Ethanol fermentation coupled with complete cell recycle pervaporation system: Dependence of glucose concentration

Summary A membrane bioreactor system comprised of a fermenter and a flat pervaporation module was developed for continuous ethanol fermentation by Saccharomyces cerevisiae. In order to obtain the guidelines for high sugar concentration fermentation, the dependence of glucose concentration on the coupled system was investigated. Fed by 158 and 290g glucose/l, the improvement in productivity was obtained with 1. 58 and 1. 86 times, and the ethanol yield was 0. 45 and 0. 395, respectively. With the fermentation proceeding, the permeate flux decreased but the selectivity kept unaltered.     

Control of ethanol production and monitoring of membrane performance by mass-spectrometric gas analysis in the coupled fermentation-pervaporation of whey permeate

A coupled fermentation-pervaporation process was operated continuously with on-line mass spectrometric gas analysis monitoring of product accumulation on both the upstream and the downstream sides of the membrane. Efficient coupling of the fermentation with pervaporation was attained when a steady state of ethanol production and removal was achieved with whey permeate containing high concentrations of lactose (>8%) or by controlled lactose additions that also compensated for loss of liquid due to pervaporation. The combined system consists of a tubular membrane pervaporation module, directly connected to a stirred fermentor to form one circulation loop, kept at 38°C, with both units operating under computer control. Mass spectrometric gas analysis of the CO₂ gas evolved in the fermentor and the ethanol and water in the pervaporate on the downstream side of the membrane enabled us to follow the production of ethanol and its simultaneous removal. Membrane selectivity was calculated on-line and served to monitor the functioning of the membrane. Batch-wise-operated fermentation-pervaporation with Candida pseudotropicalis IP-513 yielded over 120 gl^–1 of concentrated ethanol solution using supplemented whey permeate containing 16% lactose. A steady state lasting for about 20 h was achieved with ethanol productivity of 20 g h^–1 (approx. 4 g l^–1 h^–1). Membrane selectivity was over 8. Controlled feeding of concentrated lactose suspension in the whey permeate (350 g l^–1) resulted in the continuous collection of 120–140 g l^–1 of ethanol pervaporate for 5 days, by which time salt accumulation hampered the fermentation. Medium refreshment restored the fermentative activity of the yeast cells and further extended the coupled process to over 9 days (200 h), when reversible membrane fouling occurred. The membrane module was exchanged and the combined process restarted. Correspondence to: Y. Shabtai     

Acetic acid production from lactose by an anaerobic thermophilic coculture immobilized in a fibrous-bed bioreactor

An anaerobic thermophilic coculture consisting of a heterofermentative bacterium (Clostridium thermolacticum) and a homoacetogen (Moorella thermoautotrophica) was developed for acetic acid production from lactose and milk permeate. The fermentation kinetics with free cells in conventional fermentors and immobilized cells in a recycle batch fibrous-bed bioreactor were studied. The optimal conditions for the cocultured fermentation were found to be 58 degrees C and pH 6.4. In the free-cell fermentation, C. thermolacticum converted lactose to acetate, ethanol, lactate, H(2) and CO(2), and the homoacetogen then converted lactate, H(2), and CO(2) to acetate. The overall acetate yield from lactose ranged from 0.46 to 0.65 g/g lactose fermented, depending on the fermentation conditions. In contrast, no ethanol was produced in the immobilized-cell fermentation, and the overall acetate yield from lactose increased to 0.8-0.96 g/g lactose fermented. The fibrous-bed bioreactor also gave a higher final acetate concentration (up to 25. 5 g/L) and reactor productivity (0.18-0.54 g/L/h) as compared to those from the free-cell fermentation (final acetate concentration, 15 g/L; productivity, 0.06-0.08 g/L/h). The superior performance of the fibrous-bed bioreactor was attributed to the high cell density (20 g/L) immobilized in the fibrous-bed and adaptation of C. thermolacticum cells to tolerate a higher acetate concentration. The effects of yeast extract and trypticase as nutrient supplements on the fermentation were also studied. For the free-cell fermentation, nutrient supplementation was necessary for the bacteria to grow in milk permeate. For the immobilized-cell fermentation, plain milk permeate gave a high acetate yield (0.96 g/g), although the reactor productivity was lower than those with nutrient supplementation. Balanced growth and fermentation activities between the two bacteria in the coculture are important to the quantitative conversion of lactose to acetic acid. Lactate and hydrogen produced by C. thermolacticum must be timely converted to acetic acid by the homoacetogen to avoid inhibition by these metabolites.     

