Metabolomic profiling of Spathaspora passalidarum fermentations reveals mechanisms that overcome hemicellulose hydrolysate inhibitors.

Understanding the mechanisms involved in tolerance to inhibitors is the first step in developing robust yeasts for industrial second-generation ethanol (E2G) production. Here, we used ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) and MetaboAnalyst 4.0 for analysis of MS data to examine the changes in the metabolic profile of the yeast Spathaspora passalidarum during early fermentation of hemicellulosic hydrolysates containing high or low levels of inhibitors (referred to as control hydrolysate or CH and strategy hydrolysate or SH, respectively). During fermentation of SH, the maximum ethanol production was 16 g L-1 with a yield of 0.28 g g-1 and productivity of 0.22 g L-1 h-1, whereas maximum ethanol production in CH fermentation was 1.74 g L-1 with a yield of 0.11 g g-1 and productivity of 0.01 g L-1 h-1. The high level of inhibitors in CH induced complex physiological and biochemical responses related to stress tolerance in S. passalidarum. This yeast converted compounds with aldehyde groups (hydroxymethylfurfural, furfural, 4-hydroxybenzaldehyde, syringaldehyde, and vanillin) into less toxic compounds, and inhibitors were found to reduce cell viability and ethanol production. Intracellularly, high levels of inhibitors altered the energy homeostasis and redox balance, resulting in lower levels of ATP and NADPH, while that of glycolytic, pentose phosphate, and tricarboxylic acid (TCA) cycle pathways were the most affected, being the catabolism of glucogenic amino acids, the main cellular response to inhibitor-induced stress. This metabolomic investigation reveals interesting targets for metabolic engineering of ethanologenic yeast strains tolerant against multiple inhibitors for E2G production. KEY POINTS: • Inhibitors in the hydrolysates affected the yeast's redox balance and energy status. • Inhibitors altered the glycolytic, pentose phosphate, TCA cycle and amino acid pathways. • S. passalidarum converted aldehyde groups into less toxic compounds.

Improvement on D-xylose to Xylitol Biotransformation by Candida guilliermondii Using Cells Permeabilized with Triton X-100 and Selected Process Conditions.

Cells of Candida guilliermondii permeabilized with Triton X-100 were able to efficiently produce xylitol from a medium composed only by D-xylose and MgCl2·6H2O in potassium phosphate buffer, at 35 °C and pH 6.5. Under these conditions, the results were similar to those obtained when cofactor and co-substrate or nutrients were added to the medium (about 95 % D-xylose was assimilated producing 42 g/L of xylitol, corresponding to 0.80 g/g yield and 2.65 g/L h volumetric productivity). Furthermore, the permeabilized cells kept the D-xylose assimilation in about 90 % and the xylitol production in approx. 40 g/L during three bioconversion cycles of 16 h each. These values are highly relevant when compared to others reported in the literature using enzyme technology and fermentative process, thereby demonstrating the effectiveness of the proposed method. The present study reveals that the use of permeabilized cells is an interesting alternative to obtain high xylitol productivity using low cost medium formulation. This approach may allow the future development of xylitol production from xylose present in lignocellulosic biomass, with additional potential for implementation in biorefinery strategies.

Improvement of submerged culture conditions to produce colorants by Penicillium purpurogenum.

Safety issues related to the employment of synthetic colorants in different industrial segments have increased the interest in the production of colorants from natural sources, such as microorganisms. Improved cultivation technologies have allowed the use of microorganisms as an alternative source of natural colorants. The objective of this work was to evaluate the influence of some factors on natural colorants production by a recently isolated from Amazon Forest, Penicillium purpurogenum DPUA 1275 employing statistical tools. To this purpose the following variables: orbital stirring speed, pH, temperature, sucrose and yeast extract concentrations and incubation time were studied through two fractional factorial, one full factorial and a central composite factorial designs. The regression analysis pointed out that sucrose and yeast extract concentrations were the variables that influenced more in colorants production. Under the best conditions (yeast extract concentration around 10 g/L and sucrose concentration of 50 g/L) an increase of 10, 33 and 23% respectively to yellow, orange and red colorants absorbance was achieved. These results show that P. purpurogenum is an alternative colorants producer and the production of these biocompounds can be improved employing statistical tool.

