Ethanol production using hemicellulosic hydrolyzate and sugarcane juice with yeasts that converte pentoses and hexoses

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Produção de etanol a partir de mosto hidrolisado hemicelulósico de ponta e palha de cana-de-açúcar

Pós-graduação em Microbiologia Agropecuária - FCAV

CTAB, Triton X-100 and freezing-thawing treatments of Candida guilliermondii: Effects on permeability and accessibility of the glucose-6-phosphate dehydrogenase, xylose reductase and xylitol dehydrogenase enzymes

Cells of Candida guilliermondii (ATCC 201935) were permeabilised with surfactant treatment (CTAB or Triton X-100) or a freezing-thawing procedure. Treatments were monitored by in situ activities of the key enzymes involved in xylose metabolism, that is, glucose-6-phosphate dehydrogenase (G6PD), xylose reductase (XR) and xylitol dehydrogenase (XD). The permeabilising ability of the surfactants was dependent on its concentration and incubation time. The optimum operation conditions for the permeabilisation of C. guilliermondii with surfactants were 0.41 mM (CTAB) or 2.78 mM (Triton X-100), 30 degrees C, and pH 7 at 200 rpm for 50 min. The maximum permeabilisation measured in terms of the in situ G6PD activity observed was, in order, as follows: CTAB (122.4 +/- 15.7 U/g(cells)) > freezing-thawing, , (54.3 +/- 1.9 U/g(cells)) > Triton X-100 (23.5 +/- 0.0 U/g(cells)). These results suggest that CTAB surfactant is more effective in the permeabilisation of C. guilliermondii cells in comparison to the freezing-thawing and Triton X-100 treatments. Nevertheless, freezing-thawing was the only treatment that allowed measurable in situ XR activity. Therefore, freezing-thawing permeabilised yeast cells could be used as a source of xylose reductase for analytical purposes or for use in biotransformation process such as xylitol preparation from xylose. The level of in situ xylose reductase was found to be 13.2 +/- 0.1 U/g(cells).

Mikrobielle Synthese von Biopolymeren aus der nachwachsenden Rohstoffquelle Weizenstroh

