Detoxification of sugarcane bagasse hydrolysate improves ethanol production by Candida shehatae NCIM 3501

Sugarcane bagasse hydrolysis with 2.5% (v/v) HCl yielded 30.29g/L total reducing sugars along with various fermentation inhibitors such as furans, phenolics and acetic acid. The acid hydrolysate when treated with anion exchange resin brought about maximum reduction in furans (63.4%) and total phenolics (75.8%). Treatment of hydrolysate with activated charcoal caused 38.7% and 57.5% reduction in furans and total phenolics, respectively. Laccase reduced total phenolics (77.5%) without affecting furans and acetic acid content in the hydrolysate. Fermentation of these hydrolysates with Candida shehatae NCIM 3501 showed maximum ethanol yield (0.48g/g) from ion exchange treated hydrolysate, followed by activated charcoal (0.42g/g), laccase (0.37g/g), overliming (0.30g/g) and neutralized hydrolysate (0.22g/g).     

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.     

Bioconversion of water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate to motor fuel ethanol by xylose-fermenting yeast

Water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate has been utilized as a substrate for ethanol production using Pichia stipitis NRRL Y-7124. Hydrolysate fermentability was considerable improved by boiling, and overliming up to pH 10.0 with solid Ca(OH)(2) in combination with sodium sulfite. The percent total sugar utilized and ethanol yield (Y(p/s)) for the untreated hydrolysate were 20.15+/-0.17% and 0.19+/-0.003 g(p) g(s)(-1), respectively, compared with 76.0+/-0.32% and 0.35 g(p) g(s)(-1), respectively for the treated material. The fermentation was very effective at an aeration rate of 0.02 v/v/m, temperature 30+/-0.2 degrees C and pH 6.0+/-0.2. However, the volumetric productivity (Q(p)) was still considerably less than observed in a simulated synthetic hydrolysate medium with a sugar composition similar to the hemicellulose acid hydrolysate. L-Arabinose was not fermented but assimilated. The presence of acetic acid in the hydrolysate decreased the ethanol yield and productivity considerably.     

Strain improvement of thermotolerant Saccharomyces cerevisiae VS strain for better utilization of lignocellulosic substrates

AIM: Pentose-utilizing yeast development by protoplast fusion and sequential mutations and ethanol fermentation using lignocellulosic substrate. METHODS AND RESULTS: Protoplasts of thermotolerant Saccharomyces cerevisiae and mesophilic, xylose-utilizing Candida shehatae were fused by electrofusion. The fusants were selected based on their growth at 42 degrees C and ability to utilize xylose. The selected best fusant was mutated sequentially and 3 mutant fusants obtained were tested for their stability. The mutant fusant CP11 was found to be stable and used for lignocellulosic fermentation. The Prosopis juliflora wood material was hydrolysed with 1% sulphuric acid initially for 18 h at room temperature and then for 20 min at 121 degrees C. The acid hydrolysate was separated and used for detoxification by ethyl acetate and overliming. The hard cellulosic fraction was hydrolysed with Aspergillus niger crude cellulase enzyme for 18 h at 50 degrees C. The substrate (15% w/v) yielded 84 g l(-1) sugars, representing 80% (w/w) hydrolysis of carbohydrate content present in the lignocellulosic material. The acid and enzyme hydrolysates were then equally mixed and used for fermentation with the developed fusant yeast (CP11). The fusant yeast gave an ethanol yield of 0.459 +/- 0.012 g g(-1), productivity of 0.67 +/- 0.015 g l(-1) h(-1) and fermentation efficiency of 90%. CONCLUSIONS: Protoplast fusion followed by sequential mutations method gave a stable and good performing fusant with maximum utilization of reducing sugars in the media. SIGNIFICANCE AND IMPACT OF THE STUDY: This new method could be applied to develop fusants for better biotechnological applications.     

Lantana camara for fuel ethanol production using thermotolerant yeast

AIM: Evaluation of Lantana camara's use as feedstock for fuel ethanol production. METHODS AND RESULTS: Lantana camara plant material was hydrolysed with 1% sulfuric acid for 18 h at room temperature, followed by heat treatment of 121 degrees C for 20 min. Hemicellulosic hydrolyzate was separated and used for detoxification by ethyl acetate and overliming. Cellulosic fraction was hydrolysed with Aspergillus niger crude cellulase enzyme for 18 h at 55 degrees C. Using 15% (dw/v) substrate 73 g l(-1) total reducing sugars were obtained to give 78.7% hydrolysis of carbohydrate content. Acid and enzyme hydrolyzates were mixed equally and used for fermentation with thermotolerant Saccharomyces cerevisiae (VS(3)). Yeast fermented L. camara hydrolyzate well with a fermentation efficiency of 83.7% to give an ethanol yield of 0.431 +/- 0.018 g ethanol pre g sugar and productivity of 0.5 +/- 0.021 g l(-1) h(-1). CONCLUSIONS: Even though inhibitors were present in L. camara hydrolyzate, maximum sugars were utilized by thermotolerant yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of L. camara for fuel ethanol production with improved strains and detoxification can be recommended.     