Improvement of submerged culture conditions to produce colorants by Penicillium purpurogenum

Safety issues related to the employment of synthetic colorants in different industrial segments have increased the interest in the production of colorants from natural sources, such as microorganisms. Improved cultivation technologies have allowed the use of microorganisms as an alternative source of natural colorants. The objective of this work was to evaluate the influence of some factors on natural colorants production by a recently isolated from Amazon Forest, Penicillium purpurogenum DPUA 1275 employing statistical tools. To this purpose the following variables: orbital stirring speed, pH, temperature, sucrose and yeast extract concentrations and incubation time were studied through two fractional factorial, one full factorial and a central composite factorial designs. The regression analysis pointed out that sucrose and yeast extract concentrations were the variables that influenced more in colorants production. Under the best conditions (yeast extract concentration around 10 g/L and sucrose concentration of 50 g/L) an increase of 10, 33 and 23% respectively to yellow, orange and red colorants absorbance was achieved. These results show that P. purpurogenum is an alternative colorants producer and the production of these biocompounds can be improved employing statistical tool.

Detoxification of rice straw and olive tree pruning hemicellulosic hydrolysates employing Saccharomyces cerevisiae and its effect on the ethanol production by Pichia stipitis.

The aim of this work was to study the ability of Saccharomyces cerevisiae (baker's yeast) to metabolize a variety of aromatic compounds found in rice straw (RSHH) and olive tree pruning (OTHH) hemicellulosic hydrolysates, obtained by acid hydrolysis at different sugar and toxic compound concentrations. Initially, the hydrolysates were inoculated with S. cerevisiae (10 g L(-1)) and incubated at 30 °C under agitation at 200 rpm for 6 h. The results showed that this yeast was able to utilize phenolic and furan compounds in both hemicellulose hydrolysates. Next, the treated hydrolysates were inoculated with Pichia stipitis NRRL Y-7124 to evaluate the effect of biotransformation of aromatic compounds on ethanol production, and better fermentation results were obtained in this case compared to untreated ones. The untreated hemicellulose hydrolysates were not able to be fermented when they were incubated with Pichia stipitis. However, in RSHH treated hydrolysates, ethanol (Y(P/S)) and biomass (Y(X/S)) yields and volumetric ethanol productivity (Q(P)) were 0.17 g g(-1), 0.15 g g(-1) and 0.09 g L(-1) h(-1), respectively. The OTHH-treated hydrolysates showed less favorable results compared to RSHH, but the fermentation process was favored with regard to untreated hydrolysate. These results showed that the fermentation by P. stipitis in untreated hydrolysates was strongly inhibited by toxic compounds present in the media and that treatment with S. cerevisiae promoted a significant reduction in their toxicities.

Improving of red colorants production by a new Penicillium purpurogenum strain in submerged culture and the effect of different parameters in their stability.

There is a worldwide interest in the development of processes for colorants production from natural sources such as microorganism. The aim of this study was to optimize red colorants production by Penicillium purpurogenum DPUA 1275 and to evaluate the effect of pH, temperature, salts and polymers on the stability of these colorants. Under optimized conditions, a 78% increase in red colorants production was achieved. The best pH and temperature conditions were obtained at pH 8.0 and 70°C, respectively. In the presence of salts NaCl and Na2 SO4 , both at concentrations of 0.1 and 0.5 M in Mcllvaine buffer (pH 8.0), the red colorants showed good stability. In the presence of both polymers polyethylene glycol and sodium polyacrylate, the red colorants kept their color intensity. Thus, this study presents characteristics of red colorants produced by P. purpurogenum that can be applied in different industries after toxicological examination.

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

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.

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°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±15.7U/g(cells)) > freezing-thawing (54.3 ± 1.9U/g(cells))>Triton X-100 (23.5 ± 0.0U/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).

Individual and interaction effects of vanillin and syringaldehyde on the xylitol formation by Candida guilliermondii.