Weizenstroh als erneuerbare Ressource zur Produktion von Biopolymeren und wichtigen Grundchemikalien stellt eine ökologisch sinnvolle Alternative dar. Durch die vom PFI durchgeführte Thermodruckhydrolyse konnte das Weizenstroh und die darin enthaltenen Zucker fast vollständig mobilisiert werden. Ein umfangreiches Screening nach Organismen, welche die Zucker des Weizenstrohs verwerten konnten, ergab, dass einige wenige Stämme zur PHB-Bildung aus Xylose befähigt waren (10 %). Zur PHB-Synthese aus Glucose waren indes ca. doppelt so viele Organismen in der Lage (20 %). Zwei der insgesamt 118 untersuchten Organismen zeigten besonders gute PHB-Bildung sowohl mit Xylose als auch mit Glucose als Substrat. Dabei handelte es sich um die hauseigenen Stämme Bacillus licheniformis KHC 3 und Bacillus megaterium KNaC 2. Nach Enttoxifizierung der hemicellulosischen Fraktion konnte diese als C-Quelle im Mineral Medium eingesetzt werden. Burkholderia sacchari DSM 17165 und Hydrogenophaga pseudoflava DSM 1034, sowie die hauseigenen Isolate Bacillus licheniformis KHC 3 und Bacillus megaterium KNaC 2 wurden für die Synthese von PHB aus der hemicellulosischen Fraktion verwendet. Die Zucker der hemicellulosischen Fraktion (Xylose, Glucose, Arabinose) konnten durch diese Organismen zur PHB-Synthese genutzt werden. Hierbei stellte sich heraus, dass die beiden Bacillus-Stämme besser zur Produktion von PHB aus dem hemicellulosischen Hydrolysat geeignet waren als die Stämme der DSMZ. Die alternative Umsetzung der im hemicellulosischem Hydrolysat enthaltenen Zucker (Xylose, Glucose und Arabinose) in die wichtigen Grundchemikalien Lactat und Acetat konnte durch die Verwendung von heterofermentativen Milchsäurebakterien verwirklicht werden. Die Bildung dieser wichtigen Grundchemikalien stellt eine interessante Alternative zur PHB-Synthese dar. Die Menge an teuren Zusätzen wie Tomatensaft, welcher für das Wachstum der MSB essentiell war, konnte reduziert werden. Die Glucose der zweiten Fraktion des Weizenstrohs, der cellulosischen Fraktion, konnte ebenfalls durch den Einsatz von Mikroorganismen in PHB umgewandelt werden. Kommerzielle Cellulasen der Firma Novozymes konnten große Mengen an Glucose (≥10 g/l) aus der cellulosischen Fraktion freisetzen. Diese freie Glucose wurde mit Hilfe von Cupriavidus necator DSM 545, Cupriavidus necator NCIMB 11599, Bacillus licheniformis KHC 3 und Bacillus megaterium KNaC 2 zu PHB fermentiert. Wie auch beim hemicellulosischen Hydrolysat konnten hier die beiden Bacillus-Stämme die besten Ergebnisse erzielen. Bei ihnen machte die PHB mehr als die Hälfte der Trockenmasse aus. Die Abtrennung des Zielprodukts ohne die Verwendung von umweltschädlichen Lösungsmitteln wurde durch die Lyse der Zielzellen durch eigens isolierte Enzyme aus Streptomyceten verwirklicht. Die Zelllyse durch die Enzyme aus Streptomyces globisporus subsp. caucasius DSM 40814 und Streptomyces albidoflavus DSM 40233 war erfolgreich und zeigte vor allem bei den Bacillen hohe Wirkung (83 % und 99 % Zelllyse). Bei dem Gram-negativen Organismus Cupriavidus necator DSM 428 konnte die anfangs niedrige Zelllyse von 38 % durch Ultraschallbehandlung auf ca. 75 % erhöht werden.

Optimization of acid hydrolysis from the hemicellulosic fraction of Eucalyptus grandis residue using response surface methodology

Biotechnological conversion of biomass into fuels and chemicals requires hydrolysis of the polysaccharide fraction into monomeric sugars. Hydrolysis can be performed enzymatically and with dilute or concentrate mineral acids. The present study used dilute sulfuric acid as a catalyst for hydrolysis of Eucalyptus grandis residue. The purpose of this paper was to optimize the hydrolysis process in a 1.41 pilot-scale reactor and investigate the effects of the acid concentration, temperature and residue/acid solution ratio on the hemicellulose removal and consequently on the production of sugars (xylose, glucose and arabinose) as well as on the formation of by-products (furfural, 5-hydroxymethylfurfural and acetic acid). This study was based on a model composition corresponding to a 2 3 orthogonal factorial design and employed the response surface methodology (RSM) to optimize the hydrolysis conditions, aiming to attain maximum xylose extraction from hemicellulose of residue. The considered optimum conditions were: H2SO4 concentration of 0.65%, temperature of 157 degrees C and residue/acid solution ratio of 1/8.6 with a reaction time of 20 min. Under these conditions, 79.6% of the total xylose was removed and the hydrolysate contained 1.65 g/l glucose, 13.65 g/l xylose, 1.55 g/l arabinose, 3.10 g/l acetic acid, 1.23 g/l furfural and 0.20 g/l 5-hydroxymethylfurfural. (c) 2006 Published by Elsevier Ltd.