Consolidated bioprocessing strategy for ethanol production from Jerusalem artichoke tubers by Kluyveromyces marxianus under high gravity conditions

Aims: Developing an innovative process for ethanol fermentation from Jerusalem artichoke tubers under very high gravity (VHG) conditions. Methods and Results: A consolidated bioprocessing (CBP) strategy that integrated inulinase production, saccharification of inulin contained in Jerusalem artichoke tubers and ethanol production from sugars released from inulin by the enzyme was developed with the inulinase‐producing yeast Kluyveromyces marxianus Y179 and fed‐batch operation. The impact of inoculum age, aeration, the supplementation of pectinase and nutrients on the ethanol fermentation performance of the CBP system was studied. Although inulinase activities increased with the extension of the seed incubation time, its contribution to ethanol production was negligible because vigorously growing yeast cells harvested earlier carried out ethanol fermentation more efficiently. Thus, the overnight incubation that has been practised in ethanol production from starch‐based feedstocks is recommended. Aeration facilitated the fermentation process, but compromised ethanol yield because of the negative Crabtree effect of the species, and increases the risk of contamination under industrial conditions. Therefore, nonaeration conditions are preferred for the CBP system. Pectinase supplementation reduced viscosity of the fermentation broth and improved ethanol production performance, particularly under high gravity conditions, but the enzyme cost should be carefully balanced. Medium optimization was performed, and ethanol concentration as high as 94·2 g l^?1 was achieved when 0·15 g l^?1 K₂HPO₄ was supplemented, which presents a significant progress in ethanol production from Jerusalem artichoke tubers. Conclusions: A CBP system using K. marxianus is suitable for efficient ethanol production from Jerusalem artichoke tubers under VHG conditions. Significance and Impact of the Study: Jerusalem artichoke tubers are an alternative to grain‐based feedstocks for ethanol production. The high ethanol concentration achieved using K. marxianus with the CBP system not only saves energy consumption for ethanol distillation, but also significantly reduces the amount of waste distillage discharged from the distillation system.     

Consolidated bioprocessing (CBP) of AFEX™-pretreated corn stover for ethanol production using Clostridium phytofermentans at a high solids loading

Consolidated bioprocessing (CBP) using Clostridium phytofermentans (ATCC 700394) on ammonia fiber expansion (AFEX?)‐treated corn stover (AFEX?‐CS) at a low solids loading showed promising results [Jin et al. (2011) Biotechnol Bioeng 108(6): 1290–1297]. However, industrial relevant process requires high solids loading. Therefore, we studied high solids loading CBP performance on AFEX?‐CS. The factors potentially affecting the performance including solids loading, CBP products acetate and ethanol, and degradation products resulting from pretreatment were investigated. At 4% (w/w) glucan loading, C. phytofermentans performed well on AFEX?‐CS with no nutrients supplementation and reached similar sugar conversions as a fermentation with nutrients supplementation. A glucan conversion of 48.9% and a xylan conversion of 77.9% were achieved after 264 h with 7.0 g/L ethanol and 8.8 g/L acetate produced. Relatively high concentrations of acetate produced at high solids loading was found to be the major factor limiting the CBP performance. Degradation products in AFEX?‐CS helped enhance ethanol production. Biotechnol. Bioeng. 2012; 109:1929–1936. © 2012 Wiley Periodicals, Inc.     

An investigation of ethanol inhibition and other limitations occurring during the fermentation of concentrated whey permeate byKluyveromyces fragilis

Based on the well-known fact thatKluyveromyces fragilis strains show sub-optimal performance when grown in concentrated whey permeate, previously optimized medium was investigated for possible limitations appearing at high concentrations. Shaken flask cultures showed that no additional vitamin or mineral sources were required when the optimized amount of yeast extract was added to the concentrated permeate. Several aspects of the ethanol inhibition of the growth ofK. fragilis NRRL 665 were investigated in continuous culture. The maximum ethanol concentration tolerated by this yeast, i.e. 45 g/l, was much lower than commonly reported for other strains. Ethanol and biomass production were also influenced by the increased ethanol concentration of the medium. At 31 g/l of alcohol product yield was reduced to 0.23 g/g whereas biomass yield was 0.05 g/g. Some evidence suggested that residence time and residual lactose concentration played a significant role in modulating the toxic effect of ethanol.     

