Enhancement Of Ethanol Production Research Articles

The enhancement of ethanol production from NaOH-catalyzed organosolv pretreated poplar with compound additives

In this study, different additives (such as Bovine serum albumin (BSA), PEG 8000, calcium lignosulfonate, tea saponin, and Tween 80) were introduced to enhance the efficiency of simultaneous saccharification and fermentation (SSF) of NaOH-catalyzed organosolv pretreated poplar. Then the influence of two-factor additive compound on SSF was determined, only 75 mg/g BSA had a positive interaction with 150 mg/g tea saponin, which generated the maximal ethanol yield of 92.83 % with ethanol concentration of 18.82 g/L after 24 h, ascribing to the higher relative enzyme activity, free enzyme, and Zeta potential. Furthermore, with the addition of combined additives at an enzyme dosage of 8 FPU/g substrate, the NaOH-catalyzed organosolv pretreated poplar liberated higher ethanol than that without additive at an enzyme dosage of 15 FPU/g substrate. This phenomenon indicated that the use of combined additive can improve fermentation efficiency, while reducing almost 1/2 enzyme dosage. In general, this study provided a facile and efficient method for ethanol production with compound additives.

Enhancement of ethanol production and cell growth in budding yeast by direct irradiation of low-temperature plasma

Ethanol production by budding yeast was compared between direct and indirect plasma irradiation. We observed enhancement of ethanol production and cell growth not by indirect plasma irradiation but by direct plasma irradiation. Glucose consumption was increased in budding yeast by direct plasma irradiation. Extracellular flux analysis revealed that glycolytic activity in the budding yeast was elevated by direct plasma irradiation. These results suggest that direct plasma irradiation enhances ethanol production in budding yeast by elevating the glycolytic activity.

Enhancing bioproduction and thermotolerance in Saccharomyces cerevisiae via cell immobilization on biochar: Application in a citrus peel waste biorefinery

A novel method for enhancement of ethanol production and temperature tolerance of S. cerevisiae through the development of biochar-based biocatalysts (BBBs) is reported. Immobilized BBBs were applied in alcoholic fermentations of hydrolyzates generated via a citrus peel waste (CPW) biorefinery, which allowed extraction of high-purity pectin that reached 30.5% (w/w). Pistachio-nut shells, peanut shells and corks were employed for biochar generation via pyrolysis to produce the cell carriers required. All materials were highly carbonaceous with mesopore size structures (1–50 μm), while peanut shells biochar was crystalline incorporating calcite and sylvite. S. cerevisiae immobilized on pistachio-nuts biochar grown on a synthetic CPW hydrolysate, exhibited 63 g L−1 ethanol concentration and 7.9 g L−1 h−1 productivity improving substantially biosystem performance as compared to unsupported cultures. Alcoholic fermentations conducted at different elevated temperatures (37–41 °C) exhibited stable performance of the immobilized system for six repeated batch experiments. Fermentations of the CPW-hydrolyzate formed through the biorefinery at 41 °C using BBB produced 30.8 g L−1 of ethanol, while free cells achieved significantly lower concentration (13.4 g L−1). The proposed technology confers thermotolerance on S. cerevisiae, which buffers the negative impact of high temperatures on cells leading in increased bioethanol production and lower energy demand.

Improvement of ethanol production using green alga Chlorococcum minutum

ABSTRACT Incessant depletion of fossil fuels urges the governments and non-governmental organizations to invest more on renewable energy sectors including generation of biofuels such as bioethanol. Production of ethanol from algal feedstock has been an interesting area of research in recent times. In the present investigation, feedstock of a green alga Chlorococcum minutum was selected for ethanol production and compared with feedstock of model alga Chlamydomonas reinhardtii. Both the species were grown under in vitro conditions using universal tris-acetate-phosphate (TAP) medium with various concentrations and combinations of vitamins such as thiamin, biotin and cobalamin (B1, B7 and B12) to enhance the biomass in turn reducing sugars in both the algal cultures. Later, these algal feedstocks were used for the production of ethanol under fermentation conditions using yeast. Reducing sugars were more in both the algal cultures grown in Cr3 or Cm3 media (TAP with 0.8 g/L of B1, 0.004 g/L of B7 & 0.004 g/L of B12) and also in Cr2 or Cm2 media (TAP with 0.4 g/L of B1, 0.002 g/L of B7 & 0.002 g/L of B12). In extent, the enhancement of ethanol production was noticed in C. reinhardtii (33.57 g/L) and C. minutum (46.97 g/L) from the feedstocks grown in Cr3 or Cm3 media when compared with feedstocks grown in other vitamin combinations or without vitamin assistance. Specifically, feedstock of C. minutum generated more output at 48 h when compared with model alga. The present work may be useful for the production of ethanol at a commercial level.

