Effect of wood ash treatment on improving the fermentability of wood hydrolysate

Softwood hydrolysates were overlimed with wood ash to improve the fermentability of hydrolysates. It could be demonstrated in fermentation tests that wood ash treatment increases fermentability compared to the hydrolysates untreated and treated with alkaline compounds such as Ca(OH)(2), NaOH, and KOH, which are commonly used for overliming. The enhanced fermentability of the hydrolysate treated with wood ash is due to the reduction of the inhibitors of the fermentation such as furan and phenolic compounds and to nutrient effects of some inorganic components from the wood ash on the fermentation.     

Detoxification of wood hydrolysates with laccase and peroxidase from the white-rot fungus Trametes versicolor

Fermentation of wood hydrolysates to desirable products, such as fuel ethanol, is made difficult by the presence of inhibitory compounds in the hydrolysates. Here we present a novel method to increase the fermentability of lignocellulosic hydrolysates: enzymatic detoxification. Besides the detoxification effect, treatment with purified enzymes provides a new way to identify inhibitors by assaying the effect of enzymatic attack on specific compounds in the hydrolysate. Laccase, a phenol oxidase, and lignin peroxidase purified from the ligninolytic basidiomycete fungus Trametes versicolor were studied using a lignocellulosic hydrolysate from willow pretreated with steam and SO₂. Saccharomyces cerevisiae was employed for ethanolic fermentation of the hydrolysates. The results show more rapid consumption of glucose and increased ethanol productivity for samples treated with laccase. Treatment of the hydrolysate with lignin peroxidase also resulted in improved fermentability. Analyses by GC-MS indicated that the mechanism of laccase detoxification involves removal of monoaromatic phenolic compounds present in the hydrolysate. The results support the suggestion that phenolic compounds are important inhibitors of the fermentation process. Received: 3 November 1997 / Received revision: 4 February 1998 / Accepted: 6 February 1998     

Detoxification of hemicellulosic hydrolysates from extracted olive pomace by diananofiltration

Xylitol can be obtained from the pentose-rich hemicellulosic fraction of agricultural residues, such as extracted olive pomace, by fermentation. Dilute acid hydrolysis of lignocellulosic materials, produces the release of potential inhibitory compounds mainly furan derivatives, aliphatic acids, and phenolic compounds. In order to study the potential on the increase of the hydrolysate fermentability, detoxification experiments based on diananofiltration membrane separation processes were made. Two membranes, NF270 and NF90, were firstly evaluated using hydrolysate model solutions under total recirculation mode, to identify the best membrane for the detoxification. NF270 was chosen to be used in the diananofiltration experiment as it showed the lowest rejection for toxic compounds and highest permeate flux. Diananofiltration experiments, for hydrolysate model solutions and hydrolysate liquor, showed that nanofiltration is able to deplete inhibitory compounds and to obtain solutions with higher xylose content. Conversely to non-detoxified hydrolysates, nanofiltration detoxified hydrolysates enabled yeast growth and xylitol production by the yeast Debaryomyces hansenii, clearly pointing out that detoxification is an absolute requirement for extracted olive pomace dilute acid hydrolysate bioconversion.     

Biotechnological strategies to overcome inhibitors in lignocellulose hydrolysates for ethanol production: review

One of the major challenges faced in commercial production of lignocellulosic bioethanol is the inhibitory compounds generated during the thermo-chemical pre-treatment step of biomass. These inhibitory compounds are toxic to fermenting micro-organisms. The ethanol yield and productivity obtained during fermentation of lignocellulosic hydrolysates is decreased due to the presence of inhibiting compounds, such as weak acids, furans and phenolic compounds formed or released during thermo-chemical pre-treatment step such as acid and steam explosion. This review describes the application and/or effect of biological detoxification (removal of inhibitors before fermentation) or use of bioreduction capability of fermenting yeasts on the fermentability of the hydrolysates. Inhibition of yeast fermentation by the inhibitor compounds in the lignocellulosic hydrolysates can be reduced by treatment with enzymes such as the lignolytic enzymes, for example, laccase and micro-organisms such as Trichoderma reesei, Coniochaeta ligniaria NRRL30616, Trametes versicolor, Pseudomonas putida Fu1, Candida guilliermondii, and Ureibacillus thermosphaericus. Microbial and enzymatic detoxifications of lignocellulosic hydrolysate are mild and more specific in their action. The efficiency of enzymatic process is quite comparable to other physical and chemical methods. Adaptation of the fermentation yeasts to the lignocellulosic hydrolysate prior to fermentation is suggested as an alternative approach to detoxification. Increases in fermentation rate and ethanol yield by adapted micro-organisms to acid pre-treated lignocellulosic hydrolysates have been reported in some studies. Another approach to alleviate the inhibition problem is to use genetic engineering to introduce increased tolerance by Saccharomyces cerevisiae, for example, by overexpressing genes encoding enzymes for resistance against specific inhibitors and altering co-factor balance. Cloning of the laccase gene followed by heterologous expression in yeasts was shown to provide higher enzyme yields and permit production of laccases with desired properties for detoxification of lignocellulose hydrolysates. A combination of more inhibitor-tolerant yeast strains with efficient feed strategies such as fed-batch will likely improve lignocellulose-to-ethanol process robustness.     