Evaluation of Rice Bran Extract as a Nitrogen Source for Improved Hemicellulosic Ethanol Production from Sugarcane Bagasse by New Xylose-Fermenting Yeast Strains Isolated from Brazilian Forests

Xylose is the main sugar in hemicellulosic hydrolysates and its fermentation into ethanol by microorganisms is influenced by nutritional factors, such as nitrogen source, vitamins and other elements. Rice bran extract (RBE) is an inexpensive nitrogen source primarily consisting of high amount of protein. This study evaluates the potential of RBE as a nitrogen source for the hemicellulosic ethanol production from sugarcane bagasse dilute acid hydrolysate by novel yeast strains Scheffersomyces shehatae (syn. Candida shehatae) CG8-8BY and Spathaspora arborariae UFMG-HM19.1A, isolated from Brazilian forests. Two different media formulations were used for inoculum preparation and production medium, using yeast extract and RBE as nitrogen sources. S. shehatae CG8-8BY showed ethanol production of 17.0 g/l with the ethanol yield (0.33 g/g) and fermentation efficiency (64 %) from medium supplemented with RBE. On the other hand, S. arborariae presented 5.4 g/l of ethanol production with ethanol yield (0.14 g/g) and fermentation efficiency (21 %) in a fermentation medium supplemented with RBE. Appropriate media formulation is an important parameter to increase the productivity of bioconversion process and RBE proved to be an efficient and inexpensive nitrogen source to supplement sugarcane bagasse hemicellulosic hydrolysate for second generation ethanol production. © 2013 Society for Sugar Research & Promotion.

Bioprospecção de leveduras fermentadoras de xilose visando a produção de etanol a partir de bagaço de cana-de-açúcar

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Evaluation of Oat Hull Hemicellulosic Hydrolysate Fermentability Employing Pichia stipitis

Oat hull hemicellulosic hydrolysate obtained by diluted acid hydrolysis was employed as fermentation medium for Pichia stipitis cultivation. A comparison between the use of treated hydrolysate with 1% activated charcoal to reduce the toxic compounds generated during the hydrolysis process and untreated hydrolysate as a control was conducted. In the cultures using treated hydrolysate the total consumption of glucose, low xylose consumption and ethanol and glycerol formation were observed. The medium formulated with untreated hydrolysate showed morphological cell modifications with consequently cell death, no ethanol formation and formation of glycerol as byproduct of fermentative process, probably as a response to stressful conditions to yeast due to presence of high concentration of toxic compounds. Thus, further studies are suggested in order to determine the best conditions for hydrolysis and detoxification of the hydrolysate to improve the fermentative performance of P. stipitis.

Treatment of rice straw hemicellulosic hydrolysates with advanced oxidative processes: a new and promising detoxification method to improve the bioconversion process

Abstract Background The use of lignocellulosic constituents in biotechnological processes requires a selective separation of the main fractions (cellulose, hemicellulose and lignin). During diluted acid hydrolysis for hemicellulose extraction, several toxic compounds are formed by the degradation of sugars and lignin, which have ability to inhibit microbial metabolism. Thus, the use of a detoxification step represents an important aspect to be considered for the improvement of fermentation processes from hydrolysates. In this paper, we evaluated the application of Advanced Oxidative Processes (AOPs) for the detoxification of rice straw hemicellulosic hydrolysate with the goal of improving ethanol bioproduction by Pichia stipitis yeast. Aiming to reduce the toxicity of the hemicellulosic hydrolysate, different treatment conditions were analyzed. The treatments were carried out according to a Taguchi L16 orthogonal array to evaluate the influence of Fe+2, H2O2, UV, O3 and pH on the concentration of aromatic compounds and the fermentative process. Results The results showed that the AOPs were able to remove aromatic compounds (furan and phenolic compounds derived from lignin) without affecting the sugar concentration in the hydrolysate. Ozonation in alkaline medium (pH 8) in the presence of H2O2 (treatment A3) or UV radiation (treatment A5) were the most effective for hydrolysate detoxification and had a positive effect on increasing the yeast fermentability of rice straw hemicellulose hydrolysate. Under these conditions, the higher removal of total phenols (above 40%), low molecular weight phenolic compounds (above 95%) and furans (above 52%) were observed. In addition, the ethanol volumetric productivity by P. stipitis was increased in approximately twice in relation the untreated hydrolysate. Conclusion These results demonstrate that AOPs are a promising methods to reduce toxicity and improve the fermentability of lignocellulosic hydrolysates.

