial contaminants of biofuel fermentation

Bacterial contaminants can inhibit ethanol production in biofuel fermentations, and even result in stuck fermentations. Contaminants may persist in production facilities by forming recalcitrant biofilms. A two-year longitudinal study was conducted of bacterial contaminants from a Midwestern dry grind corn fuel ethanol facility. Among eight sites sampled in the facility, the combined liquefaction stream and yeast propagation tank were consistently contaminated, leading to contamination of early fermentation tanks. Among 768 contaminants isolated, 92% were identified as Lactobacillus sp., with the most abundant species being Lactobacillus plantarum, Lactobacillus casei, Lactobacillus mucosae, and Lactobacillus fermentum. Seven percent of total isolates showed the ability to form biofilms in pure cultures, and 22% showed the capacity to significantly inhibit ethanol production. However, these traits were not correlated. Ethanol inhibition appeared to be related to acetic acid production by contaminants, particularly by obligately heterofermentative species such as L. fermentum and L. mucosae.

Protease increases fermentation rate and ethanol yield in dry-grind ethanol production

The effects of acid protease and urea addition during the fermentation step were evaluated. The fermentations were also tested with and without the addition of urea to determine if protease altered the nitrogen requirements of the yeast. Results show that the addition of the protease had a statistically significant effect on the fermentation rate and yield. Fermentation rates and yields were improved with the addition of the protease over the corresponding controls without protease. Protease addition either with or with added urea resulted in a higher final ethanol yield than without the protease addition. Urea addition levels >1200 ppm of supplemental nitrogen inhibited ethanol production. The economic effects of the protease addition were evaluated by using process engineering and economic models developed at the Eastern Regional Research Center. The decrease in overall processing costs from protease addition was as high as $0.01/L (4 ¢/gal) of denatured ethanol produced.

Fermentation of xylose into ethanol by a new fungus strain Pestalotiopsis sp. XE-1

A new fungus, Pestalotiopsis sp. XE-1, which produced ethanol from xylose with yield of 0.47 g ethanol/g of consumed xylose was isolated. It also produced ethanol from arabinose, glucose, fructose, mannose, galactose, cellobiose, maltose, and sucrose with yields of 0.38, 0.47, 0.45, 0.46, 0.31, 0.25, 0.31, and 0.34 g ethanol/g of sugar consumed, respectively. It produced maximum ethanol from xylose at pH 6.5, 30°C under a semi-aerobic condition. Acetic acid produced in xylose fermenting process inhibited ethanol production of XE-1. The ethanol yield in the pH-uncontrolled batch fermentation was about 27% lower than that in the pH-controlled one. The ethanol tolerance of XE-1 was higher than most xylose-fermenting, ethanol-producing microbes, but lower than Saccharomyces cerevisiae and Hansenula polymorpha. XE-1 showed tolerance to high concentration of xylose, and was able to grow and produce ethanol even when it was cultivated in 97.71 g/l xylose.

Effect of orange peel oil on ethanol production by Zymomonas mobilis

Abstract The effect of orange peel oil on ethanol production by the ethanologenic bacterium Zymomonas mobilis was investigated. Orange peel oil was added in various amounts to determine its effects on ethanol production. Fermentation of model sugar solutions was conducted at 30 and 37 °C. The minimum orange peel oil concentration that inhibited ethanol production by Z. mobilis was determined after 24, 48, 72 and 96 h for both temperatures. Minimum inhibitory orange peel oil concentrations for ethanol production at 30 °C were 0.05% after 24 h, 0.10% after 48 h, 0.15% after 72 h, and 0.20% after 96 h. Minimum inhibitory orange peel oil concentrations for ethanol production at 37 °C were 0.05% after 24 h, 0.10% after 48 h, and 0.20% after 72 h. Orange peel oil did not inhibit ethanol production after 96 h at a temperature of 37 °C.