Ethanol production from wheat straw hemicellulose hydrolysate by Pichia stipitis

Ethanol production was evaluated from wheat straw (WS) hemicellulose acid hydrolysate using an adapted and parent strain of Pichia stipitis. NRRL Y-7124. The treatment by boiling and overliming with Ca(OH)(2) significantly improved the fermentability of the hydrolysate. Ethanol yield (Yp/s) and productivity (Qp av) were increased 2.4+/-0.10 and 5.7+/-0.24 folds, respectively, compared to neutralized hydrolysate. Adaptation of the yeast to the hydrolysate resulted further improvement in yield and productivity. The maximum yield was 0.41+/-0.01 g(p) g(s)(-1), equivalent to 80.4+/-0.55% theoretical conversion efficiency. Acetic acid, furfurals and lignins present in the hydrolysate were inhibitory to microbial growth and ethanol production. The addition of these inhibitory components individually or in various combinations at a concentrations similar to that found in hydrolysate to simulated medium resulted a reduction in ethanol yield (Yp/s) and productivity (Qp av). The hydrolysate used had the following composition (expressed in g x l(-1)): xylose 12.8+/-0.25; glucose 1.7+/-0.3; arabinose 2.6+/-0.21 and acetic acid 2.7+/-0.33.     

Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass

An overview of the different inhibitors formed by pre-treatment of lignocellulosic materials and their inhibition of ethanol production in yeast and bacteria is given. Different high temperature physical pre-treatment methods are available to render the carbohydrates in lignocellulose accessible for ethanol fermentation. The resulting hydrolyzsates contain substances inhibitory to fermentation—depending on both the raw material (biomass) and the pre-treatment applied. An overview of the inhibitory effect on ethanol production by yeast and bacteria is presented. Apart from furans formed by sugar degradation, phenol monomers from lignin degradation are important co-factors in hydrolysate inhibition, and inhibitory effects of these aromatic compounds on different ethanol producing microorganisms is reviewed. The furans and phenols generally inhibited growth and ethanol production rate (Q_EtOH) but not the ethanol yields (Y_EtOH) in Saccharomyces cerevisiae. Within the same phenol functional group (aldehyde, ketone, and acid) the inhibition of volumetric ethanol productivity was found to depend on the amount of methoxyl substituents and hence hydrophobicity (log P). Many pentose-utilizing strains Escherichia coli, Pichia stipititis, and Zymomonas mobilis produce ethanol in concentrated hemicellulose liquors but detoxification by overliming is needed. Thermoanaerobacter mathranii A3M3 can grow on pentoses and produce ethanol in hydrolysate without any need for detoxification.     

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.     

Soluble and insoluble solids contributions to high-solids enzymatic hydrolysis of lignocellulose

The rates and extents of enzymatic cellulose hydrolysis of dilute acid pretreated corn stover (PCS) decline with increasing slurry concentration. However, mass transfer limitations are not apparent until insoluble solids concentrations approach 20% w/w, indicating that inhibition of enzyme hydrolysis at lower solids concentrations is primarily due to soluble components. Consequently, the inhibitory effects of pH-adjusted pretreatment liquor on the enzymatic hydrolysis of PCS were investigated. A response surface methodology (RSM) was applied to empirically model how hydrolysis performance varied as a function of enzyme loading (12-40mg protein/g cellulose) and insoluble solids concentration (5-13%) in full-slurry hydrolyzates. Factorial design and analysis of variance (ANOVA) were also used to assess the contribution of the major classes of soluble components (acetic acid, phenolics, furans, sugars) to total inhibition. High sugar concentrations (130g/L total initial background sugars) were shown to be the primary cause of performance inhibition, with acetic acid (15g/L) only slightly inhibiting enzymatic hydrolysis and phenolic compounds (9g/L total including vanillin, syringaldehyde, and 4-hydroxycinnamic acid) and furans (8g/L total of furfural and hydroxymethylfurfural, HMF) with only a minor effect on reaction kinetics. It was also demonstrated that this enzyme inhibition in high-solids PCS slurries can be approximated using a synthetic hydrolyzate composed of pure sugars supplemented with a mixture of acetic acid, furans, and phenolic compounds, which indicates that generally all of the reaction rate-determining soluble compounds for this system can be approximated synthetically.     