Urea-Appended Amino Acid To Vitalize Yeast Growth, Enhance Fermentation, and Promote Ethanol Production.

The development of sustainable protocols for enhancing the production of ethanol is highly important for its utilization in automotive transportation and energy sector. Up to 15% ethanol can blend with diesel to make e-diesel that can be used in fuel compression ignition engine. Urea-modified amino acids can be used as a very good vitalizer for yeast (Saccharomyces cerevisiae, Baker’s yeast (ATCC 204508)) growth and thus promote ethanol production. A simple, one-step, room-temperature synthetic procedure has been developed for urea-appended α-amino acids from amino acid by treatment with KCNO. Single-crystal X-ray studies confirm the successful synthesis and molecular structures of the urea-appended α-amino acids. Out of 20 urea-appended amino acids, Arg-, Pro-, His-, and Gln-containing compounds promote yeast growth significantly after 12 h at pH 6.8 and 38 °C. These compounds are nontoxic. The urea-appended Arg shows 2-fold increase of yeast growth. However, urea-appended m-aminobenzoic acid and p-aminobenzoic acid inhibit yeast growth. NMR experiments confirmed the enhanced production of ethanol by glucose fermentation in the presence of 2.5 μmol urea-appended Arg. Not only glucose but also commercially available sugars and feedstock of the starch slurry drained out after boiling of rice exhibit significant enhancement of ethanol production under same conditions.

Enhancement of Ethanol Production in Electrochemical Cell by Saccharomyces cerevisiae (CDBT2) and Wickerhamomyces anomalus (CDBT7)

Bioethanol (a renewable resource), blended with gasoline, is used as liquid transportation fuel worldwide and produced from either starch or lignocellulose. Local production and use of bioethanol supports local economies, decreases country’s carbon footprint and promotes self-sufficiency. The latter is especially important for bio-resource-rich land-locked countries like Nepal that are seeking alternative transportation fuels and technologies to produce them. In that regard, in the present study, we have used two highly efficient ethanol producing yeast strains, viz., Saccharomyces cerevisiae (CDBT2) and Wickerhamomyces anomalous (CDBT7), in an electrochemical cell to enhance ethanol production. Ethanol production by CDBT2 (anodic chamber) and CDBT7 (cathodic chamber) control cultures, using 5% glucose as substrate, were 12.6 ± 0.42 and 10.1 ± 0.17 mg•mL-1 respectively. These cultures in the electrochemical cell, when externally supplied with 4V, the ethanol production was enhanced by 19.8 ± 0.50 % and 23.7 ± 0.51% respectively as compared to the control cultures. On the other hand, co-culturing of those two yeast strains in both electrode compartments resulted only 3.96 ± 0.83% enhancement in ethanol production. Immobilization of CDBT7 in the graphite cathode resulted in lower enhancement of ethanol production (5.30 ± 0.82 mg•mL-1) less than free cell culture of CDBT7. CDBT2 and CDBT7 when cultured in platinum nano particle coated platinum anode and neutral red-coated graphite cathode, respectively, ethanol production was substantially enhanced (52.8 ± 0.44%). The above experiments when repeated using lignocellulosic biomass hydrolysate (reducing sugar content was 3.3%) as substrate, resulted in even better enhancement in ethanol production (61.5 ± 0.12%) as compared to glucose. The results concluded that CDBT2 and CDBT7 yeast strains produced ethanol efficiently from both glucose and lignocellulosic biomass hydrolysate. . Ethanol production was enhanced in the presence of low levels of externally applied voltage. Ethanol production was further enhanced with the better electron transport provision ie; when neutral red was deposited on cathode and fine platinum nanoparticles were coated on the platinum anode.