Performance of a continuous bioreactor with immobilized yeast cells in the ethanol fermentation of molasses-stillage medium

Summary It is feasible to produce ethanol by continuous fermentation of molasses-stillage medium, without any supplementation, employing calcium alginate immobilized cells of Saccharomyces cerevisiae. High flow rates generated high values for the productivity; however, the percent substrate conversion decreases with the dilution rate.     

Ethanol production from biodiesel-derived crude glycerol by newly isolated Kluyvera cryocrescens

The rapidly expanding market for biodiesel has increased the supply and reduced the cost of glycerol, making it an attractive sustainable feed stock for the fuel and chemical industry. Glycerol-based biorefinery is the microbial fermentation of crude glycerol to produce fuels and chemicals. A major challenge is to obtain microbes tolerant to inhibitors such as salts and organic solvents present in crude glycerol. Microbial screening was attempted to isolate novel strain capable of growing on crude glycerol as a sole carbon source. The newly isolated bacteria, identified as nonpathogenic Kluyvera cryocrescens S26 could convert biodiesel-derived crude glycerol to ethanol with high yield and productivity. The supplementation of nutrients such as yeast extract resulted in distinguished enhancement in cell growth as well as ethanol productivity under anaerobic condition. When glycerol fermentation is performed under microaerobic condition, there is also a remarkable improvement in cell growth, ethanol productivity and yield, compared with those under strict anaerobic condition. In batch fermentation under microaerobic condition, K. cryocrescens S26 produced 27 g/l of ethanol from crude glycerol with high molar yield of 80% and productivity of 0.61 g/l/h.     

Divergent Effects of Chaperone Overexpression and Ethanol Supplementation on Inclusion Body Formation in RecombinantEscherichia coli

The proper folding of aggregation-prone recombinant proteins inEscherichia colican be facilitated by co-overexpressing specific molecular chaperones or by culturing the cells in the presence of ethanol or other agents that upregulate the synthesis of all heat-shock proteins (hsps). We have investigated the effect of combining direct chaperone overproduction with ethanol supplementation on the cytoplasmic folding of two aggregation-prone model proteins, preS2-S′-β-galactosidase and human SPARC. In 25-ml shake flask cultures grown at 30°C, addition of 3% (v/v) ethanol to the growth medium prior to inoculation improved the chaperone-mediated increase in the yields of active preS2-S′-β-galactosidase 1.5- to 2-fold. When cultures overexpressing thednaKJoperon were grown in the presence of ethanol, the levels of enzymatic activity were 5-fold higher relative to control cells and preS2-S′-β-galactosidase aggregation was almost entirely abolished. Combining DnaK–DnaJ overexpression and growth of the cells at temperatures lower than 30°C did not result in a comparable increase in activity. Although the individual effects of ethanol supplementation anddnaKJoverproduction were more limited when the culture volume was raised, a synergistic improvement in preS2-S′-β-galactosidase activity was observed when the two approaches were used in concert. In contrast, ethanol supplementation promoted the aggregation of human SPARC, a protein exhibiting a chaperone dependency similar to that of preS2-S′-β-galactosidase. Our results show that ethanol can exert complex and divergent effects on inclusion body formation and that the beneficial effect of the solvent on recombinant protein folding cannot simply be explained by an increase in the intracellular concentration of molecular chaperones.     

Uncoupling glucose sensing from GAL metabolism for heterologous lactose fermentation in Saccharomyces cerevisiae

Objectives

Development of a system for direct lactose to ethanol fermentation provides a market for the massive amounts of underutilized whey permeate made by the dairy industry. For this system, glucose and galactose metabolism were uncoupled in Saccharomyces cerevisiae by deleting two negative regulatory genes, GAL80 and MIG1, and introducing the essential lactose hydrolase LAC4 and lactose transporter LAC12, from the native but inefficient lactose fermenting yeast Kluyveromyces marxianus.