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.     

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     

Overcoming inhibitors in a hemicellulosic hydrolysate: improving fermentability by feedstock detoxification and adaptation of <Emphasis Type="Italic">Pichia stipitis</Emphasis>

In order to improve the fermentative efficiency of sugar maple hemicellulosic hydrolysates for fuel ethanol production, various methods to mitigate the effects of inhibitory compounds were employed. These methods included detoxification treatments utilizing activated charcoal, anion exchange resin, overliming, and ethyl acetate extraction. Results demonstrated the greatest fermentative improvement of 50% wood hydrolysate (v/v) by Pichia stipitis with activated charcoal treatment. Another method employed to reduce inhibition was an adaptation procedure to produce P. stipitis stains more tolerant of inhibitory compounds. This adaptation resulted in yeast variants capable of improved fermentation of 75% untreated wood hydrolysate (v/v), one of which produced 9.8 g/l ± 0.6 ethanol, whereas the parent strain produced 0.0 g/l ± 0.0 within the first 24 h. Adapted strains RS01, RS02, and RS03 were analyzed for glucose and xylose utilization and results demonstrated increased glucose and decreased xylose utilization rates in comparison to the wild type. These changes in carbohydrate utilization may be indicative of detoxification or tolerance activities related to proteins involved in glucose and xylose metabolism.     

Laccase detoxification of steam-exploded wheat straw for second generation bioethanol

In this work we compared the efficiency of a laccase treatment performed on steam-exploded wheat straw pretreated under soft conditions (water impregnation) or harsh conditions (impregnation with diluted acid). The effect of several enzymatic treatment parameters (pH, time of incubation, laccase origin and loading) was analysed. The results obtained indicated that severity conditions applied during steam explosion have an influence on the efficiency of detoxification. A reduction of the toxic effect of phenolic compounds by laccase polymerization of free phenols was demonstrated. Laccase treatment of steam-exploded wheat straw reduced sugar recovery after enzymatic hydrolysis, and it should be better performed after hydrolysis with cellulases. The fermentability of hydrolysates was greatly improved by the laccase treatment in all the samples. Our results demonstrate the action of phenolic compounds as fermentation inhibitors, and the advantages of a laccase treatment to increase the ethanol production from steam-exploded wheat straw.     

Production of biobutanol from acid-pretreated corncob using Clostridium beijerinckii TISTR 1461: Process optimization studies

Corncob is a potential feedstock in Thailand that can be used for fermentable sugar production through dilute sulfuric acid pretreatment and enzymatic hydrolysis. To recover high amounts of monomeric sugars from corncob, the sulfuric pretreatment conditions were optimized by using response surface methodology with three independent variables: sulfuric acid concentration, temperature, and time. The highest response of total sugars, 48.84 g/L, was found at 122.78°C, 4.65 min, and 2.82% (v/v) H₂SO₄. With these conditions, total sugars from the confirmation experiment were 46.29 g/L, with 5.51% error from the predicted value. The hydrolysate was used as a substrate for acetone–butanol–ethanol fermentation to evaluate its potential for microbial growth. The simultaneous saccharification and fermentation (SSF) showed that C. beijerinckii TISTR 1461 can generate acetone–butanol–ethanol products at 11.64 g/L (5.29 g/L acetone, 6.26 g/L butanol, and 0.09 g/L ethanol) instantly using sugars from the hydrolysed corncob with Novozymes 50013 cellulase enzyme without an overliming process.     

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.     

发酵性丝孢酵母HWZ004利用水稻秸秆水解液发酵产油脂

为高效利用水稻秸秆中的纤维素和半纤维素产油脂,采用稀酸预处理和酶水解两步法对水稻秸秆进行水解,然后以水解液为碳源,培养发酵性丝孢酵母Trichosporon fermentans HWZ004产微生物油脂。结果表明,经简单overliming法脱毒后水稻秸秆水解液中乙酸、糠醛和5-羟甲基糠醛的浓度分别为0.4 g/L、0.1 g/L和0.05 g/L。只需添加少量氮源和微量CuSO4?5H2O,该水解液即可满足T. fermentans HWZ004发酵产油脂的要求。发酵最适接种量、初始pH和温度分别是5.0％、7.0和25 ℃。T. fermentans HWZ004在优化条件下培养7 d的生物量、油脂含量和油脂产量分别是26.4 g/L,52.2％和13.8 g/L;油脂得率系数为17.0,大大高于驯化前菌株T. fermentans CICC 1368在脱毒水稻秸秆半纤维素水解液中的对应值 (11.9)。所产油脂的脂肪酸组成与植物油相似,不饱和脂肪酸含量达70％以上,宜作为生物柴油的生产原料。     