The effect of lignin degradation products liberated during chemical hydrolysis of lignocellulosic materials on xylose-to-xylitol bioconversion by Candida guilliermondii FTI 20037 was studied. Two aromatic aldehydes (vanillin and syringaldehyde) were selected as model compounds. A two-level factorial design was employed to evaluate the effects of pH (5.5-7.0), cell concentration (1.0-3.0 g l(-1)), vanillin concentration (0-2.0 g l(-1)) and syringaldehyde concentration (0-2.0 g l(-1)) on this bioprocess. The results showed that in the presence of vanillin or syringaldehyde (up to 2.0 g l(-1)) the cell growth was inhibited to different degrees with a complete inhibition of the yeast growth when the mixture of both (at 2.0 g l(-1) each) was added to the fermentation medium. The xylitol yield was not significantly influenced by vanillin, but was strongly reduced by syringaldehyde, which showed a more pronounced inhibitor effect at pH 7.0. The yeast was also able to convert vanillin and syringaldehyde to the corresponding aromatic acids or alcohols and their formation was dependent of the experimental conditions employed.

Micelas reversas de lecitina de soja: uma alternativa para purificação de proteínas / Soybean lecithin reversed micelles: an alternative for protein purification

No presente trabalho, estudaram-se os efeitos de diversos parâmetros sobre a extração das proteínas caseína e albumina de soro bovino empregando micelas reversas de lecitina de soja. Independentemente da condição empregada, a extração da albumina apresentou baixo rendimento (variando de 0 por cento a 4 por cento, aproximadamente), resultado de um significativo efeito de exclusão por tamanho. Com relação à caseína, o rendimento da extração aumentou 23 vezes com o aumento do tempo de agitação, ou seja, com o maior tempo de contato entre a proteína e o sistema de micelas reversas. A adição de 1-hexanol ao sistema, usado como co-solvente, foi efetiva, aumentando a solubilização da caseína em 36 por cento, sendo os rendimentos da extração desta proteína muito influenciados pelo pH. Os valores máximos de eficiência obtidos foram de 20 por cento em pH 7,9, 80 por cento em pH 5,4 e 100 por cento em pH 5,0 (pH próximo ao pI da proteína).

In this work, the effect of different parameters for extraction of casein and bovine serum albumin (BSA) were studied. Such proteins were extracted by soybean lecithin reversed micelles. BSA extraction was not effective, independent of the extraction conditions employed. Owing to its molar mass, the effect of exclusion by size was clearly observed. The casein extraction yield increased about 23-fold as a function of agitation time. In other words, the increase occurred by using higher contact time between protein and reversed micelles. The use of hexanol as a co-solvent was effective, and increased casein extraction to 36 percent. The extraction values were strongly influenced by pH, and the high extraction yield was obtained under the following conditions: 20 percent at pH 7.9, 80 percent at pH 5.4 e 100 percent at pH 5.0 (close to casein isoelectrical point).

Partial purification of glucose-6-phosphate dehydrogenase by aqueous two-phase poly(ethyleneglycol)/phosphate systems

Glucose-6-phosphate dehydrogenase (G6PDH) is an important enzyme used in biochemical and medical studies and in several analytical methods that have industrial and commercial application. This work evaluated the extraction of G6PDH in aqueous two-phase system (ATPS) of poly(ethyleneglycol) (PEG)/phosphate buffer, using as enzyme source a medium prepared through commercial baker's yeast disruption. Firstly, the effects of PEG molar mass on the enzyme partition and of homogenization and rest on the system equilibrium were investigated. Afterwards, several ATPS were prepared using statistical analysis (2² factorial design). The results, including kinetic and thermodynamic parameters for the G6PDH activity, showed partial purification of this enzyme in ATPS composed of 17.5 percent (w/w) PEG400 and 15.0 percent (w/w) phosphate. A high enzymatic recovery value (97.7 percent), a high partition coefficient (351), and an acceptable purification factor (2.28 times higher than in cell homogenate) were attained from the top phase. So, it was possible to attain an effective enzyme pre-purification by separating some contaminants with a simple method such as liquid-liquid extraction in aqueous two-phase systems (ATPS).

