Solid-State Fermentation of Chestnut Shells and Effect of Explanatory Variables in Predictive Saccharification Models.

In this study, chestnut shells (CNS), a recalcitrant and low-value agro-industrial waste obtained during the peeling of Castanea sativa fruits, were subjected to solid-state fermentation by six white-rot fungal strains (Irpex lacteus, Ganoderma resinaceum, Phlebia rufa, Bjerkandera adusta and two Trametes isolates). After being fermented, CNS was subjected to hydrolysis by a commercial enzymatic mix to evaluate the effect of fermentation in saccharification yield. After 48 h hydrolysis with 10 CMCase U mL−1 enzymatic mix, CNS fermented with both Trametes strains was recorded with higher saccharification yield (around 253 mg g−1 fermented CNS), representing 25% w/w increase in reducing sugars as compared to non-fermented controls. To clarify the relationships and general mechanisms of fungal fermentation and its impacts on substrate saccharification, the effects of some independent or explanatory variables in the production of reducing sugars were estimated by general predictive saccharification models. The variables considered were lignocellulolytic activities in fungal fermentation, CNS hydrolysis time, and concentration of enzymatic hydrolysis mix. Multiple linear regression analysis revealed a very high significant effect (p < 0.0001) of fungal laccase and xylanase activities in the saccharification models, thus proving the key potential of these enzymes in CNS solid-state fermentation.

Removal pattern of vinasse phenolics by Phlebia rufa, characterization of an induced laccase and inhibition kinetics modeling.

Vinasse from the distillation of winemaking residues is a wastewater characterized by high levels of aromatic compounds. Batch cultures of Phlebia rufa showed a significant (p < 0.05) correlation between laccase activity and initial vinasse concentration. The pattern of biodegradation of hydroxybenzoic acids, hydroxycinnamic acids and flavonoids, assessed by HPLC-DAD, revealed that p-hydroxybenzoic acid is the most recalcitrant compound. Vinasse-induced laccase showed electrophoretic homogeneity and molecular weight of 62 kDa after being purified 21-fold. Optimum pH for oxidation of 2,6-dimethoxyphenol (2,6-DMP) was 3.5 and optimum temperature was 50 °C, with an activation energy of 42.8 kJ mol-1. Catalytic efficiency of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) oxidation is about two orders of magnitude higher than 2,6-DMP oxidation, being their Km values 36.2 ± 2.6 µM and 303.0 ± 44.7 µM, respectively and kcat values 486.1 s-1 and 179.6 s-1, respectively. Akaike information criterion and Akaike weights were used to discriminate inhibition models that best fitted 2,6-DMP oxidation in the presence of inhibitors. Inhibition constants of mixed-type inhibitors azide and fluoride, and competitive-type inhibitor chloride, showed the following inhibitors potency: azide > fluoride > chloride. Taken together, this study is consistent with the assumption that P. rufa could be a useful tool for aerobic degradation of phenolic-rich wastewaters.

Pretreatment of Grape Stalks by Fungi: Effect on Bioactive Compounds, Fiber Composition, Saccharification Kinetics and Monosaccharides Ratio.

Grape stalks, an inedible lignocellulosic residue from winemaking and agro-industrial grape juice production, can be valorized as a source of bioactive compounds and as feedstock for the saccharification and bioconversion of soluble sugars. Solid-state fermentation (SSF) by six white-rot fungi was applied as pretreatment. Fiber composition, free radical scavenging activity, four ligninolytic, and three hydrolytic enzyme activities were determined. Saccharification kinetics, yield, and productivity were evaluated and complemented with scanning electron microscopy (SEM), high performance liquid chromatography (HPLC) quantification of monosaccharides, and principal component analysis (PCA). After SSF, the biomass exhibited a drastic free radical scavenging activity decrease and the main enzymes produced were manganese-dependent peroxidase and xylanase. Scanning electron microscopy revealed the erosion of cell walls, and PCA exhibited a negative correlation between saccharification, and neutral detergent fiber and acid detergent lignin. Phlebia rufa pretreated biomass gave the highest sugars yield and productivity, representing a nearly three-fold increase compared to untreated samples. Also, monosaccharides quantification revealed that the 1:1 ratio of glucose to the sum of xylose plus galactose changes to the value of 2:1 after pretreatment. In this work, and for the first time, P. rufa proved to be an effective pretreatment of grape stalks for the saccharification and further bioconversion into value-added chemicals. In addition, lignocellulolytic enzymes were also produced through SSF.