Evaluation of oat hull hemicellulosic hydrolysate fermentability employing Pichia stipitis

Oat hull hemicellulosic hydrolysate obtained by diluted acid hydrolysis was employed as fermentation medium for Pichia stipitis cultivation. A comparison between the use of treated hydrolysate with 1% activated charcoal to reduce the toxic compounds generated during the hydrolysis process and untreated hydrolysate as a control was conducted. In the cultures using treated hydrolysate the total consumption of glucose, low xylose consumption and ethanol and glycerol formation were observed. The medium formulated with untreated hydrolysate showed morphological cell modifications with consequently cell death, no ethanol formation and formation of glycerol as byproduct of fermentative process, probably as a response to stressful conditions to yeast due to presence of high concentration of toxic compounds. Thus, further studies are suggested in order to determine the best conditions for hydrolysis and detoxification of the hydrolysate to improve the fermentative performance of P. stipitis.

Understanding mild acid pretreatment of sugarcane bagasse through particle scale modeling

Sugarcane bagasse is an abundant and sustainable resource, generated as a by-product of sugarcane milling. The cellulosic material within bagasse can be broken down into glucose molecules and fermented to produce ethanol, making it a promising feedstock for biofuel production. Mild acid pretreatment hydrolyses the hemicellulosic component of biomass, thus allowing enzymes greater access to the cellulosic substrate during saccharification. A particle-scale mathematical model describing the mild acid pretreatment of sugarcane bagasse has been developed, using a volume averaged framework. Discrete population-balance equations are used to characterise the polymer degradation kinetics, and diffusive effects account for mass transport within the cell wall of the bagasse. As the fibrous material hydrolyses over time, variations in the porosity of the cell wall and the downstream effects on the reaction kinetics are accounted for using conservation of volume arguments. Non-dimensionalization of the model equations reduces the number of parameters in the system to a set of four dimensionless ratios that compare the timescales of different reaction and diffusion events. Theoretical yield curves are compared to macroscopic experimental observations from the literature and inferences are made as to constraints on these “unknown” parameters. These results enable connections to be made between experimental data and the underlying thermodynamics of acid pretreatment. Consequently, the results suggest that data-fitting techniques used to obtain kinetic parameters should be carefully applied, with prudent consideration given to the chemical and physiological processes being modeled.

Isolation and Culture of a Thermophilic Fungus, Melanocarpus albomyces, and Factors Influencing the Production and Activity of Xylanase

Summary: An uncommon thermophilic fungus, Melanocarpus albomyces, was isolated from soil and compost by incubating samples in a glucose/sorbose/asparagine liquid medium, followed by enrichment culture in medium containing sugarcane bagasse as carbon source. The culture filtrate protein of the fungus grown in the presence of bagasse or xylose hydrolysed xylan and some other polysaccharides but cellulose was not hydrolysed. High extracellular xylanase (EC 3.2.1.8) activity was produced by cultures grown on xylose or hemicellulosic materials. The enzyme was induced in glucose-grown washed mycelia in response to addition of xylose or xylan but not by alkyl or aryl β-D-xylosides. Cultures produced higher enzyme yields in shaken flasks than in a fermenter. Gel-filtration chromatography of culture filtrate protein showed the presence of two isoenzymes of xylanase, whose relative proportions varied with the carbon source used for growth. The extent of hydrolysis of heteroxylans or the hemicellulosic fraction of bagasse by culture filtrate protein preparations was greater when the cultures had been grown on bagasse rather than xylose as the inducing substrate. The activity of xylanase preparations was increased when an exogenous β-glucosidase was added.