Glicose-6-fosfato desidrogenase (G6PDH) é uma importante enzima usada em estudos bioquímicos e médicos, bem como em diversos métodos analíticos com aplicação comercial e industrial. Neste trabalho foi avaliado a extração da G6PDH em sistemas de duas fases aquosas (ATPS) constituídos por poli(etilenoglicol) (PEG)/tampão fosfato, usando como fonte de enzima um meio preparado por rompimento de leveduras de panificação comercial. Inicialmente foram investigados os efeitos da massa molar do PEG na partição da enzima e da homogeneização e repouso no equilíbrio do sistema. Na sequência, diversos ATPS foram preparados usando análise estatística (planejamento fatorial 2²). Os resultados, incluindo parâmetros cinéticos e termodinâmicos para a atividade da G6PDH, indicaram parcial purificação desta enzima em ATPS constituídos por 17,5 por cento (p/p) PEG400 e 15,0 por cento (p/p) fosfato. Um alto valor de recuperação enzimática (97,7 por cento), um alto coeficiente de partição (351), e um fator de purificação aceitável (2,28 vezes maior que em homogenato celular) foram obtidos na fase superior do sistema. Assim, foi possível alcançar uma pré-purificação eficaz da enzima separando alguns contaminadores aplicando um método simples tal como a extração líquido-líquido em sistemas bifásicos (ATPS).

Response surface methodology for the evaluation of glucose-6-phosphate dehydrogenase enrichment process by soybean lecithin reversed micelles.

Glucose-6-phosphate dehydrogenase (G6PD) present in Saccahromyces cerevisiae is an enzyme of the pentose pathway. An effective enrichment of this intracellular enzyme can be achieved with the reversed micellar methodology. In this work, this methodology was employed with soybean lecithin, a biocompatible surfactant. A factorial design was used to evaluate the influence of pH (A) and extraction runs (B) on the G6PD purification factor. After statistical analysis and process optimization, a mathematical model representing G6PD enrichment was obtained: Y=4.89-0.83A+0.092B+0.27AB-1.37B2 with an enzyme purification factor of about 5.2.

Caracterização da xilose redutase extraída por micelas reversas-CTAB a partir de homogenato de Candida guilliermondii / Characterization of xylose reductase extracted by CTAB-reversed micelles from Candida guilliermondii homogenate

Xylosereductase (XR) (E.C.1.1.1.21), produced by Candida guilliermondii, grown in sugar cane bagasse hydrolysate, was separated directly from the cell homogenate by reversed micelles of cetyl trimethyl ammonium bromide (CTAB), attaining a recovery yield of 100% and enrichment factor of 5.6 fold. The extraction conditions were: pH=7.0, electrical conductivity= 14 mS/cm, T=5 degrees C, 5% (w/w) of hexanol, 22% (w/w) of butanol and 0.15 M CTAB. The XR after extraction was stable in pH interval of 6.0-6.5 and its heat inactivation constant was about 6.5 fold higher than that before extraction. The 'V IND. max' values against both xylose and NADPH for XR before and after extraction by reversed-micelles differed about 6%, whereas the difference on KM values were more pronounced. The ('K IND. m') 'IND. xylose' for XR after extraction was about 35% higher than before extraction, meanwhile ('K IND. m') 'IND. NADPH' was about 30% lower after than before extraction. As the KM variations indirectly signaled, the XR affinity simultaneously diminishes for xylose and increases for NADPH. This could probably explain why the 'V IND. max' values for XR before and after extraction were quite similar

A xilose redutase (XR) (E.C.1.1.1.21), produzida por Candida guilliermondii cultivada em hidrolisado de bagaço de cana de açúcar, foi separada diretamente do homogenato livre de células através da técnica de micelas reversas feitas com cetil trimetil brometo de amônio (CTAB). Obteve-se um rendimento de recuperação da enzima de 100% e um fator de enriquecimento de 5,6 vezes. As condições de extração foram: pH=7,0, condutividade elétrica = 14 mS/cm, T= 5 ºC, 5% (w/w) de hexanol, 22% (w/w) de butanol e 0.15M CTAB. A XR após a extração manteve-se estável no intervalo de pH entre 6.0 e 6.5, sendo a constante de inativação térmica cerca de 6,5 vezes maior do que aquela antes da extração. Os valores de Vmax da XR frente à xilose e NADPH antes e após a extração por micelas reversas diferiram cerca de 6%, enquanto que as diferenças nos valores de KM foram mais pronunciadas. O (KM)xilose para a XR após a extração foi cerca de 35% maior do que antes da extração, enquanto que (KM)NADPH foi 30% menor após do que antes da extração. As variações nos valores de KM indicam, indiretamente, que a afinidade da XR simultaneamente diminui para a xilose e aumenta para o NADPH. Este resultado poderia explicar a razão pela qual os valores de Vmax antes e após a extração terem sido praticamente iguais

