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.     

Butanol production from sweet sorghum bagasse with high solids content: Part I—comparison of liquid hot water pretreatment with dilute sulfuric acid

In these studies, we pretreated sweet sorghum bagasse (SSB) using liquid hot water (LHW) or dilute H₂SO₄ (2 g L^?1) at 190°C for zero min (as soon as temperature reached 190°C, cooling was started) to reduce generation of sugar degradation fermentation inhibiting products such as furfural and hydroxymethyl furfural (HMF). The solids loading were 250–300 g L^?1. This was followed by enzymatic hydrolysis. After hydrolysis, 89.0 g L^?1 sugars, 7.60 g L^?1 acetic acid, 0.33 g L^?1 furfural, and 0.07 g L^?1 HMF were released. This pretreatment and hydrolysis resulted in the release of 57.9% sugars. This was followed by second hydrolysis of the fibrous biomass which resulted in the release of 43.64 g L^?1 additional sugars, 2.40 g L^?1 acetic acid, zero g L^?1 furfural, and zero g L^?1 HMF. In both the hydrolyzates, 86.3% sugars present in SSB were released. Fermentation of the hydrolyzate I resulted in poor acetone‐butanol‐ethanol (ABE) fermentation. However, fermentation of the hydrolyzate II was successful and produced 13.43 g L^?1 ABE of which butanol was the main product. Use of 2 g L^?1 H₂SO₄ as a pretreatment medium followed by enzymatic hydrolysis resulted in the release of 100.6–93.8% (w/w) sugars from 250 to 300 g L^?1 SSB, respectively. LHW or dilute H₂SO₄ were used to economize production of cellulosic sugars from SSB. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:960–966, 2018     

Detoxification of dilute acid hydrolysates of lignocellulose with lime

The hydrolysis of hemicellulose to monomeric sugars by dilute acid hydrolysis is accompanied by the production of inhibitors that retard microbial fermentation. Treatment of hot hydrolysate with Ca(OH)(2) (overliming) is an effective method for detoxification. Using ethanologenic Escherichia coli LY01 as the biocatalyst, our results indicate that the optimal lime addition for detoxification varies and depends on the concentration of mineral acids and organic acids in each hydrolysate. This optimum was shown to be readily predicted on the basis of the titration of hydrolysate with 2 N NaOH at ambient temperature to either pH 7.0 or pH 11.0. The average composition of 15 hydrolysates prior to treatment was as follows (per L): 95.24 +/- 7.29 g sugar, 5.3 +/- 2.99 g acetic acid, 1.305 +/- 0.288 g total furans (furfural and hydroxymethylfurfural), and 2.86 +/- 0.34 g phenolic compounds. Optimal overliming resulted in a 51 +/- 9% reduction of total furans, a 41 +/- 6% reduction in phenolic compounds, and a 8.7 +/- 4.5% decline in sugar. Acetic acid levels were unchanged. Considering the similarity of microorganisms, it is possible that the titration method described here may also prove useful for detoxification and fermentation processes using other microbial biocatalysts.     

Kinetic study of detoxification of dilute-acid hydrolyzates by Ca(OH)2

Detoxification of dilute-acid hydrolyzates by addition of Ca(OH)(2) (overliming) and cultivation of the detoxified hydrolyzates by Saccharomyces cerevisiae were examined. The examined overliming involves increasing the pH of the hydrolyzates to 9, 10, 11 or 12, keeping up to 90 min at different temperatures of 30, 45 and 60 degrees C, followed by readjustment of the pH to 5. Increasing the pH, time and/or temperature resulted in more effective degradation of furans and resulted in better fermentability for both of the tested hydrolyzates, but higher loss of the sugars was observed as well. Overliming of glucose and furfural solution at pH 12 showed a rapid decrease in concentration of these chemicals followed by a slow degradation process. Therefore, a kinetic model was proposed for the detoxification, where the sugars or furans make transient complexes with calcium ions and this complex will then be converted to the degradation product. The ANOVA analysis of the model resulted in an average R(2) of 0.99 for the model fitted to all the experimental data points.     