Isolation and characterization of Cupriavidus basilensis HMF14 for biological removal of inhibitors from lignocellulosic hydrolysate

The formation of toxic fermentation inhibitors such as furfural and 5-hydroxy-2-methylfurfural (HMF) during acid (pre-)treatment of lignocellulose, calls for the efficient removal of these compounds. Lignocellulosic hydrolysates can be efficiently detoxified biologically with microorganisms that specifically metabolize the fermentation inhibitors while preserving the sugars for subsequent use by the fermentation host. The bacterium Cupriavidus basilensis HMF14 was isolated from enrichment cultures with HMF as the sole carbon source and was found to metabolize many of the toxic constituents of lignocellulosic hydrolysate including furfural, HMF, acetate, formate and a host of aromatic compounds. Remarkably, this microorganism does not grow on the most abundant sugars in lignocellulosic hydrolysates: glucose, xylose and arabinose. In addition, C. basilensis HMF14 can produce polyhydroxyalkanoates. Cultivation of C. basilensis HMF14 on wheat straw hydrolysate resulted in the complete removal of furfural, HMF, acetate and formate, leaving the sugar fraction intact. This unique substrate profile makes C. basilensis HMF14 extremely well suited for biological removal of inhibitors from lignocellulosic hydrolysates prior to their use as fermentation feedstock.     

Effects of Ca(OH)(2) treatments ("overliming") on the composition and toxicity of bagasse hemicellulose hydrolysates

Hemicellulose syrups from dilute sulfuric acid hydrolysates of hemicellulose contain inhibitors that prevent efficient fermentation by yeast or bacteria. It is well known that the toxicity of these hydrolysate syrups can be ameliorated by optimized "overliming" with Ca(OH)(2). We have investigated the optimization of overliming treatments for sugar cane bagasse hydrolysates (primarily pentose sugars) using recombinant Escherichia coli LY01 as the biocatalyst. A comparison of composition before and after optimal overliming revealed a substantial reduction in furfural, hydroxymethylfurfural, and three unidentified high-performance liquid chromatography (HPLC) peaks. Organic acids (acetic, formic, levulinic) were not affected. Similar changes have been reported after overliming of spruce hemicellulose hydrolysates (Larsson et al., 1999). Our studies further demonstrated that the extent of furan reduction correlated with increasing fermentability. However, furan reduction was not the sole cause for reduced toxicity. After optimal overliming, bagasse hydrolysate was rapidly and efficiently fermented (>90% yield) by LY01. During these studies, titration, and conductivity were found to be in excellent agreement as methods to estimate sulfuric acid content. Titration was also found to provide an estimate of total organic acids in hydrolysate, which agreed well with the sum of acetic, levulinic, and formic acids obtained by HPLC. Titration of acids, measurement of pH before and after treatment, and furan analyses are proposed as relatively simple methods to monitor the reproducibility of hydrolysate preparations and the effectiveness of overliming treatments.     