Perspectives on bioenergy and biotechnology in Brazil.

Brazil is one of the world's largest producers of alcohol from biomass at low cost and is responsible for more than 1 million direct jobs. In 1973, the Brazilian Program of Alcohol (Proalcool) stimulated the creation of a bioethanol industry that has led to large economic, social, and scientific improvements. In the year 1984, 94.5% of Brazil's cars used bioethanol as fuel. In 2003/2004, 350.3 million of sugarcane produced 24.2 million t of sugar and 14.4 billion L of ethanol for an average 4.3 million cars using ethanol. Since its inception, cumulative investment in Proalcool totals US$11 billion, and Brazil has saved US$27 billion in oil imports. The ethanol production industry from sugarcane gene-rates 152 times more jobs than would have been the case if the same amount of fuel was produced from petroleum, and the use of ethanol as a fuel is advantageous for environmental reasons. In 2003, one of the biggest Brazilian ethanol industries started consuming 50% of the residual sugarcane bagasse to produce electrical energy (60 MW), a new alternative use of bioenergy for the Brazilian market. Other technologies for commercial uses of bagasse are in development, such as in the production of natural fibers, sweeteners (glucose and xylitol), single-cell proteins, lactic acid, microbial enzymes, and many other products based on fermentations (submerged and semisolid). Furthermore, studies aimed at the increase in the biosynthesis of sucrose and, consequently, ethanol productivity are being conducted to understand the genetics of sugarcane. Although, at present, there remain technical obstacles to the economic use of some ethanol industry residues, several research projects have been carried out and useful data generated. Efficient utilization of ethanol industry residues has created new opportunities for new value-added products, especially in Brazil, where they are produced in high quantities.

Evaluation of nutrient supplementation to charcoal-treated and untreated rice straw hydrolysate for xylitol production by Candida guilliermondii

Este trabalho avaliou a produção de xilitol pela levedura Candida guilliermondii, a partir de hidrolisado hemicelulósico de palha de arroz não tratado e tratado com carvão ativo, ambos suplementados ou não com nutrientes (sulfato de amônio, cloreto de cálcio e extrato de farelo de arroz). Os resultados mostraram que tanto o rendimento como a produtividade volumétrica em xilitol diminuíram quando os nutrientes foram adicionados em ambos hidrolisados, tratado e não tratado. Em hidrolisado tratado, a eficiência de conversão de xilose em xilitol foi de 79% quando em ausência de nutrientes. Estes resultados mostram que o hidrolisado hemicelulósico de palha de arroz tratado com carvão ativo é uma fonte barata de xilose e outros nutrientes, para a produção de xilitol por Candida guilliermondii. A não necessidade de adicionar nutrientes ao meio a base de hidrolisado é muito vantajosa, uma vez que o processo se torna mais econômico.

Optimized extraction by cetyl trimethyl ammonium bromide reversed micelles of xylose reductase and xylitol dehydrogenase from Candida guilliermondii homogenate.