Fungal biodegradation and multi-level toxicity assessment of vinasse from distillation of winemaking by-products.

The wastewaters from distilleries of winemaking by-products, a scarcely studied type of vinasse, were treated by white-rot fungal strains from species Irpex lacteus, Ganoderma resinaceum, Trametes versicolor, Phlebia rufa and Bjerkandera adusta. The main objectives of this study were to evaluate fungal performance during vinasse biodegradation, their enzyme patterns and ecotoxicity evolution throughout treatment. Despite all strains were able to promote strong (>80%) dephenolization and reduction of total organic carbon (TOC), P. rufa was less affected by vinasse toxicity and exhibit better decolorization. In batch cultures at 28 °C and pH 4.0, the first phase of P. rufa biodegradation kinetics was characterized by strong metabolic activity with simultaneous depletion of TOC, phenolics and sugars. The main events of second phase are the increase of peroxidases production after the peak of laccase activity, and strong color removal. At the end of treatment, it was observed highly significant (p < 0.001) abatement of pollution parameters (83-100% removal). Since water reclamation and reuse for e.g. crop irrigation is a priority issue, vinasse ecotoxicity was assessed with bioindicators representing three different phylogenetic and trophic levels: a marine bacterium (Aliivibrio fischeri), a freshwater microcrustacean (Daphnia magna) and a dicotyledonous macrophyte (Lepidium sativum). It was observed significant (p < 0.05) reduction of initial vinasse toxicity, as evaluated by these bioindicators, deserving special mention an almost complete phytotoxicity elimination.

A Kinetic Process to Determine the Interaction Type Between Two Compounds, One of Which Is a Reaction Product, Using Alkaline Phosphatase Inhibition as a Case Study.

This study describes the development of a new methodology based on a new integrated equation which allows the determination of the kinetic parameters for two mutually non-exclusive inhibitors when one of which is produced during the time-course reaction. Alkaline phosphatase simultaneously inhibited by phosphate and urea is used to illustrate this methodology, including the evaluation of interaction effects between them. Data analyses were carried out using two integrated velocity equations: exclusive linear mixed inhibition (EMI) and non-exclusive linear mixed inhibition (NEMI). Kinetic parameters are estimated using non-linear regression and results show that (i) the interaction between enzyme and the inhibitors urea and phosphate exhibit a mutually non-exclusive behavior; (ii) more specifically, these inhibitors are non-exclusive only in free enzyme (E) species; (iii) the inhibitors also show an interaction with enzyme classified as facilitation; (iv) phosphate is a competitive inhibitor and urea a mixed inhibitor; (v) the inhibition constant for phosphate is much lower than that determined for urea. In addition, a functional Excel Spreadsheet which can be adapted to any kinetic study is also included as a supplement.

Mediterranean forested wetlands are yeast hotspots for bioremediation: a case study using azo dyes.

Forested wetlands are interfaces between terrestrial and aquatic environments. These ecosystems play an important role in the hydrology, chemistry and biodiversity maintenance. Despite their high microbial diversity, there has been a lack of attention to the potential of their yeast communities. The purpose of this study is to evaluate the potential of yeasts isolated from a Mediterranean forested wetlands in decolorizing azo dyes. Azo dyes are synthetic, and highly recalcitrant to degradation. Ninety-two out of 560 isolates were randomly chosen to assess their ability to decolorize five azo dyes. Hierarchical clustering based on medium color changes during incubations was used to evaluate the isolates' decolorization performance. All of the isolates that best degraded the 5 dyes tested were identified as Basidiomycota (Filobasidiales, Tremellales and Sporidiobolales). This work identifies new azo dye-degrading yeast species, and supports the hypothesis that forested wetlands are a niche for yeasts with bioremediation potential - namely azo dyes removal.