Results

Previously, integration of the LAC4 and LAC12 genes into the MIG1 and NTH1 loci was achieved to construct strain AY-51024M. Low rates of lactose conversion led us to generate the Δmig1Δgal80 diploid mutant strain AY-GM from AY-5, which exhibited loss of diauxic growth and glucose repression, subsequently taking up galactose for consumption at a significantly higher rate and yielding higher ethanol concentrations than strain AY-51024M. Similarly, in cheese whey permeate powder solution (CWPS) during three, repeated, batch processes in a 5L bioreactor containing either 100 g/L or 150 g/L lactose, the lactose uptake and ethanol productivity rates were both significantly greater than that of AY-51024M, while the overall fermentation times were considerably lower.

Conclusions

Using the Cre-loxp system for deletion of the MIG1 and GAL80 genes to relieve glucose repression, and LAC4 and LAC12 overexpression to increase lactose uptake and conversion provides an efficient basis for yeast fermentation of whey permeate by-product into ethanol.

     

Rapid production of high concentrations of ethanol bySaccharomyces bayanus: Mechanisms of action of soy flour supplementation

Summary The supplementation of a simple medium with 2% soy flour increased the final ethanol concentration and the rate of fermentation bySaccharomyces bayanus. This improvement could not be attributed to an increase of ethanol tolerance of yeast cells but to the satisfaction of nutritional deficiencies.     

Utilization of dry distiller’s grain and solubles as nutrient supplement in the simultaneous saccharification and ethanol fermentation at high solids loading of corn stover

Dry distiller’s grain and solubles (DDGS) is a major by-product of corn-based ethanol production and is usually used as animal feed. Here, it was added to the simultaneous saccharification and ethanol fermentation (SSF) carried out at high solids loading of steam explosion pretreated corn stover using a mutant strain Saccharomyces cerevisiae DQ1. The performance of SSF process with DDGS was comparable to those using the expensive yeast extract supplementation. With 30% (w/w) solids plus the addition of cellulase and 1 g DDGS l⁻¹, the final ethanol reached 55 g l⁻¹ (7% v/v). The results indicated that the expensive supplement of yeast extract could be replaced by DDGS.     

Mitogenic signaling mediated by oxidants in retinol treated sertoli cells

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol-induced oxidative stress is accompanied by cellular proliferation. Retinol (7 μM) significantly induced thiobarbituric acid reactive species (TBARS) formation, which was inhibited by trolox, superoxide dismutase, N-acetylcysteine and ethanol. This was accompanied by an increase in DNA synthesis and focus formation in cultured rat Sertoli cells. Antioxidants and ethanol inhibited retinol-induced DNA synthesis. Our findings suggest that retinol-induced oxidative stress was associated with cellular proliferation complementing our understanding of the significance of retinol supplementation in neoplastic transformation.     

Comparison of ethanol production by Zymomonas mobilis from sugar beet substrates

Summary The potential of four sugar beet substrates from the sugar industry [syrup (S), crystallizer effluent 1 (CE1), crystallizer effluent 2 (CE2) and molasses (M)] were compared for ethanol production using an osmotolerant mutant strain of the bacterium Zymomonas mobilis. Sucrose of the substrates was enzymatically hydrolysed to avoid levan formation during fermentation. Nutrient supplementation experiments have shown that reproducible growth and ethanol production could be obtained on the four substrates supplemented only with magnesium sulphate (CE2 and M) or additionally with ammonium sulphate (S and CE1). Thus, addition of costly yeast extract could be avoided. All 20% (w/v) substrates showed nearly complete sugar conversion (>94.9%), good growth (0.16 h^–1) and ethanol production (>40 g 1^–1). However, sorbitol formation reduced the ethanol yield (73–79% of the theoretical value) significantly. Batch kinetic parameters and studies of instantaneous parameters showed that enhanced osmolality of substrates (SZ. mobilis with appropriate supplementation. Offprint requests to: J. Baratti     

Fermentation of Agave tequilana juice by Kloeckera africana: influence of amino-acid supplementations