Ethanol Production from Saccharum Spontanum Biomass Native to Nepal Using Bioelectrochemical Cell

Bioethanol can be used as an octane enhancer and alternative replacement to blend with petroleum fuels. Using an electrochemical cell for the production of bioethanol facilitates the enhancement in ethanol production exploiting the electrochemical redox reactions occuring inside the cell. The externally supplied voltage is used to drive the chemical reactions to generate the metabolite, i.e; ethanol. A microbial electrochemical cell was designed with porous carbon fiber coated with neutral red as cathode and platinum wire coated with fine platinum as anode. Saccharum spontanum biomass pretreated with hot water at 100oC for 2 hours followed by acid hydrolysis was neutralized and used for production of ethanol by Saccharomyces cereviseae in electrochemical cell. A total supply of 4V was found to be best for maximum ethanol production in 300 ml fermentation volume. Key words: ethanol, microbial electrochemical cell, voltage.

Enhancement of ethanol production in very high gravity fermentation by reducing fermentation-induced oxidative stress in Saccharomyces cerevisiae

During fermentation, yeast cells encounter a number of stresses, including hyperosmolarity, high ethanol concentration, and high temperature. Previous deletome analysis in the yeast Saccharomyces cerevisiae has revealed that SOD1 gene encoding cytosolic Cu/Zn-superoxide dismutase (SOD), a major antioxidant enzyme, was required for tolerances to not only oxidative stress but also other stresses present during fermentation such as osmotic, ethanol, and heat stresses. It is therefore possible that these fermentation-associated stresses may also induce endogenous oxidative stress. In this study, we show that osmotic, ethanol, and heat stresses promoted generation of intracellular reactive oxygen species (ROS) such as superoxide anion in the cytosol through a mitochondria-independent mechanism. Consistent with this finding, cytosolic Cu/Zn-SOD, but not mitochondrial Mn-SOD, was required for protection against oxidative stress induced by these fermentation-associated stresses. Furthermore, supplementation of ROS scavengers such as N-acetyl-L-cysteine (NAC) alleviated oxidative stress induced during very high gravity (VHG) fermentation and enhanced fermentation performance at both normal and high temperatures. In addition, NAC also plays an important role in maintaining the Cu/Zn-SOD activity during VHG fermentation. These findings suggest the potential role of ROS scavengers for application in industrial-scale VHG ethanol fermentation.

Simultaneous Saccharification and Fermentation of Corn Husk by Co-Culture Strategy

Lignocellulosic biofuel production mainly carried out by two ways: simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF). In the present study, simultaneous saccharification and fermentation (SSF) was carried out using microwave assisted thermochemically pretreated (0.5 M NaOH for 20 minutes at 120°C in preheated oven) corn husk. Using co-cultures of Saccharomyces cerevisiae and Fusarium oxysporum, SSF process was optimized. Maximum ethanol production (6.24%, v/v) was observed after 24 h of incubation. Further for enhanced ethanol production, effect of different surfactant was carried out on SSF using co-culture strategy. It was found that addition of Tween 60 enhanced the ethanol production upto 6.38% (v/v). Further for addition enhancement of ethanol production, different co-culture strategy was adopted. It was found that maximum ethanol production (6.58% v/v) was obtained when ethanol fermentation was carried out by Fusarium oxysporum followed by Saccharomyces cerevisiae.

Enhancement of ethanol production from synthetic medium model of hydrolysate of macroalgae

Abstract Among biomass materials available, macroalgae is a promising alternative to traditional energy crops. The absence of lignin, a high growth rate and a richness of fermentable sugars and nitrogen, are real gains for a competitive ethanol production. But the presence of salts can be an obstacle to obtain relevant performances. Experiments were carried out with a synthetic medium adjusted on algal hydrolysate composition in order to reduce resource limitations and variations of composition. The behavior of four yeast strains for ethanol production was investigated: Candida guilliermondii, Scheffersomyces stipitis, Kluyveromyces marxianus and Saccharomyces cerevisiae. Glucose, which is the most abundant sugar in the targeted algal hydrolysate (Ulva spp), was completely assimilated by all of the considered strains, even in the presence of salts at levels found in macroalgal hydrolysates (0.25 M of sodium chloride and 0.21 M of sulfate). The use of peptone as nitrogen source enhanced kinetics of consumption and production. For instance, the rate of ethanol production by S. cerevisiae in the presence of peptone was six times higher than that obtained using ammonium, 0.6 and 0.1 g L−1 h−1 respectively. In the presence of salts, the rates of glucose consumption and ethanol production were lowered for the considered strains, except for K. marxianus. Nevertheless, S. cerevisiae could be the most promising strain to valorize Ulva spp hydrolysate in bioethanol, in terms of ethanol produced (7.5–7.9 g L−1) whether in the presence or in absence of salts.