Evaluation of oil production from oil palm empty fruit bunch by oleaginous micro-organisms

Oil palm empty fruit bunch (EFB) is a readily available, lignocellulosic biomass that has potential to be utilized as a carbon substrate for microbial oil production. In order to evaluate the production of microbial oil from EFB, a technical study was performed through the cultivation of oleaginous micro-organisms (Rhodotorula mucilaginosa, Aspergillus oryzae, and Mucor plumbeus) on EFB hydrolyzates. EFB hydrolyzates were prepared through dilute acid pre-treatment of the biomass, where the liquid fraction of pre-treatment was detoxified and used as an EFB liquid hydrolyzate (EFBLH). The solid residue was enzymatically hydrolyzed prior to be used as an EFB enzymatic hydrolyzate (EFBEH). The highest oil concentrations were obtained from M. plumbeus (1.9 g/L of oil on EFBLH and 4.7 g/L of oil on EFBEH). In order to evaluate the feasibility of large-scale microbial oil production, a techno-economic study was performed based on the oil yields of M. plumbeus per hectare of plantation, followed by the estimation of the feedstock cost for oil production. Other oil palm biomasses (frond and trunk) were also included in this study, as it could potentially improve the economics of large-scale microbial oil production. Microbial oil from oil palm biomasses was estimated to potentially increase oil production in the palm oil industry up to 25%, at a cheaper feedstock cost. The outcome of this study demonstrates the potential integration of microbial oil production from oil palm biomasses with existing palm oil industry (biodiesel, food and oleochemicals production), that could potentially enhance sustainability and profitability of microbial oil production.

Feasibility study of biofuel production from freshwater fern, Azolla sp in Anzali Wetland

Production of bioethanol through acidic and enzymatic hydrolysis of aquatic Azolla sp., as a new source of bio-mass, has been performed, as a means to control increasing growth and reducing undesirable effects of this plant in Anzali lagoon. After sampling, drying and crushing, Azolla was hydrolyzed, using diluted acid and enzyme. Diluted acid hydrolysis was done using both autoclave and a high-pressure system (Batch Synth® Microwave synthesizer). The effects of temperature and time (in autoclave) and concentration of acid (in both) were compared. Cellubrix®, a ommercial cellulase source, was used for enzymatic hydrolysis process. The amounts of reducing sugars, glucose and furfural, released from hydrolyzate, were measured. To produce alcohol, Sacchromyces cerevisiae (to ferment sixcarbon sugars), Zygowilliopsis californica and Pichia stipitis (to ferment five-carbon and sixcarbon sugars) were used. Maximum amounts of glucose (4.83% w/w) and reducing sugars (14.15% w/w) were obtained using acid hydrolysis in autoclave. In the microwave oven, maximum glucose (5.04% w/w) and reducing sugars (13.27 w/w) were obtained at 180 and 200 °C, respectively. Under these conditions, maximum produced furfural was 1.54 g/L. The difference between amounts of furfural obtained from acid hydrolysis of Azolla in microwave oven compared to autoclave was statistically significant. Amounts of alcohol produced and its yields were 3.99 g/L and 33.13% for S. cerevisiae in 48 hours, 3.73 g/L and 30.45% for Pichia stipites in 48 hours, and 3.73 g/L and 30.45% for Z. californica in 24 hours after inoculation, respectively, with significant differences. Statistical comparison of results showed significant differences (P<0.05) in glucose production, at different conditions. Amounts of reducing sugars and glucose increased after optimization of levels of acid, time, and temperature. The overall optimum released sugar and glucose were obtained with 1.67% (w/v) acid using autoclave. Higher temperatures in microwave oven caused a significant increase (P<0.05) in furfural. Furfural severely inhibits fermentation. Hence, regarding the issues of energy consumption and time, amounts of inhibiting substances and sugar production, autoclave is found to be superior to the high temperature and pressure, generated in microwave oven, for hydrolyzing Azolla. Furthermore, given the amounts of Azolla in Anzali lagoon, it may be recommendable to use this plant as a biomass resource.