Detoxification of a lignocellulosic biomass slurry by soluble polyelectrolyte adsorption for improved fermentation efficiency

This study investigated the detoxification of a dilute acid pretreated Ponderosa pine slurry using the polyelectrolyte polyethyleneimine (PEI). The addition of polyelectrolyte to remove enzymatic and/or fermentation inhibitory compounds, that is, acetic acid, furfural, and 5-hydroxymethylfurfural (HMF), was performed either before or after enzymatic hydrolysis to determine the optimal process sequence. Negligible acetic acid, glucose, and xylose were removed regardless of where in the process the polymer addition was made. Maximum furfural and HMF separation was achieved with the addition of PEI to a clarified pre-enzymatic hydrolysis liquor, which showed that 88.3% of furfural and 66.4% of HMF could be removed. On the other hand, only 23.1% and 13.4% of furfural and HMF, respectively, were removed from a post-enzymatic hydrolysis sample; thus, the effects of enzymes, glucose, and wood solids on inhibitor removal were also investigated. The presence of solid particles >0.2 μm and unknown soluble components <10 kDa reduced inhibitory compound removal, but the presence of elevated glucose levels and enzymes (cellulases) did not affect the separation. The fermentability of detoxified versus undetoxified hydrolysate was also investigated. An ethanol yield of 92.6% of theoretical was achieved with Saccharomyces cerevisiae fermenting the detoxified hydrolyzate, while no significant ethanol was produced in the undetoxified hydrolyzate. These results indicate that PEI may provide a practical alternative for furan removal and detoxification of lignocellolosic hydrolysates, and that application before enzymatic hydrolysis minimizes separation interferences.     

High solid fed‐batch butanol fermentation with simultaneous product recovery: Part II—process integration

In these studies, liquid hot water (LHW) pretreated and enzymatically hydrolyzed Sweet Sorghum Bagasse (SSB) hydrolyzates were fermented in a fed‐batch reactor. As reported in the preceding paper, the culture was not able to ferment the hydrolyzate I in a batch process due to presence of high level of toxic chemicals, in particular acetic acid released from SSB during the hydrolytic process. To be able to ferment the hydrolyzate I obtained from 250 g L^?1 SSB hydrolysis, a fed‐batch reactor with in situ butanol recovery was devised. The process was started with the hydrolyzate II and when good cell growth and vigorous fermentation were observed, the hydrolyzate I was slowly fed to the reactor. In this manner the culture was able to ferment all the sugars present in both the hydrolyzates to acetone butanol ethanol (ABE). In a control batch reactor in which ABE was produced from glucose, ABE productivity and yield of 0.42 g L^?1 h^?1 and 0.36 were obtained, respectively. In the fed‐batch reactor fed with SSB hydrolyzates, these productivity and yield values were 0.44 g L^?1 h^?1 and 0.45, respectively. ABE yield in the integrated system was high due to utilization of acetic acid to convert to ABE. In summary we were able to utilize both the hydrolyzates obtained from LHW pretreated and enzymatically hydrolyzed SSB (250 g L^?1) and convert them to ABE. Complete fermentation was possible due to simultaneous recovery of ABE by vacuum. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:967–972, 2018     

Wood hydrolyzate treatments for improved fermentation of wood sugars to 2,3-butanediol

Acid-hydrolyzed hardwood contains compounds inhibitory to microorganisms that convert wood sugars to fermentation products such as fuels and chemicals. Several methods of treating acid-hydrolyzed hardwood (hydrolyzate) to reduce the levels of potential microbial inhibitors (acetate, furfural, sulfate, and phenolics) were evaluated. The methods evaluated were precipitation with calcium hydroxide, extraction with organic solvents, treatment with ion-exchange resins, adsorption resins, and activated charcoal. Treatment of the hydrolyzate with an anion exchange resin (Amberlite IRA-400) was the most effective method for removing potential inhibitors. Non-treated hydrolyzate adjusted to pH 6 inhibited growth of a 2,3-butanediol-producing culture of Klebsiella pneumoniae. However, hydrolyzate treated with Amberlite IRA-400 was not inhibitory and resulted in yields of 2,3-butanediol that were greater than 90% of theoretical.     

On-line control of fed-batch fermentation of dilute-acid hydrolyzates

Dilute-acid hydrolyzates from lignocellulose are, to a varying degree, inhibitory to yeast. In the present work, dilute-acid hydrolyzates from spruce, birch, and forest residue, as well as synthetic model media, were fermented by Saccharomyces cerevisiae in fed-batch cultures. A control strategy based on on-line measurement of carbon dioxide evolution (CER) was used to control the substrate feed rate in a lab scale bioreactor. The control strategy was based solely on the ratio between the relative increase in CER and the relative increase in feed rate. Severely inhibiting hydrolyzates could be fermented without detoxification and the time required for fermentation of moderately inhibiting hydrolyzates was also reduced. The feed rate approached a limiting value for inhibiting media, with a corresponding pseudo steady-state value for CER. However, a slow decrease of CER with time was found for media containing high amounts of 5-hydroxymethyl furfural (HMF). The success of the control strategy is explained by the conversion of furfural and HMF by the yeast during fed-batch operation. The hydrolyzates contained between 1.4 and 5 g/l of furfural and between 2.4 and 6.5 g/l of HMF. A high conversion of furfural was obtained (between 65-95%) at the end of the feeding phase, but the conversion of HMF was considerably lower (between 12-40%).     

Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol

Wheat straw consists of 48.57 ± 0.30% cellulose and 27.70 ± 0.12% hemicellulose on dry solid (DS) basis and has the potential to serve as a low cost feedstock for production of ethanol. Dilute acid pretreatment at varied temperature and enzymatic saccharification were evaluated for conversion of wheat straw cellulose and hemicellulose to monomeric sugars. The maximum yield of monomeric sugars from wheat straw (7.83%, w/v, DS) by dilute H₂SO₄ (0.75%, v/v) pretreatment and enzymatic saccharification (45 °C, pH 5.0, 72 h) using cellulase, β-glucosidase, xylanase and esterase was 565 ± 10 mg/g. Under this condition, no measurable quantities of furfural and hydroxymethyl furfural were produced. The yield of ethanol (per litre) from acid pretreated enzyme saccharified wheat straw (78.3 g) hydrolyzate by recombinant Escherichia coli strain FBR5 was 19 ± 1 g with a yield of 0.24 g/g DS. Detoxification of the acid and enzyme treated wheat straw hydrolyzate by overliming reduced the fermentation time from 118 to 39 h in the case of separate hydrolysis and fermentation (35 °C, pH 6.5), and increased the ethanol yield from 13 ± 2 to 17 ± 0 g/l and decreased the fermentation time from 136 to 112 h in the case of simultaneous saccharification and fermentation (35 °C, pH 6.0).     

On-line estimation of sugar concentration for control of fed-batch fermentation of lignocellulosic hydrolyzates by Saccharomyces cerevisiae

A feed control strategy, based on estimated sugar concentrations, was developed with the purpose of avoiding severe inhibition of the yeast Saccharomyces cerevisiae during fermentation of spruce hydrolyzate. The sum of the fermentable hexose sugars, glucose and mannose, was estimated from on-line measurements of carbon dioxide evolution rate and biomass concentration by use of a simple stoichiometric model. The feed rate of the hydrolyzate was controlled to maintain constant sugar concentration during fed-batch fermentation, and the effect of different set-point concentrations was investigated using both untreated and detoxified hydrolyzates. The fed-batch cultivations were evaluated with respect to cellular physiology in terms of the specific ethanol productivities, ethanol yields, and viability of the yeast. The simple stoichiometric model used resulted in a good agreement between estimated sugar concentrations and off-line determinations of sugar concentrations. Furthermore, the control strategy used made it possible to maintain a constant sugar concentration without major oscillations in the feed rate or the sugar concentration. For untreated hydrolyzates the average ethanol productivity could be increased by more than 130% compared to batch fermentation. The average ethanol productivity was increased from 0.12 to 0.28 g/g h. The productivity also increased for detoxified hydrolyzates, where an increase of 16% was found (from 0.50 to 0.58 g/g h).     

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.     

Optimization of the pretreatment of rice straw hemicellulosic hydrolyzates for microbial production of xylitol

Hemicellulosic hydrolyzate obtained from rice straw was evaluated to determine if it was a suitable fementation medium for the production of xylitol byCandida mogii ATCC 18364. To obtain xylose selectively from rice straw, it is important to establish rapid hydrolysis conditions that yield xylose-rich substrates. The results of hydrolysis experiments indicated that the optimal reaction conditions for the recovery of xylose from rice straw hemicellulose were obtained using a sulfuric acid concentration of 1.5%, a reaction temperature of 130°C, a reaction time of 20 min and a solid to liquid ratio of 1∶10. Because the fermentation of concentrated acid hydrolyzates can be inhibited by compounds present in the raw material or produced during the hydrolysis process, various methods were tested to determine if they could detoxify the hydrolyzates and thus improve xylitol production. The greatest xylitol yield (0.53 g/g) and volumetric productivity (0.38 g/L·h) were obtained when an overlimed hydrolyzate was treated with activated charcoal.     

Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran

Renewable lignocellulosic materials are attractive low-cost feedstocks for bioethanol production. Furfural and 5-hydroxymethylfurfural (HMF) are among the most potent inhibitory compounds generated from acid hydrolysis of lignocelluloses to simple sugars for fermentation. In Saccharomyces cerevisiae ATCC 211239 and NRRL Y-12632 and Pichia stipitis NRRL Y-7124, furfural and HMF inhibition were determined to be dose-dependent at concentrations from 10 to 120 mM. The yeast strains were more sensitive to inhibition by furfural than HMF at the same concentration, while combined treatment of furfural and HMF synergistically suppressed cell growth. A metabolite transformed from HMF by strain NRRL Y-12632 was isolated from the culture supernatant, and conclusively identified as 2,5-bis-hydroxymethylfuran, a previously postulated HMF alcohol, with a composition of C₆H₈O₃ and a molecular weight of 128. It is proposed that, in the presence of HMF, the yeast reduces the aldehyde group on the furan ring of HMF into an alcohol, in a similar manner as for furfural. The accumulation of this biotransformed metabolite may be less toxic to yeast cultures than HMF, as evidenced by the rapid yeast fermentation and growth rates associated with HMF conversion. The ability of yeasts to adapt to and transform furfural and HMF offers the potential for in situ detoxification of these inhibitors and suggests a genetic basis for further development of highly tolerant strains for biofuel production.     

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.     

Potential of biotechnological conversion of lignocellulose hydrolyzates by Pseudomonas putida KT2440 as a model organism for a bio‐based economy

Lignocellulose‐derived hydrolyzates typically display a high degree of variation depending on applied biomass source material as well as process conditions. Consequently, this typically results in variable composition such as different sugar concentrations as well as degree and the presence of inhibitors formed during hydrolysis. These key obstacles commonly limit its efficient use as a carbon source for biotechnological conversion. The gram‐negative soil bacterium Pseudomonas putida KT2440 is a promising candidate for a future lignocellulose‐based biotechnology process due to its robustness and versatile metabolism. Recently, P. putida KT2440_xylAB which was able to metabolize the hemicellulose (HC) sugars, xylose and arabinose, was developed and characterized. Building on this, the intent of the study was to evaluate different lignocellulose hydrolyzates as platform substrates for P. putida KT2440 as a model organism for a bio‐based economy. Firstly, hydrolyzates of different origins were evaluated as potential carbon sources by cultivation experiments and determination of cell growth and sugar consumption. Secondly, the content of major toxic substances in cellulose and HC hydrolyzates was determined and their inhibitory effect on bacterial growth was characterized. Thirdly, fed‐batch bioreactor cultivations with hydrolyzate as the carbon source were characterized and a diauxic‐like growth behavior with regard to different sugars was revealed. In this context, a feeding strategy to overcome the diauxic‐like growth behavior preventing accumulation of sugars is proposed and presented. Results obtained in this study represent a first step and proof‐of‐concept toward establishing lignocellulose hydrolyzates as platform substrates for a bio‐based economy.     

Valorization of Hemicelluloses: Production of Bioxylitol from Poplar Wood Prehydrolyzates by <Emphasis Type="Italic">Candida guilliermondii</Emphasis> FTI 20037

In this study on the valorization of hemicelluloses (a co-product generated during cellulosic bioethanol production), prehydrolyzates obtained from poplar woodchips pretreated in an industrial experimental steam-explosion pilot-plant facility were evaluated for the production of bioxylitol using the yeast, Candida guilliermondii FTI 20037, employing both batch and fed-batch fermentation modes in shake flasks on defined nutrient medium. The prehydrolyzates consisted of monosaccharides (pentose and hexose sugars) as well as xylo-oligosaccharides and undegraded hemicellulose. Xylose (31.6?±?0.57 g/L) was the major sugar in the prehydrolyzates that also contained acetic acid and degradation products of lignin and sugars (phenolic and furanic compounds). Xylose in the prehydrolyzates could be further increased (106.4?±?0.02 g/L) through an acid hydrolysis step (0.6 % (w/v) H₂SO₄). Compounds of a toxic nature in both the acid hydrolyzates and prehydrolyzates were removed by treatment with Amberlite IRA-400 resin (chloride form). Batch fermentation of pure xylose and poplar prehydrolyzate resulted in bioxylitol production of 9.9?±?0.01 and 4.9?±?0.17 g/L, respectively, indicating that the poplar prehydrolyzates exhibited an inhibitory effect on fermentation. After detoxification of the poplar prehydrolyzates, bioxylitol production increased to 8.9?±?0.01 g/L. Fed-batch fermentation of the prehydrolyzate increased the bioxylitol production to 12.39?±?0.33 g/L, while acid hydrolysis followed by detoxification resulted in a maximum bioxylitol production of 22.0?±?0.01 g/L, a 348 % increase. The results demonstrated that acid hydrolysis and detoxification followed by fed-batch fermentation was an efficient way to produce bioxylitol from poplar prehydrolyzates.     