Enhanced ethanol production by Clostridium ragsdalei from syngas by incorporating biochar in the fermentation medium

Biochar contains minerals and metals that can serve as nutrients for acetogens to produce ethanol via syngas fermentation. In this study, four fermentation media containing biochar from switchgrass (SGBC), forage sorghum (FSBC), red cedar (RCBC) and poultry litter (PLBC) were compared with standard yeast extract (YE) medium for syngas fermentation using Clostridium ragsdalei. Fermentations were performed in 250mL bottle reactors at 150rpm and 37°C with syngas containing CO:H2:CO2 (40:30:30) by volume. Results showed that media containing RCBC and PLBC improved ethanol production by 16.3% and 58.9%, respectively, compared to YE medium. C. ragsdalei consumed 69% more H2 and 40% more CO in PLBC medium compared to YE medium. However, no enhancement of ethanol production was observed in SGBC and FSBC media. The highest release of Na, K, Ca, Mg, S and P was from PLBC, which was considered to contribute in enhancement of ethanol production.

Enhancement of ethanol production by novel morpholinium ionic liquids

Two novel morpholinium ionic liquids, N-allyl-N-methylmorpholinium acetate ([AMMorph][Ac]) and morpholinium acetate ([Morph][Ac]), were synthesized and successfully used for pretreatment of rice straw. While imidazolium liquids are the most efficient solvents for the pretreatment of lignocelluloses, the main advantages of morpholinium liquids are their lower toxicity and price. Different concentrations of dimethyl sulfoxide (DMSO), as an inexpensive industrial co-solvent, were mixed with the synthesized ionic liquids to assist the pretreatment efficiency. The pretreatment was performed at 90, 100, and 120 °C for 2, 3, and 5 h. Then, the pretreated materials were subjected to enzymatic hydrolysis followed by fermentation. Moreover, the efficiency of the synthesized ionic liquids was compared with those of four widely used solvents, i.e., 85% N-methylmorpholine-N-oxide (NMMO), 1-butyl-3-methylimidazolium acetate ([Bmim][Ac]), 8% NaOH solution, and 85% H3PO4 solution. The pretreatment with [AMMorph][Ac] resulted in the significant improvement of enzymatic hydrolysis, which was as efficient as the best widely used pretreatments. The glucose yields of 98.4 ± 1.3% were achieved by pretreatment with mixtures of [AMMorph][Ac]-DMSO as well as NMMO, [Bmim][Ac], and NaOH. The highest ethanol yields of 90.8% and 94.8% were achieved through 70:30 (v/v) [AMMorph][Ac]-DMSO solution and NMMO, respectively. Moreover, using DMSO as a co-solvent could minimize the ionic liquids usage without lowering their performance and enhanced the pretreatment efficiency by viscosity reduction.

Enhancement of ethanol production from green liquor\u2013ethanol-pretreated sugarcane bagasse by glucose\u2013xylose cofermentation at high solid loadings with mixed Saccharomyces cerevisiae strains

BackgroundEfficient cofermentation of glucose and xylose is necessary for economically feasible bioethanol production from lignocellulosic biomass. Here, we demonstrate pretreatment of sugarcane bagasse (SCB) with green liquor (GL) combined with ethanol (GL–Ethanol) by adding different GL amounts. The common Saccharomyces cerevisiae (CSC) and thermophilic S. cerevisiae (TSC) strains were used and different yeast cell mass ratios (CSC to TSC) were compared. The simultaneous saccharification and cofermentation (SSF/SSCF) process was performed by 5–20% (w/v) dry substrate (DS) solid loadings to determine optimal conditions for the co-consumption of glucose and xylose.ResultsCompared to previous studies that tested fermentation of glucose using only the CSC, we obtained higher ethanol yield and concentration (92.80% and 23.22 g/L) with 1.5 mL GL/g-DS GL–Ethanol-pretreated SCB at 5% (w/v) solid loading and a CSC-to-TSC yeast cell mass ratio of 1:2 (w/w). Using 10% (w/v) solid loading under the same conditions, the ethanol concentration increased to 42.53 g/L but the ethanol yield decreased to 84.99%. In addition, an increase in the solid loading up to a certain point led to an increase in the ethanol concentration from 1.5 mL GL/g-DS-pretreated SCB. The highest ethanol concentration (68.24 g/L) was obtained with 15% (w/v) solid loading, using a CSC-to-TSC yeast cell mass ratio of 1:3 (w/w).ConclusionsGL–Ethanol pretreatment is a promising pretreatment method for improving both glucan and xylan conversion efficiencies of SCB. There was a competitive relationship between the two yeast strains, and the glucose and xylose utilization ability of the TSC was better than that of the CSC. Ethanol concentration was obviously increased at high solid loading, but the yield decreased as a result of an increase in the viscosity and inhibitor levels in the fermentation system. Finally, the SSCF of GL–Ethanol-pretreated SCB with mixed S. cerevisiae strains increased ethanol concentration and was an effective conversion process for ethanol production at high solid loading.