Enzyme inhibition studies by integrated Michaelis-Menten equation considering simultaneous presence of two inhibitors when one of them is a reaction product.

To determine initial velocities of enzyme catalyzed reactions without theoretical errors it is necessary to consider the use of the integrated Michaelis-Menten equation. When the reaction product is an inhibitor, this approach is particularly important. Nevertheless, kinetic studies usually involved the evaluation of other inhibitors beyond the reaction product. The occurrence of these situations emphasizes the importance of extending the integrated Michaelis-Menten equation, assuming the simultaneous presence of more than one inhibitor because reaction product is always present. This methodology is illustrated with the reaction catalyzed by alkaline phosphatase inhibited by phosphate (reaction product, inhibitor 1) and urea (inhibitor 2). The approach is explained in a step by step manner using an Excel spreadsheet (available as a template in Appendix). Curve fitting by nonlinear regression was performed with the Solver add-in (Microsoft Office Excel). Discrimination of the kinetic models was carried out based on Akaike information criterion. This work presents a methodology that can be used to develop an automated process, to discriminate in real time the inhibition type and kinetic constants as data (product vs. time) are achieved by the spectrophotometer.

Winery wastewater treatment by combination of Cryptococcus laurentii and Fenton's reagent.

Winery wastewaters (WW) have high levels of organic matter, resulting in high COD and BOD and suspended solids. This paper studies the combination of biological and chemical processes in WW treatment. Among 10 yeast isolates, Filobasidium sp. (AGG 577) and Cryptococcus laurentii (AGG 726) were selected due to their superior performance in COD removal. During WW degradation, COD and total polyphenols (TPP) content removal of 89-90% for Filobasidium sp. and 90-93% for C. laurentii were obtained. However, despite similar degradation efficiency for both yeasts, COD kinetics and pH evolution during treatment reveals that C. laurentii presents a faster response than Filobasidium sp. The toxicity (inhibition of Vibrio fischeri luminescence) of C. laurentii treated WW decreases to an inhibition value below 2.5%. However, treated WW exceeds the legal limits, making necessary an additional treatment. In this case, the selection of Fenton's reagent as a chemical final polish step process is a good compromise between efficiency and lower practical complexity. The best results for both COD and TPP removal were obtained with H2O2 initial concentration of 39.2mM and a H2O2:Fe(2+) molar ratio of 15:1. The combined C. laurentii - Fenton's reagent treatment of WW achieved a total reduction of 98% and 96%, for COD and TPP, respectively.

Utilization of integrated Michaelis-Menten equations for enzyme inhibition diagnosis and determination of kinetic constants using Solver supplement of Microsoft Office Excel.

Enzyme kinetic parameters are usually determined from initial rates nevertheless, laboratory instruments only measure substrate or product concentration versus reaction time (progress curves). To overcome this problem we present a methodology which uses integrated models based on Michaelis-Menten equation. The most severe practical limitation of progress curve analysis occurs when the enzyme shows a loss of activity under the chosen assay conditions. To avoid this problem it is possible to work with the same experimental points utilized for initial rates determination. This methodology is illustrated by the use of integrated kinetic equations with the well-known reaction catalyzed by alkaline phosphatase enzyme. In this work nonlinear regression was performed with the Solver supplement (Microsoft Office Excel). It is easy to work with and track graphically the convergence of SSE (sum of square errors). The diagnosis of enzyme inhibition was performed according to Akaike information criterion.

Influence of ligninolytic enzymes on straw saccharification during fungal pretreatment.

Solid state and submerged fermentations in the presence of white-rot basidiomycetes (Bjerkandera adusta, Fomes fomentarius, Ganoderma resinaceum, Irpex lacteus, Phanerochaete chrysosporium, Trametes versicolor and basidiomycete Euc-1) and the litter-decomposing basidiomycete Lepista nuda were evaluated as a pretreatment to increase enzymatic saccharification of wheat straw. Enzymatic hydrolysis of holocellulose after solid state pretreatment showed a significant (P<0.05) increase of saccharification process for T. versicolor, Euc-1, G. resinaceum and I. lacteus, being T. versicolor (strain Tv2) the best one with a sugar yield increase of 91% compared with untreated straw. In submerged medium the pretreatment with I. lacteus, Euc-1 and P. chrysosporium enhanced saccharification but at a lesser extent. Covariance analysis was used to evaluate the relationships between ligninolytic enzymes (lignin peroxidase, manganese-dependent peroxidase and laccase) and saccharification increase. Results showed that only the presence of lignin peroxidase during pretreatment can lead to a significant (P<0.05) increase in the saccharification yield.