Biological detoxification of waste house wood hydrolysate using Ureibacillus thermosphaericus for bioethanol production

Hydrolysates of lignocelluloses hydrolyzed by diluted sulfuric acid contain toxic compounds that inhibit ethanol production by Saccharomyces cerevisiae and the ethanologenic recombinant Escherichia coli KO11. We investigated the biological detoxification of a hydrolysate of waste house wood (WHW) by a thermophilic bacterium, Ureibacillus thermosphaericus. When the hydrolysate was treated with this bacterium at 50 degrees C for 24 h, the ethanol production rate by S. cerevisiae increased markedly and was comparable to that for the hydrolysate treated with an excess amount of calcium hydroxide (overliming). Chromatographic analysis of synthetic hydrolysates containing furfural or 5-hydroxymethyl furfural that are considered to be major toxic compounds in hydrolysates revealed that U. thermosphaericus degrades these compounds. In the WHW hydrolysates, however, the concentrations of these compounds were not decreased markedly by the bacterium. These results suggest that the bacterium degrades minor but more toxic compounds or phenolic compounds in the WHW hydrolysates. The combination of bacterial and overliming treatments of hydrolysates minimized significantly the decrease in ethanol production rate by E. coli KO11 as fermentation proceeded. Because the bacterium grows rapidly and does not consume sugars, our biological detoxification should be useful for bioethanol production from acid hydrolysates of lignocelluloses.

Ethanol production by Saccharomyces cerevisiae and Kluyveromyces marxianus in the presence of orange-peel oil

Two ethanologenic yeasts, Saccharomyces cerevisiae and Kluyveromyces marxianus, were used to ferment sugar solutions modeling hydrolyzed Valencia orange peel waste at 37°C. Orange stripper oil produced from orange peel was added in various amounts to determine its effect on ethanol production. The minimum peel oil concentration that inhibited ethanol production was determined after 24, 48 and 72 h and the two yeasts were compared to one another in terms of ethanol yield. Minimum inhibitory peel oil concentrations for ethanol production were 0.05% at 24 h, 0.10% at 48 h, and 0.15% at 72 h for both yeasts. S. cerevisiae produced more ethanol than K. marxianus at each time point.

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 < or =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 > or =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 alcohol compounds found in hemicellulose hydrolysate on the growth and fermentation of ethanologenic Escherichia coli.

Lignocellulose can be readily hydrolyzed into a mixture of sugars using dilute mineral acids. During hydrolysis, a variety of inhibitors are also produced which include aromatic alcohols from lignin and furfuryl alcohol from pentose destruction. Seven compounds were investigated individually and in binary combinations (catechol, coniferyl alcohol, furfuryl alcohol, guaiacol, hydroquinone, methylcatechol, and vanillyl alcohol). Aromatic alcohols and furfuryl alcohol inhibited ethanol production from xylose in batch fermentations primarily by inhibiting the growth of Escherichia coli LY01, the biocatalyst. The toxicities of these compounds were directly related to their hydrophobicity. Methylcatechol was the most toxic compound tested (MIC = 1.5 g/L). In binary combination, the extent of growth inhibition was roughly additive for most compounds tested. However, combinations with furfuryl alcohol and furfural (furaldehyde) appear synergistic in toxicity. When compared individually, alcohol components which are formed during hemicellulose hydrolysis are less toxic for growth than the aldehydes and organic acids either on a weight basis or a molar basis.

Effect of selected aldehydes on the growth and fermentation of ethanologenic Escherichia coli.

Bioethanol production from lignocellulosic raw-materials requires the hydrolysis of carbohydrate polymers into a fermentable syrup. During the hydrolysis of hemicellulose with dilute acid, a variety of toxic compounds are produced such as soluble aromatic aldehydes from lignin and furfural from pentose destruction. In this study, we have investigated the toxicity of representative aldehydes (furfural, 5-hydroxymethlyfurfural, 4-hydroxybenzaldehyde, syringaldehyde, and vanillin) as inhibitors of growth and ethanol production by ethanologenic derivatives of Escherichia coli B (strains KO11 and LY01). Aromatic aldehydes were at least twice as toxic as furfural or 5-hydroxymethylfurfural on a weight basis. The toxicities of all aldehydes (and ethanol) except furfural were additive when tested in binary combinations. In all cases, combinations with furfural were unexpectedly toxic. Although the potency of these aldehydes was directly related to hydrophobicity indicating a hydrophobic site of action, none caused sufficient membrane damage to allow the leakage of intracellular magnesium even when present at sixfold the concentrations required for growth inhibition. Of the aldehydes tested, only furfural strongly inhibited ethanol production in vitro. A comparison with published results for other microorganisms indicates that LY01 is equivalent or more resistant than other biocatalysts to the aldehydes examined in this study.