Ethanolic fermentation of hydrolysates from ammonia fiber expansion (AFEX) treated corn stover and distillers grain without detoxification and external nutrient supplementation

External nutrient supplementation and detoxification of hydrolysate significantly increase the production cost of cellulosic ethanol. In this study, we investigated the feasibility of fermenting cellulosic hydrolysates without washing, detoxification or external nutrient supplementation using ethanologens Escherichia coli KO11 and the adapted strain ML01 at low initial cell density (16 mg dry weight/L). The cellulosic hydrolysates were derived from enzymatically digested ammonia fiber expansion (AFEX)-treated corn stover and dry distiller's grain and solubles (DDGS) at high solids loading (18% by weight). The adaptation was achieved through selective evolution of KO11 on hydrolysate from AFEX-treated corn stover. All cellulosic hydrolysates tested (36-52 g/L glucose) were fermentable. Regardless of strains, metabolic ethanol yields were near the theoretical limit (0.51 g ethanol/g consumed sugar). Volumetric ethanol productivity of 1.2 g/h/L was achieved in fermentation on DDGS hydrolysate and DDGS improved the fermentability of hydrolysate from corn stover. However, enzymatic hydrolysis and xylose utilization during fermentation were the bottlenecks for ethanol production from corn stover at these experimental conditions. In conclusion, fermentation under the baseline conditions was feasible. Utilization of nutrient-rich feedstocks such as DDGS in fermentation can replace expensive media supplementation.     

Dilute acid pretreatment, enzymatic saccharification, and fermentation of rice hulls to ethanol

Rice hulls, a complex lignocellulosic material with high lignin (15.38 +/- 0.2%) and ash (18.71 +/- 0.01%) content, contain 35.62 +/- 0.12% cellulose and 11.96 +/- 0.73% hemicellulose and has the potential to serve as a low-cost feedstock for production of ethanol. Dilute H2SO4 pretreatments at varied temperature (120-190 degrees C) and enzymatic saccharification (45 degrees C, pH 5.0) were evaluated for conversion of rice hull cellulose and hemicellulose to monomeric sugars. The maximum yield of monomeric sugars from rice hulls (15%, w/v) by dilute H2SO4 (1.0%, v/v) pretreatment and enzymatic saccharification (45 degrees C, pH 5.0, 72 h) using cellulase, beta-glucosidase, xylanase, esterase, and Tween 20 was 287 +/- 3 mg/g (60% yield based on total carbohydrate content). Under this condition, no furfural and hydroxymethyl furfural were produced. The yield of ethanol per L by the mixed sugar utilizing recombinant Escherichia colistrain FBR 5 from rice hull hydrolyzate containing 43.6 +/- 3.0 g fermentable sugars (glucose, 18.2 +/- 1.4 g; xylose, 21.4 +/- 1.1 g; arabinose, 2.4 +/- 0.3 g; galactose, 1.6 +/- 0.2 g) was 18.7 +/- 0.6 g (0.43 +/- 0.02 g/g sugars obtained; 0.13 +/- 0.01 g/g rice hulls) at pH 6.5 and 35 degrees C. Detoxification of the acid- and enzyme-treated rice hull hydrolyzate by overliming (pH 10.5, 90 degrees C, 30 min) reduced the time required for maximum ethanol production (17 +/- 0.2 g from 42.0 +/- 0.7 g sugars per L) by the E. coli strain from 64 to 39 h in the case of separate hydrolysis and fermentation and increased the maximum ethanol yield (per L) from 7.1 +/- 2.3 g in 140 h to 9.1 +/- 0.7 g in 112 h in the case of simultaneous saccharification and fermentation.     

Detoxification requirements for bioconversion of softwood dilute acid hydrolyzates to succinic acid

In this work an Escherichia coli metabolically engineered to ferment lignocellulosic biomass sugars to succinic acid was tested for growth and fermentation of detoxified softwood dilute sulfuric acid hydrolyzates, and the minimum detoxification requirements were investigated with activated carbon and/or overliming treatments. Detoxified hydrolyzates supported fast growth and complete fermentation of all hydrolyzate sugars to succinate at yields comparable to pure sugar, while untreated hydrolyzates were unable to support either growth or fermentation. Activated carbon treatment was able to remove significantly more HMF and phenolics than overliming. However, in some cases, overliming treatment was capable of generating a fermentable hydrolyzate where activated carbon treatment was not. The implications of this are that in addition to the known organic inhibitors, the changes in the inorganic content and/or composition due to overliming are significant to the hydrolyzate toxicity. It was also found that any HMF remaining after detoxification was completely metabolized during aerobic cell growth on the hydrolyzates that were capable of supporting growth.     

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.