This study aimed to improve the fermentation efficiency of Kloeckera africana K1, in tequila fermentations. We investigated organic and inorganic nitrogen source requirements in continuous K. africana fermentations fed with Agave tequilana juice. The addition of a mixture of 20 amino-acids greatly improved the fermentation efficiency of this yeast, increasing the consumption of reducing sugars and production of ethanol, compared with fermentations supplemented with ammonium sulfate. The preference of K. africana for each of the 20 amino-acids was further determined in batch fermentations and we found that asparagine supplementation increased K. africana biomass production, reducing sugar consumption and ethanol production (by 30, 36.7 and 45%, respectively) over fermentations supplemented with ammonium sulfate. Therefore, asparagine appears to overcome K. africana nutritional limitation in Agave juice. Surprisingly, K. africana produced a high concentration of ethanol. This contrasts to poor ethanol productivities reported for other non-Saccharomyces yeasts indicating a relatively high ethanol tolerance for the K. africana K1 strain. Kloeckera spp. strains are known to synthesize a wide variety of volatile compounds and we have shown that amino-acid supplements influenced the synthesis by K. africana of important metabolites involved in the bouquet of tequila. The findings of this study have revealed important nutritional limitations of non-Saccharomyces yeasts fermenting Agave tequilana juice, and have highlighted the potential of K. africana in tequila production processes.     

Continuous propionate production from whey permeate using a novel fibrous bed bioreactor

Continuous production of propionate from whey lactose by Propionibacterium acidipropionici immobilized in a novel fibrous bed bioreactor was studied. In conventional batch propionic acid fermentation, whey permeate without nutrient supplementation was unable to support cell growth and failed to give satisfactory fermentation results for over 7 days. However, with the fibrous bed bioreactor, a high fermentation rate and high conversion were obtained with plain whey permeate and de-lactose whey permeate. About 2% (wt/vol) propionic acid was obtained from a 4.2% lactose feed at a retention time of 35 to 45 h. The propionic acid yield was approximately 46% (wt/vol) from lactose. The optimal pH for fementation was 6.5, and lower fermentation rates and yields were obtained at lower pH values. The optimal temperature was 30 degrees C, but the temperature effect was not dramatic in the range of 25 to 35 degrees C. Addition of yeast extract and trypticase to whey permeate hastened reactor startup and increased the fermentation rate and product yields, but the addition was not required for long-term reactor performance. The improved fermentation results with the immobilized cell bioreactor can be attributed to the high cell density, approximately 50 g/L, attained in the bioreactor, Cells were immobilized by loose attachement to fiber surfaces and entrapment in the void spaces within the fibrous matrix, thus allowing constant renewal of cells. Consequently, this bioreactor was able to operate continuously for 6 months without encountering any clogging, degeneration, or contamination problems. Compared to conventional batch fermentors, the new bioreactor offers many advantages for industrial fermentation, including a more than 10-fold increase in productivity, acceptance of low-nutrient feedstocks such as whey permeate, and resistance to contamination. (c) 1994 John Wiley & Sons, Inc.     

Combined effects of temperature and medium composition on exopolysaccharide production by Lactobacillus rhamnosus RW-9595M in a whey permeate based medium

The effects of temperature (22-42 degrees C), whey permeate concentration (WP, 1.6-8.4%), and supplementation level with yeast nitrogen base (YNB, 0-2.0%) on exopolysaccharide (EPS) production was studied during 20 pH-controlled (pH = 6.0) batch cultures with Lactobacillus rhamnosus RW-9595M, using a central composite design (CCD). The EPS production was measured using both the conventional method based on ethanol precipitation of EPS and a new ultrafiltration (UF) method. EPS production was not growth-associated for high temperatures (32-42 degrees C) and WP concentrations (7.0-8.4%). In contrast, at suboptimal temperature (22-26 degrees C), EPS production was growth-associated. Maximal EPS production measured with the UF method was approximately 2-fold higher than those measured with the conventional method and varied from 125 to 477 mg/L. This parameter was significantly influenced by WP and YNBWP interaction, whereas ANOVA for maximal EPS production measured by the conventional method did not show significant factor effects. EPS volumetric productivities varied from 3.0 to 16.4 mg EPS/L small middle doth. YNB supplementation did not promote cell growth but did increase EPS production at high WP concentrations. Our data indicate the potential of L. rhamnosus RW-9595M for producing EPS in a supplemented WP medium and suggest that this production could be further increased by the addition of a growth-limiting nutrient in the medium.     