Sugar recovery and fermentability of hemicellulose hydrolysates from steam-exploded softwoods containing bark

The hemicellulose sugar recovery and ethanol production obtained from SO2-catalyzed steam explosion of a mixed white fir (70%) and ponderosa pine (30%) feedstock containing bark (9% dry weight/dry weight) was assessed. More than 90% of the available hemicellulose sugars could be recovered in the hydrolysate obtained after steam explosion at 195 degrees C, 2.38 min, and 3.91% SO2, with 59% of the original hemicellulose sugars detected in a monomeric form. Despite this high sugar recovery, this hydrolysate showed low ethanol yield (64% of theoretical yield) when fermented with a spent sulfite liquor-adapted strain of Saccharomyces cerevisiae. In contrast, most hydrolysates prepared at higher steam explosion severity showed comparable or higher ethanol yields. Furthermore, the hydrolysates prepared from bark-free feedstock showed better fermentability (87% of theoretical yield) despite containing higher concentration of known inhibitors. The ethanol yield from the hydrolysate prepared from a bark-containing wood sample could be improved to 81% by an extra stage acid hydrolysis (121 degrees C for 1 h in 3% sulfuric acid). This extra stage acid hydrolysis and steam explosion at higher severity conditions seem to improve the fermentability of the hydrolysates by transforming certain inhibitory compounds present in the hydrolysates prepared from the bark-containing feedstock and thus lowering their inhibitory effect on the yeast used for the ethanol fermentation.     

Detoxification of Lignocellulose Hydrolysates: Biochemical and Metabolic Engineering Toward White Biotechnology

Chemical hydrolysis of lignocellulosic biomass (LB) produces a number of inhibitors in addition to sugars. These inhibitors include lignin-derived phenolics, carbohydrate-derived furans, and weak acids that have shown a marked effect on the productivities of various metabolites and the growth of biocatalysts in the fermentative reaction. In the past, a number of physicochemical and biological approaches have been proposed to overcome these fermentation inhibitors, including modified fermentative strategies. Additionally, the timely intervention of genetic engineering has provided an impetus to develop suitable technologies for the detoxification of lignocellulosics in biorefineries. However, the improvements in detoxification strategies for lignocellulose hydrolysates have resulted in significant loss of sugars after detoxification. Hydrolysis of myco-LB (LB after fungal pretreatment) has been recognized as a promising approach to avoid fermentation inhibitors and improve total sugar recovery. Biotechnological inventions have also made it possible to widen the range of suitable biocatalysts for biorefineries by microbial-routed induction of enzymatic expression for the elimination of inhibitors, eventually improving ethanol production from acid hydrolysates. This article aims to highlight the strategies that have been adopted to detoxify lignocellulosic hydrolysates and their effects on the chemical composition of the hydrolysates to improve the fermentability of lignocellulosics. In addition, genetic manipulation could widen the availability and variety of substrates and modify the metabolic routes to produce bioethanol or other value-added compounds in an efficient manner.     

Influence of the propagation strategy for obtaining robust Saccharomyces cerevisiae cells that efficiently co-ferment xylose and glucose in lignocellulosic hydrolysates

Development of xylose-fermenting yeast strains that are tolerant to the inhibitors present in lignocellulosic hydrolysates is crucial to achieve efficient bioethanol production processes. In this study, the importance of the propagation strategy for obtaining robust cells was studied. Addition of hydrolysate during propagation of the cells adapted them to the inhibitors, resulting in more tolerant cells with shorter lag phases and higher specific growth rates in minimal medium containing acetic acid and vanillin than unadapted cells. Addition of hydrolysate during propagation also resulted in cells with better fermentation capabilities. Cells propagated without hydrolysate were unable to consume xylose in wheat straw hydrolysate fermentations, whereas 40.3% and 97.7% of the xylose was consumed when 12% and 23% (v/v) hydrolysate, respectively, was added during propagation. Quantitative polymerase chain reaction revealed changes in gene expression, depending on the concentration of hydrolysate added during propagation. This study highlights the importance of using an appropriate propagation strategy for the optimum performance of yeast in fermentation of lignocellulosic hydrolysates.     