Improved bioethanol production from metabolic engineering of Enterobacter aerogenes ATCC 29007

This study investigates the enhancement of bioethanol production using a genetic engineering approach. The bioethanol-producing strain, E. aerogenes ATCC 29007, was engineered by deleting the D-lactate dehydrogenase (ldhA) gene to block the production of lactic acid. The Open-reading frame coding region of ldhA gene was replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method to inactivate chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. The colony PCR was used to confirm the deleted gene. Glycerol, a useful byproduct in the biodiesel industry, was employed to convert into bioethanol, using engineered E. aerogenes SUMI014. Under optimal conditions of fermentation (34°C, pH 7.5, 78h), bioethanol production by the mutant strain was 34.54g/L, 1.5 times greater than that produced by its wild type (13.09g/L). Subsequent overexpression of alcohol dehydrogenase (adhE) gene in the mutant strain; increased the production of bioethanol up to 38.32g/L. By the combination of gene deletion and overexpression, the bioethanol yield was 0.48g/g when employing 80g/L glycerol. Hence, a significant enhancement in ethanol production was observed. These results may provide valuable guidelines for further engineering bioethanol producers.

Improved Ethanol Production from Xylose by Candida shehatae Induced by Dielectric Barrier Discharge Air Plasma

Xylose fermentation is essential for ethanol production from lignocellulosic biomass. Exposure of the xylose-fermenting yeast Candida shehatae (C. shehatae) CICC1766 to atmospheric pressure dielectric barrier discharge (DBD) air plasma yields a clone (designated as C81015) with stability, which exhibits a higher ethanol fermentation rate from xylose, giving a maximal enhancement in ethanol production of 36.2% compared to the control (untreated). However, the biomass production of C81015 is lower than that of the control. Analysis of the NADH (nicotinamide adenine dinucleotide)- and NADPH (nicotinamide adenine dinucleotide phosphate)-linked xylose reductases and NAD+-linked xylitol dehydrogenase indicates that their activities are enhanced by 34.1%, 61.5% and 66.3%, respectively, suggesting that the activities of these three enzymes are responsible for improving ethanol fermentation in C81015 with xylose as a substrate. The results of this study show that DBD air plasma could serve as a novel and effective means of generating microbial strains that can better use xylose for ethanol fermentation.

Optimization of Simultaneous Saccharification and Fermentation for Ethanol Production from Steam-Exploded Cotton Stalk

Steam-exploded cotton stalk was used as raw material in ethanol production through simultaneous saccharification and fermentation by Penicillium Q59 and Saccharomyces cerevisiae P1. The fermentative conditions were firstly examined by single factor experiments to determine the central point in Box-Behnken design, which was explored with expectation to get optimized fermentative conditions for enhancement of ethanol production. The results of optimized fermentative conditions were determined as follows: fermentation time was 10.5 days, bran added percent was 15%, initial pH value was 5.5. Under the optimal conditions, the experimental yield of ethanol was 99.85 ± 4.21 g·kg-1SECS (steam-exploded cotton stalk), which was close to the theoretical predicting value, it showed the model was feasible. The research results will provide technical reference for further exploitation of cotton stalk.