Bioethanol fermentation by recombinant E. coli FBR5 and its robust mutant FBHW using hot-water wood extract hydrolyzate as substrate

Hemicellulose is a potential by-product currently under-utilized in the papermaking industry. It is a hetero-carbohydrate polymer. For hardwood hemicelluloses, D-xylose is the major component upon depolymerization. At SUNY-ESF, wood extracts were obtained by extracting sugar maple wood chips with hot water at an elevated temperature. The wood extracts were then concentrated and acid hydrolyzed. Ethanologenic bacteria, E. coli FBR5, had a good performance in pure xylose medium for ethanol production. However, FBR5 was strongly inhibited in dilute sulfuric acid hydrolyzate of hot-water wood extract. FBR5 was challenged by hot-water wood extract hydrolyzate in this study. After repeated strain adaptation, an improved strain: E. coli FBHW was obtained. Fermentation experiments indicated that FBHW was resistant to the toxicity of hydrolyzate in the fermentation media of concentrated hydrolyzate, and xylose was completely utilized by the strain to produce ethanol. FBHW was grown in the concentrated hydrolyzate without any detoxification treatment and has yielded 36.8 g/L ethanol.     

Comparative hydrolysis and fermentation of sugarcane and agave bagasse

Sugarcane and agave bagasse samples were hydrolyzed with either mineral acids (HCl), commercial glucanases or a combined treatment consisting of alkaline delignification followed by enzymatic hydrolysis. Acid hydrolysis of sugar cane bagasse yielded a higher level of reducing sugars (37.21% for depithed bagasse and 35.37% for pith bagasse), when compared to metzal or metzontete (agave pinecone and leaves, 5.02% and 9.91%, respectively). An optimized enzyme formulation was used to process sugar cane bagasse, which contained Celluclast, Novozyme and Viscozyme L. From alkaline–enzymatic hydrolysis of sugarcane bagasse samples, a reduced level of reducing sugar yield was obtained (11–20%) compared to agave bagasse (12–58%). Selected hydrolyzates were fermented with a non-recombinant strain of Saccharomyces cerevisiae. Maximum alcohol yield by fermentation (32.6%) was obtained from the hydrolyzate of sugarcane depithed bagasse. Hydrolyzed agave waste residues provide an increased glucose decreased xylose product useful for biotechnological conversion.     

Use of dynamic step response for control of fed-batch conversion of lignocellulosic hydrolyzates to ethanol

Optimization of fed-batch conversion of lignocellulosic hydrolyzates by the yeast Saccharomyces cerevisiae was studied. The feed rate was controlled using a step response strategy, in which the carbon dioxide evolution rate was used as input variable. The performance of the control strategy was examined using both an untreated and a detoxified dilute acid hydrolyzate, and the performance was compared to that obtained with a synthetic medium. In batch cultivation of the untreated hydrolyzate, only 23% of the hexose sugars were assimilated. However, by using the feed-back controlled fed-batch technique, it was possible to obtain complete conversion of the hexose sugars. Furthermore, the maximal specific ethanol productivity (q(E,max)) increased more than 10-fold, from 0.06 to 0.70 g g(-1) h(-1). In addition, the viability of the yeast cells decreased by more than 99% in batch cultivation, whereas a viability of more than 40% could be maintained during fed-batch cultivation. In contrast to untreated hydrolyzate, it was possible to convert the sugars in the detoxified hydrolyzate also in batch cultivation. However, a 50% higher specific ethanol productivity was obtained using fed-batch cultivation. During batch cultivation of both untreated and detoxified hydrolyzate a gradual decrease in specific ethanol productivity was observed. This decrease could largely be avoided in fed-batch cultivations.     

Improved 2,3-butanediol production from corncob acid hydrolysate by fed-batch fermentation using Klebsiella oxytoca

Corncob acid hydrolysate, detoxed by sequently boiling, overliming and activated charcoal adsorption, was used for 2,3-butanediol production by Klebsiella oxytoca ACCC 10370. The effects of acetate in hydrolysate and pH on 2,3-butanediol production were investigated. It was found that acetic acid in hydrolysate inhibited the growth of K. oxytoca while benefited the 2,3-butanediol yield. With the increase in acetic acid concentration in medium from 0 to 4 g/l, the lag phase was prolonged and the specific growth rate decreased. The acetic acid inhibition on cell growth can be alleviated by adjusting pH to 6.3 prior to fermentation and a substrate fed-batch strategy with a low initial acetic acid concentration. Under the optimum condition, a maximal 2,3-butanediol concentration of 35.7 g/l was obtained after 60 h of fed-batch fermentation, giving a yield of 0.5 g/g reducing sugar and a productivity of 0.59 g/h l.