Removal of phenolic inhibitors from lignocellulose hydrolysates using laccases for the production of fuels and chemicals

Lignocellulose is the most abundant biopolymer in the biosphere. It is inexpensive and therefore considered an attractive feedstock to produce biofuels and other biochemicals. Thermochemical and/or enzymatic pretreatment is used to release fermentable monomeric sugars. However, a variety of inhibitory by-products such as weak acids, furans, and phenolics that inhibit cell growth and fermentation are also released. Phenolic compounds are among the most toxic components in lignocellulosic hydrolysates and slurries derived from lignin decomposition, affecting overall fermentation processes and production yields and productivity. Ligninolytic enzymes have been shown to lower inhibitor concentrations in these hydrolysates, thereby enhancing their fermentability into valuable products. Among them, laccases, which are capable of oxidizing lignin and a variety of phenolic compounds in an environmentally benign manner, have been used for biomass delignification and detoxification of lignocellulose hydrolysates with promising results. This review discusses the state of the art of different enzymatic approaches to hydrolysate detoxification. In particular, laccases are used in separate or in situ detoxification steps, namely in free enzyme processes or immobilized by cell surface display technology to improve the efficiency of the fermentative process and consequently the production of second-generation biofuels and bio-based chemicals.     

Supercritical carbon dioxide extraction,fluorimetric and electrochemical high performance liquid chromatrographic detection of vitamin E from Hordeum vulgare L.

A supercritical fluid extraction (SFE), method has been applied to extract the natural vitamin E components, tocopherols and tocotrienols, from fruits of Hordeum vulgare L. The fractions were monitored by analytical high performance liquid chromatography using fluorimetric and electrochemical detectors. High yields, and clear and purified extracts were obtained by SFE; compared with the n-hexane extract, the SFE extract was lighter in colour and more concentrated in vitamin E components. © 1998 John Wiley & Sons, Ltd.     

Application of supercritical CO2 for extraction of polyisoprenoid alcohols and their esters from plant tissues

In this study, a method of supercritical fluid extraction (SFE) with carbon dioxide of polyisoprenoids from plant photosynthetic tissues is described. SFE was an effective extraction method for short- and medium-chain compounds with even higher yield than that observed for the “classical extraction” method with organic solvents. Moreover, SFE-derived extracts contained lower amounts of impurities (e.g., chlorophylls) than those obtained by extraction of the same tissue with organic solvents. Elevated temperature and extended extraction time of SFE resulted in a higher rate of extraction of long-chain polyisoprenoids. Ethanol cofeeding did not increase the extraction efficiency of polyisoprenoids; instead, it increased the content of impurities in the lipid extract. Optimization of SFE time and temperature gives the opportunity of prefractionation of complex polyisoprenoid mixtures accumulated in plant tissues. Extracts obtained with application of SFE are very stable and free from organic solvents and can further be used directly in experimental diet supplementation or as starting material for preparation of semisynthetic polyisoprenoid derivatives, e.g., polyisoprenoid phosphates.     

Production of xylitol by a Coniochaeta ligniaria strain tolerant of inhibitors and defective in growth on xylose

In conversion of biomass to fuels or chemicals, inhibitory compounds arising from physical–chemical pretreatment of the feedstock can interfere with fermentation of the sugars to product. Fungal strain Coniochaeta ligniaria NRRL30616 metabolizes the furan aldehydes furfural and 5‐hydroxymethylfurfural, as well as a number of aromatic and aliphatic acids and aldehydes. Use of NRRL30616 to condition biomass sugars by metabolizing the inhibitors improves their fermentability. Wild‐type C. ligniaria has the ability to grow on xylose as sole source of carbon and energy, with no accumulation of xylitol. Mutants of C. ligniaria unable to grow on xylose were constructed. Xylose reductase and xylitol dehydrogenase activities were reduced by approximately two thirds in mutant C8100. The mutant retained ability to metabolize inhibitors in biomass hydrolysates. Although C. ligniaria C8100 did not grow on xylose, the strain converted a portion of xylose to xylitol, producing 0.59 g xylitol/g xylose in rich medium and 0.48 g xylitol/g xylose in corn stover dilute acid hydrolysate. 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016 © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:606–612, 2016     