Enzymatic saccharification of biologically pre-treated wheat straw with white-rot fungi.

Wheat straw was submitted to a pre-treatment by the basidiomycetous fungi Euc-1 and Irpex lacteus, aiming to improve the accessibility of cellulose towards enzymatic hydrolysis via previous selective bio-delignification. This allowed the increase of substrate saccharification nearly four and three times while applying the basidiomycetes Euc-1 and I. lacteus, respectively. The cellulose/lignin ratio increased from 2.7 in the untreated wheat straw to 5.9 and 4.6 after the bio-treatment by the basidiomycetes Euc-1 and I. lacteus, respectively, thus evidencing the highly selective lignin biodegradation. The enzymatic profile of both fungi upon bio-treatment of wheat straw have been assessed including laccase, manganese-dependent peroxidase, lignin peroxidase, carboxymethylcellulase, xylanase, avicelase and feruloyl esterase activities. The difference in efficiency and selectivity of delignification within the two fungi treatments was interpreted in terms of specific lignolytic enzyme profiles and moderate xylanase and cellulolytic activities.

Modification of wheat straw lignin by solid state fermentation with white-rot fungi.

The potential of crude enzyme extracts, obtained from solid state cultivation of four white-rot fungi (Trametes versicolor, Bjerkandera adusta, Ganoderma applanatum and Phlebia rufa), was exploited to modify wheat straw cell wall. At different fermentation times, manganese-dependent peroxidase (MnP), lignin peroxidase (LiP), laccase, carboxymethylcellulase (CMCase), avicelase, xylanase and feruloyl esterase activities were screened and the content of lignin as well as hydroxycinnamic acids in fermented straw were determined. All fungi secreted feruloyl esterase while LiP was only detected in crude extracts from B. adusta. Since no significant differences (P>0.05) were observed in remaining lignin content of fermented straw, LiP activity was not a limiting factor of enzymatic lignin removal process. The levels of esterified hydroxycinnamic acids degradation were considerably higher than previous reports with lignocellulosic biomass. The data show that P. rufa, may be considered for more specific studies as higher ferulic and p-coumaric acids degradation was observed for earlier incubation times.

Utilization of integrated Michaelis-Menten equation to determine kinetic constants.

Students of biochemistry and related biosciences are urged to solve problems where kinetic parameters are calculated from initial rates obtained at different substrate concentrations. Troubles begin when they go to the laboratory to perform kinetic experiments and realize that usual laboratory instruments do not measure initial rates but only substrate or product concentrations as a function of reaction time. To overcome this problem we present a methodology which uses the integrated form of Michaelis-Menten equation. The method presented has a theoretical and pedagogic basis which is not as arbitrary as other approaches. Here we present and describe the methodology for analyzing time course data together with some examples of the essential computer procedures to implement these analyses. To simplify the understanding of this methodology the experimental examples are confined to linear inhibitions and experimental points utilized are the same from which the initial rates are determined.

Simultaneous ethanol and cellobiose inhibition of cellulose hydrolysis studied with integrated equations assuming constant or variable substrate concentration.