The use of monochloroacetic acid for improved ethanol production by immobilized Saccharomyces cerevisiae

Saccharomyces cerevisiae adsorbed on acid-treated glass beads produced 5.4 g I -l ethanol at 96 h in batch process. Precoating the acid-treated glass beads with gelatin (25 g I -l ) before immobilization of the cells increased ethanol production to 26.3 g I -1 at 72 h. Cell leakage into the medium was decreased when the immobilized cells were crosslinked with increasing concentrations of glutaraldehyde (0-100 g I -1 ). Although monochloroacetic acid inhibited ethanol production and cell multiplication at concentrations greater than 0.1 g I -1 , at a concentration of 0.01 g I -1 it not only increased the ethanol production to 52.1 g I -1 but also shortened the ethanol production time to 48 h. In a semi-continuous batch process with the feed containing 0.01 g I -1 monochloroacetic acid, immobilized cells showed no significant change in ethanol-producing ability for 40 days when incubated with nutrient medium intermittently.

Influence of the oxygenation level on d-xylose fermentation by free and agar-entrapped cultures of Candida shehatae

This paper investigates the effects of the oxygenation level on the performance of d-xylose alcoholic fermentation by free- and immobilized-cell batch cultures of Candida shehatae (ATCC 22984). Yeast cells were immobilized in composite agar layer/microporous membrane structures. Fermentations were performed under varying oxygenation levels corresponding to different O2 flow rates (OFRs). Low OFRs enhanced the fermentation performance of free and immobilized yeasts. The best ethanol yield coefficient, obtained at an OFR of 5 mmol O2 h−1 dm−3 for both culture modes, was slightly higher (0.425 g g−1) for immobilized cultures than for their free counterparts (0.39 g g−1). More sustained aeration inhibited ethanol production by free and immobilized organisms. However, this inhibition was more pronounced for agar-entrapped cultures. Xylitol production of free cultures normally decreased as the OFR increased. At high OFR, however, immobilized organisms surprisingly produced more xylitol than at lower OFR or in anaerobiosis. This effect is discussed by referring to the mass transfer limitations that occur inside the immobilized-cell structures. Gel-entrapped cultures displayed higher specific and volumetric production rates of ethanol and xylitol than free-cell cultures.

The effects of oxygen on fermentation in Giardia lamblia.

Detailed study of the effects of oxygen on the carbohydrate metabolism of Giardia lamblia revealed that low concentrations of oxygen (< 0.25 microM) produced profound alterations in the carbon balance of this organism. Although this concentration of oxygen could not be detected by mass spectrometry, a marked stimulation of ethanol production was observed. Associated with this was an inhibition of alanine production and oxidation of the intracellular NAD(P)H pool. Higher concentrations of oxygen inhibited ethanol production and further reduced levels of alanine. These results suggest that this stimulation is due to changes in carbon flux. Analysis of cell and medium hydrolysates after the growth of trophozoites in [U-14C]glucose suggests that G. lamblia does not synthesise detectable levels of labelled amino acids, except alanine and to a lesser extent valine, from this sugar. Trophozoites of G. lamblia have both glutamate dehydrogenase and alanine aminotransferase activity. As glutamate is taken up from the medium, it is suggested that glutamate dehydrogenase and alanine aminotransferase cooperate to convert pyruvate to alanine, with the concomitant oxidation of NAD(P)H.

Effects of T‐2 toxin on ethanol production by Saccharomyces cerevisiae

A trichothecene mycotoxin, T-2 toxin, inhibits several aspects of cellular physiology in Saccharomyces cerevisiae, including protein synthesis and mitochondrial functions. We have studied growth of, glucose utilization by, and ethanol production by S. cerevisiae and show that they are inhibited by T-2 toxin between 20 and 200 micrograms/ml in a dose-dependent manner. At 200 micrograms/ml, T-2 toxin causes cell death. This apparent inhibition of ethanol production was found to be the result of growth inhibition. On the basis of biomass or glucose consumption, T-2 toxin increased the amount of ethanol present in the culture. This suggests that T-2 inhibits oxidative but not fermentative energy metabolism by inhibiting mitochondrial function and shifting glucose catabolism toward ethanol formation. As T-2 toxin does not directly inhibit ethanol production by S. cerevisiae, this system could be used for ethanol production from trichothecene-contaminated grain products.