Conversion of corn fiber to ethanol by recombinant E. coli strain FBR3

Escherichia coli strain FBR3 that is an efficient biocatalyst for converting mixed sugar streams (eg, arabinose, glucose, and xylose) into ethanol. In this report, the strain was tested for conversion of corn fiber hydrolysates into ethanol. Corn fiber hydrolysates with total sugar concentrations of 7.5% (w/v) were prepared by reacting corn fiber with dilute sulfuric acid at 145°C. Initial fermentations of the hydrolysate by strain FBR3 had lag times of approximately 30 h judged by ethanol production. Further experiments indicated that the acetate present in the hydrolysate could not solely account for the long lag. The lag phase was greatly reduced by growing the pre-seed and seed cultures on corn fiber hydrolysate. Ethanol yields for the optimized fermentations were 90% of theoretical. Maximum ethanol concentrations were 2.80% w/v, and the fermentations were completed in approximately 50 h. The optimal pH for the fermentation was 6.5. Below this pH, sugar consumption was incomplete and above it, excess base addition was required throughout the fermentation. Two alternative neutralization methods (overliming and overliming with sulfite addition) have been reported for improving the fermentability of lignocellulosic hydrolysates. These methods further reduced the lag phase of the fermentation, albeit by a minor amount. Received 29 September 1998/ Accepted in revised form 20 February 1999     

Fungal metabolism of fermentation inhibitors present in corn stover dilute acid hydrolysate

Use of agricultural residues for ethanol production requires pretreatment of the material to facilitate release of sugars. Physical–chemical pretreatment of lignocellulosic biomass can, however, give rise to side-products that may be toxic to fermenting microorganisms and hinder utilization of sugars obtained from biomass. Potentially problematic compounds include furan aldehydes formed by degradation of sugars, organic acids released from hemicellulose side-groups, and aldehydes and phenolics released from lignin. A fungal isolate, Coniochaeta ligniaria NRRL30616, metabolizes furfural and 5-hydroxymethylfurfural (HMF) as well as aromatic and aliphatic acids and aldehydes. NRRL30616 grew in corn stover dilute-acid hydrolysate, and converted furfural to both furfuryl alcohol and furoic acid. Hydrolysate was inoculated with NRRL30616, and the fate of pretreatment side-products was followed in a time-course study. A number of aromatic and aliphatic acids, aldehydes, and phenolic compounds were quantitated by analytical extraction of corn stover hydrolysate, followed by HPLC–UV–MS/MS analysis. Compounds representing all of the classes of inhibitory side-products were removed during the course of fungal growth. Biological abatement of hydrolysates using C. ligniaria improved xylose utilization in subsequent ethanol fermentations.     

Preparation of hydrolysates from tobacco stalks and ethanolic fermentation by Saccharomyces cerevisiae

Chipped tobacco stalks were subjected to steam pretreatment at 205 °C for either 5 or 10 min before enzymatic hydrolysis. Glucose (15.4–17.1 g/l) and xylose (4.5–5.0 g/l) were the most abundant monosaccharides in the hydrolysates. Mannose, galactose and arabinose were also detected. The hydrolysate produced by pretreatment for 10 min contained higher levels of all sugars than the 5 min-pretreated hydrolysate. The amounts of inhibitory compounds found in the hydrolysates were relatively low and increased with increasing pretreatment time. The hydrolysates were fermented with baker's yeast. Ethanol yield, maximum volumetric productivity and specific productivity were used as criteria of fermentability of the hydrolysates. The fermentation of the hydrolysates was only slightly inhibited compared to reference solutions having a similar composition of fermentable sugars. The ethanol yield in the hydrolysates was 0.38–0.39 g/g of initial fermentable sugars, whereas it was 0.42 g/g in the reference. The biomass yield was twofold lower in the hydrolysates than in the reference. The fermentation inhibition caused by the tobacco stalk hydrolysates was less than that caused by sugarcane bagasse hydrolysates obtained under the same hydrolysis conditions.     

Effect of sulfur oxyanions on lignocellulose-derived fermentation inhibitors

Recent results show that treatments with reducing agents, including the sulfur oxyanions dithionite and hydrogen sulfite, efficiently improve the fermentability of inhibitory lignocellulose hydrolysates, and that the treatments are effective when the reducing agents are added in situ into the fermentation vessel at low temperature. In the present investigation, dithionite was added to medium with model inhibitors (coniferyl aldehyde, furfural, 5-hydroxymethylfurfural, or acetic acid) and the effects on the fermentability with yeast were studied. Addition of 10 mM dithionite to medium containing 2.5 mM coniferyl aldehyde resulted in a nine-fold increase in the glucose consumption rate and a three-fold increase in the ethanol yield. To investigate the mechanism behind the positive effects of adding sulfur oxyanions, mixtures containing 2.5 mM of a model inhibitor (an aromatic compound, a furan aldehyde, or an aliphatic acid) and 15 mM dithionite or hydrogen sulfite were analyzed using mass spectrometry (MS). The results of the analyses, which were performed by using UHPLC-ESI-TOF-MS and UHPLC-LTQ/Orbitrap-MS/MS, indicate that the positive effects of sulfur oxyanions are primarily due to their capability to react with and sulfonate inhibitory aromatic compounds and furan aldehydes at low temperature and slightly acidic pH (such as 25°C and pH 5.5).     