The integrated forms of the Michaelis-Menten equation assuming variable substrate (depletion) or constant substrate concentration were used to study the effect of the simultaneous presence of two exoglucanase Cel7A inhibitors (cellobiose and ethanol) on the kinetics of cellulose hydrolysis. The kinetic parameters obtained, assuming constant substrate (K(m) = 21 mM, Kic = 0.035 mM; K(icl) = 1.5 x 1,015 mM; k(cat) = 12 h-1) or assuming variable substrate (K(m) = 16 mM, Kic = 0.037 mM; K(icl) = 5.8 x 1,014 mM; k(cat) = 9 h-1), showed a good similarity between these two alternative methodologies and pointed out that both ethanol and cellobiose are competitive inhibitors. Nevertheless, ethanol is a very weak inhibitor, as shown by the large value estimated for the kinetic constant K(icl). In addition, assuming different concentrations of initial accessible substrate present in the reaction, both inhibition and velocity constants are at the same order of magnitude, which is consistent with the obtained values. The possibility of using this kind of methodology to determine kinetic constants in general kinetic studies is discussed, and several integrated equations of different Michaelis-Menten kinetic models are presented. Also examined is the possibility of determining inhibition constants without knowledge of the true accessible substrate concentration.

Simulaaneous ethanol and cellobiose inhibition of cellulose hydrolysis studied with integrated equations assuming constant or variable substrate concentration.

The integrated forms of the Michaelis-Menten equation assuming variable substrate (depletion) or constant substrate concentration were used to study the effect of the simultaneous presence of two exoglucanase Cel7A inhibitors (cellobiose and ethanol) on the kinetics of cellulose hydrolysis. The kinetic parameters obtained, assuming constant substrate (K m =21 mM, K ic =0.035 mM; K icl =1.5×1015mM; kcat=12 h-1) or assuming variable substrate (K m =16 mM, K ic =0.037 mM; K icl =5.8×1014 mM; kcat=9 h-1), showed a good similarity between these two alternative methodologies and pointed out that bothethanol and cellobiose are competitive inhibitors. Nevertheless, ethanol is a very weak inhibitor, as shown by the large value estimated for the kinetic constant K icl . In addition, assuming different concentrations of initial accessible substrate present in the reaction, both inhibition and velocity constants are at the same order of magnitude, which is consistent with the obtained values. The possibility of using this kind of methodology to determine kinetic constants in general kinetic studies is discussed, and several integrated equations of different Michaelis-Menten kinetic models are presented. Also examined is the possibility of determining inhibition constants without knowledge of the true accessible substrate concentration.

Enzymatic kinetic of cellulose hydrolysis: inhibition by ethanol and cellobiose.

The ethanol effect on the Trichoderma reesei cellulases was studied to quantify and clarify this inhibition type. To determine inhibition parameters of crude cellulase and purified exoglucanase Cel7A, integrated Michaelis-Menten equations were used assuming the presence of two inhibitors: cellobiose as the reaction product and ethanol as a possible bioproduct of cellulose fermentation. It was found that hydrolysis of cellulose by crude enzyme follows a model that considers noncompetitive inhibition by ethanol, whereas Cel7A is very slightly competitively inhibited. Crude cellulase is much more inhibited (K(iul) = K(icl) = 151.9 mM) than exoglucanase Cel7A (K(icl) = 1.6 x 1015 mM). Also, calculated inhibition constants showed that cellobiose inhibition is more potent than ethanol inhibition both for the crude enzyme as well as exoglucanase Cel7A.

Discrimination among eight modified michaelis-menten kinetics models of cellulose hydrolysis with a large range of substrate/enzyme ratios: inhibition by cellobiose.

The kinetics of exoglucanase (Cel7A) from Trichoderma reesei was investigated in the presence of cellobiose and 24 different enzyme/Avicel ratios for 47 h, in order to establish which of the eight available kinetic models best explained the factors involved. The heterogeneous catalysis was studied and the kinetic parameters were estimated employing integrated forms of Michaelis-Menten equations through the use of nonlinear least squares. It was found that cellulose hydrolysis follows a model that takes into account competitive inhibition by cellobiose (final product) with the following parameters: Km = 3.8 mM, Kic = 0.041 mM, kcat = 2 h-1 (5.6 x 10-4 s-1). Other models, such as mixed type inhibition and those incorporating improvements concerning inhibition by substrate and parabolic inhibition, increased the modulation performance very slightly. The results support the hypothesis that nonproductive enzyme substrate complexes, parabolic inhibition, and enzyme inactivation (Selwyn test) are not the principal constraints in enzymatic cellulose hydrolysis. Under our conditions, the increment in hydrolysis was not significant for substrate/enzyme ratios <6.5.