The intracellular enzymes xylose reductase (XR, EC 1.1.1.21) and xylitol dehydrogenase (XD, EC 1.1.1.9) from Candida guilliermondii, grown in sugar cane bagasse hydrolysate, were separated by reversed micelles of cetyl trimethyl ammonium bromide (CTAB) cationic surfactant. An experimental design was employed to optimize the extraction conditions of both enzymes. Under these conditions (temperature = 5 degree C, hexanol: isooctane proportion = 5% (v/v), 22 %, surfactant concentration = 0.15M, pH = 7.0 and electrical conductivity = 14 mScm(-1)) recovery values of about 100 and 80% were achieved for the enzymes XR and XD, respectively. The purity of XR and XD increased 5.6- and 1.8-fold, respectively. The extraction process caused some structural modifications in the enzymes molecules, as evidenced by the alteration of K(M) values determined before and after extraction, either in regard to the substrate (up 35% for XR and down 48% for XD) or cofactor (down 29% for XR and up 11% for XD). However, the average variation of V(max) values for both enzymes was not higher than 7%, indicating that the modified affinity of enzymes for their respective substrates and cofactors, as consequence of structural modifications suffered by them during the extraction, are compensated in some extension. This study demonstrated that liquid-liquid extraction by CTAB reversed micelles is an efficient process to separate the enzymes XR and XD present in the cell extract, and simultaneously increase the enzymatic activity and the purity of both enzymes produced by C. guilliermondii.

Liquid-liquid extraction of xylitol dehydrogenase from Candida guilliermondii homogenate by reversed micelles.

The intracellular enzyme xylitol dehydrogenase (XD, EC 1.1.1.9) from Candida guilliermondii, grown in sugarcane bagasse hydrolysate, was separated by reversed micelles of BDBAC [N-benzyl-N-dodecyl-N-bis (2-hydroxyethyl) ammonium chloride] cationic surfactant. An experimental design was employed to evaluate the influence of the following factors on the enzyme separation: temperature, co-solvent concentration and surfactant concentration. The results showed that just the temperature did not show significant effect on XD recovery. A model was used to represent the activity recovery and fit the experimental data. Under optimized conditions, the recovery of total activity was about 121%, and the purity increased 2.3-fold.

Alternatives for detoxification of diluted-acid lignocellulosic hydrolyzates for use in fermentative processes: a review.

Acid hydrolysis of lignocellulosic materials produces several inhibitory compounds, such as sugar and lignin degradation products, compounds derived from the lignocellulosic structure, and heavy metal ions. Their toxicity is a major factor limiting bioconversion processes that utilize hydrolyzates. The identification of these compounds and the choice of the best hydrolyzate detoxification method are important for improving the efficiency of the fermentative processes. A variety of biological, physical, and chemical techniques have been proposed to reduce the concentration of these compounds in lignocellulose hydrolyzates. However, the efficiency of any detoxification method depends both on the hydrolyzate composition, which differs according to the raw material used, and on the hydrolysis conditions employed. This review describes the effects of the inhibitory compounds on fermentation yield and productivity, as well as various detoxification methods for treating the hydrolyzates.

Kinetics of the solid state fermentation of sugarcane bagasse by Thermoascus aurantiacus for the production of xylanase.

Xylanase was produced by solid-state fermentation using Thermoascus aurantiacus. Maximum production (500 U g(-1) bagasse) was achieved on the sixth day of cultivation on solid sugarcane bagasse medium supplemented with 15% (v/w) rice bran extract. The fungal biomass, determined from its glucosamine content, reached 28 mg g(-1) on the 8th day of cultivation. The cell yield against O2 (Y(x/o) = 0.18 g(cell)/g(O2)) and maintenance coefficient (m0 = 0.013 g(O2)/g(cell)h) were determined with the low Y(x/o) value for T. aurantiacus agreeing with the calculated value.

Partition behavior and partial purification of hexokinase in aqueous two-phase polyethylene glycol/citrate systems.

This study dealt with the partition behavior and partial purification of hexokinase (HK) from baker's yeast by liquid-liquid extraction using aqueous two-phase polyethylene glycol (PEG)/citrate systems. First, we investigated the effect of agitation type (vortex and 8 rpm rotation) on the stability of the system, and then the effects of sodium citrate concentration, PEG concentration, and molar mass of PEG on the partition coefficient of this enzyme by using a 25 factorial experimental design. The results of this factorial experiment showed the possibility of a partial purification of HK by using two extraction steps, since the enzyme preferentially migrated to the top phase and the total proteins (mainly contaminants) remained in the bottom phase. The purification factor (PurTOP) of the enzyme in the top phase was 1.87, and the partition coefficient of the total proteins (KProt) was 0.47.