The activity of beta-galactosidase and lactose metabolism in Kluyveromyces lactis cultured in cheese whey as a function of growth rate

Aims: Kluyveromyces lactis was cultured in cheese whey permeate on both batch and continuous mode to investigate the effect of time course and growth rate on β‐galactosidase activity, lactose consumption, ethanol production and protein profiles of the cells. Methods and Results: Cheese whey was the substrate to grow K. lactis as a batch or continuous culture. In order to precise the specific growth rate for maximum β‐galactosidase activity a continuous culture was performed at five dilution (growth) rates ranging from 0·06, 0·09, 0·12, 0·18 to 0·24 h^?1. The kinetics of lactose consumption and ethanol production were also evaluated. On both batch and continuous culture a respirofermentative metabolism was detected. The growth stage for maximum β‐gal activity was found to be at the transition between late exponential and entrance of stationary growth phase of batch cultures. Fractionating that transition stage in several growth rates at continuous culture a maximum β‐galactosidase activity at 0·24 h^?1 was observed. Following that stage β‐gal activity undergoes a decline which does not correlate to the density of its corresponding protein band on the gel prepared from the same samples. Conclusion: The maximum β‐galactosidase activity per unit of cell mass was found to be 341·18 mmol ONP min^?1 g^?1 at a dilution rate of 0·24 h^?1. Significance and Impact of the Study: The physiology of K. lactis growing in cheese whey permeate can proven useful to optimize the conversion of that substrate in biomass rich in β‐gal or in ethanol fuel. In addition to increasing the native enzyme the conditions established here can be set to increase yields of recombinant protein production based on the LAC4 promoter in K. lactis host.     

Effects of by-products of fermentation of banana pseudostem on ethanol separation by pervaporation

In this work, we performed recovery of ethanol from a fermentation broth of banana pseudostem by pervaporation (PV) as a lower-energy-cost alternative to traditional separation processes such as distillation. As real fermentation systems generally contain by-products, it was investigated the effects of different components from the fermentation broth of banana pseudostem on PV performance for ethanol recovery through commercial flat sheet polydimethylsiloxane (PDMS) membrane. The experiments were compared to a binary solution (ethanol/water) to determine differences in the results due to the presence of fermentation by-products. A real fermented broth of banana pseudostem was also used as feed for the PV experiments. Seven by-products from fermented broth were identified: propanol, isobutanol, methanol, isoamyl alcohol, 1-pentanol, acetic acid, and succinic acid. Moreover, the residual sugar content of 3.02 g/L¹ was obtained. The presence of methanol showed the best results for total permeate flux (0.1626 kg·m⁻²·h⁻¹) and ethanol permeate flux (0.0391 kg·m⁻²·h⁻¹) during PV at 25°C and 3 wt% ethanol, also demonstrated by the selectivity and enrichment factor. The lowest total fluxes of permeate were observed in the experiments containing the acids. Better permeance of 0.1171 from 0.0796 kg·m⁻²·h⁻¹ and membrane selectivity of 9.77 from 9.30 were obtained with real fermentation broth than with synthetic solutions, possibly due to the presence of by-products in the multicomponent mixtures, which contributed to ethanol permeation. The results of this work indicate that by-products influence pervaporation of ethanol with hydrophobic flat sheet membrane produced from the fermented broth of banana pseudostem.     

Ascorbic acid supplementation causes faster restoration of reduced glutathione content in the regression of alcohol-induced hepatotoxicity in male guinea pigs

Abstract

Alcoholic liver disease is caused mainly by free radicals. Ascorbic acid (AA) and glutathione (GSH) are the major water-soluble antioxidants in the liver. The impact of AA supplementation on GSH, AA and activities of GSH-dependent enzymes in alcoholic guinea pigs was studied and was compared with alcohol abstention. Guinea pigs were administered ethanol at a dose of 4 g/kg body weight (b.wt)/day for 90 days. After 90 days, alcohol administration was stopped and one-half of the ethanol-treated animals were supplemented with AA (25 mg/100 g b.wt) for 30 days and the other half was maintained as the abstention group. There was a significant increase in the activities of alanine aminotransferase, aspartate aminotransferase, and gamma-glutamyl transpeptidase in the serum of the ethanol group. In addition, a significant decrease in the GSH content, activities of GSH peroxidase, GSH reductase, and increased activity of GSH-S-transferase were observed in the liver of the ethanol group. Histopathological analysis and triglycerides content in the liver of the ethanol group showed induction of steatosis. But AA supplementation and abstention altered the changes caused by ethanol. However, maximum protective effect was observed in the AA-supplemented group indicating the ameliorative effect of AA in the liver.