Detoxification of acidic catalyzed hydrolysate of Kappaphycus alvarezii (cottonii)

Red seaweed, Kappaphycus alvarezii, holds great promise for use in biofuel production due to its high carbohydrate content. In this study, we investigated the effect of fermentation inhibitors to the K. alvarezii hydrolysate on cell growth and ethanol fermentation. In addition, detoxification of fermentation inhibitors was performed to decrease the fermentation inhibitory effect. 5-Hydroxymethylfurfural and levulinic acid, which are liberated from acidic hydrolysis, was also observed in the hydrolysate of K. alvarezii. These compounds inhibited ethanol fermentation. In order to remove these inhibitors, activated charcoal and calcium hydroxide were introduced. The efficiency of activated charcoals was examined and over-liming was used to remove the inhibitors. Activated charcoal was found to be more effective than calcium hydroxide to remove the inhibitors. Detoxification by activated charcoal strongly improved the fermentability of dilute acid hydrolysate in the production of bioethanol from K. alvarezii with Saccharomyces cerevisiae. The optimal detoxifying conditions were found to be below an activated charcoal concentration of 5%.     

Biotransformation of furfural and 5-hydroxymethyl furfural (HMF) by Clostridium acetobutylicum ATCC 824 during butanol fermentation

The ability of fermenting microorganisms to tolerate furan aldehyde inhibitors (furfural and 5-hydroxymethyl furfural (HMF)) will enhance efficient bioconversion of lignocellulosic biomass hydrolysates to fuels and chemicals. The effect of furfural and HMF on butanol production by Clostridium acetobutylicum 824 was investigated. Whereas specific growth rates, μ, of C. acetobutylicum in the presence of furfural and HMF were in the range of 15-85% and 23-78%, respectively, of the uninhibited Control, μ increased by 8-15% and 23-38% following exhaustion of furfural and HMF in the bioreactor. Using high performance liquid chromatography and spectrophotometric assays, batch fermentations revealed that furfural and HMF were converted to furfuryl alcohol and 2,5-bis-hydroxymethylfuran, respectively, with specific conversion rates of 2.13g furfural and 0.50g HMF per g (biomass) per hour, by exponentially growing C. acetobutylicum. Biotransformation of these furans to lesser inhibitory compounds by C. acetobutylicum will probably enhance overall fermentation of lignocellulosic hydrolysates to butanol.     

Inhibition of <Emphasis Type="Italic">Pichia stipitis</Emphasis> fermentation of hydrolysates from olive tree cuttings

The ethanolic fermentation of liquid fractions (hydrolysates) issued from dilute acid pre-treatment of olive tree biomass by Pichia stipitis is reported for the first time. On the one side, P. stipitis has been reported as the most promising naturally occurring C5 fermenting microorganism; on the other side, olive tree biomass is a renewable, low cost, and lacking of alternatives agricultural residue especially abundant in Mediterranean countries. The study was performed in two steps. First, the fermentation performance of P. stipitis was evaluated on a fermentation medium also containing the main inhibitors found in these hydrolysates (acetic acid, formic acid, and furfural), as well as glucose and xylose as carbon sources. The effect of inhibitors, individually or in a mixture, on kinetic and yield parameters was calculated. In a second step, hydrolysates obtained from 1% (w/w) sulfuric acid pre-treatment of olive tree biomass at 190°C for 10 min were used as a real fermentation medium with the same microorganism. Due to inhibition, effective fermentation required dilution of the hydrolysate and either overliming or activated charcoal treatment. Results show that ethanol yields obtained from hydrolysates, ranging from 0.35 to 0.42 g/g, are similar to those from synthetic medium, although the process proceeds at lower rates. Inhibiting compounds affect the fermentation performance in a synergistic way. Furfural is rapidly assimilated by the yeast; acetic acid and formic acid concentrations decrease slowly during the process. Activated charcoal or overliming detoxification improve the fermentability of diluted hydrolysates.