Enhancement of ethanol production from potato-processing wastewater by engineering Escherichia coli using Vitreoscilla haemoglobin

Ethanologenic Escherichia coli strain FBR5 was transformed with the Vitreoscilla haemoglobin (VHb) gene (vgb) in two constructs (resulting in strains TS3 and TS4). Strains FBR5, TS3 and TS4 were grown at two scales in LB medium supplemented with potato-processing wastewater hydrolysate. Aeration was varied by changes in the medium volume to flask volume ratio. Parameters measured included culture pH, cell growth, VHb levels and ethanol production. VHb expression in strains TS3 and TS4 was consistently correlated with increases in ethanol production (5-18%) under conditions of low aeration, but rarely did this occur with normal aeration. The increase in ethanol yields under low aeration conditions was the result of enhancement of ethanol produced per unit of biomass rather than enhancement of growth. 'VHb technology' may be a useful adjunct in the production of biofuels from food-processing wastewater. Genetic engineering using Vitreoscilla haemoglobin (VHb) has been shown previously to increase ethanol production by Escherichia coli from fermentation of the sugars in corn fibre hydrolysate. The study reported here demonstrates a similar VHb enhancement of ethanol production by fermentation of the glucose from potato waste water hydrolysate and thus extends the list of sugar containing waste products from which ethanol production may be enhanced by this strategy.

Enhancement of stress tolerance and ethanol production in Saccharomyces cerevisiae by heterologous expression of a trehalose biosynthetic gene from Streptomyces albus

The ability to grow and produce ethanol under stressful conditions is an important factor in industrial bioethanol production. Trehalose is found in many organisms including Saccharomyces cerevisiae, and has been known to play an important role in enhancing various types of stress tolerance. In this study, Streptomyces albus trehalose-6-phosphate synthase gene (salC) was expressed in Saccharomyces cerevisiae, and the recombinant strain with salC gene showed significantly improved stress resistances and ethanol production. The stress sensitivity and viability tests indicated that the recombinant had a greater resistance to ethanol than the control. At elevated temperatures, the results of flask cultures showed that the expression of salC played a positive role in protecting cells from heat stress. The recombinant strain was found to consume 100 g/L glucose and to produce 39 g/L ethanol at 40°C with an ethanol yield 6% higher than that of the control strain. In the fed-batch experiment in a bioreactor the recombinant strain produced 69 g/L ethanol with about 16% higher yield and about 13% higher productivity than the control strain. This demonstrated the enhancement of ethanol production capabilities of the recombinant strain under a high-ethanol stress condition.

Enhancement of ethanol production from spruce wood chips by ionic liquid pretreatment

Pretreatment with three green cellulose solvents, N-methylmorpholine-N-oxide (NMMO), 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]), and 1-butyl-3-methylimidazolium acetate ([BMIM][OAc]) were used for improvement of ethanol production. Spruce chips and powder were pretreated at 120°C for 1, 3, and 15h. In order to investigate the efficiency of the pretreatments, the pretreated materials were subjected to enzymatic hydrolysis at 45°C for 72h, followed by fermentation with Saccharomyces cerevisiae at 32°C for 24h. The best results were obtained from 15h pretreatment with [EMIM][OAc]. The ethanol yield from the untreated spruce chips and powder were 2.7% and 9.7% of the maximum theoretical yield, respectively, whereas pretreatment of these materials with [EMIM][OAc] improved the ethanol yield to 66.8% and 81.5%, respectively.

Vitreoscilla hemoglobin enhances ethanol production by Escherichia coli in a variety of growth media

Abstract In a previous study, we showed that expression of Vitreoscilla hemoglobin (VHb) in ethanologenic Escherichia. coli strain FBR5 enhanced ethanol production in a limited range of nutrient concentrations. Here enhancement of ethanol production in the VHb expressing strain (TS3) was investigated in a wider variety of growth media. For nutrient (LB) concentrations from 0.25X to 2X (supplemented with 80 g dm −3 xylose), ethanol production was always better in TS3 than in FBR5. The greatest relative advantage of TS3 over FBR5 occurred at 0.25X LB, while the greatest yield for both strains relative to LB concentration occurred at 0.5X LB. In these experiments, the enhancement in ethanol production of TS3 was determined to be due to both an increased ethanol metabolic efficiency and increased cell growth. 2X LB was also supplemented with purified biomass hydrolysate produced by acid treatment and overliming; additional treatment with activated carbon decreased the fermentability of the hydrolysate. Strain TS3 outperformed strain FBR5 in 2X LB at hydrolysate concentrations of 0.2, 0.4 and 0.8 m 3 m −3 ; the greatest yield by TS3 relative to the level of hydrolysate occurred at 0.4 m 3 m −3 . The concentrations of NAD, NADH, and ATP were measured in both strains to investigate the biochemical basis of the ethanol production enhancement by VHb. The most significant difference found was in the NAD/NADH ratio; this was generally lower for TS3 than for FBR5 earlier in the fermentation, while this pattern was reversed later in the fermentation.