Review of lignocellulolytic enzyme activity analyses and scale-down to microplate-based assays.

With the increasing use of enzymes in environmental applications, there is a need for analytical methods adapted to large factorial experiments. Existing reference methods are chemical and labor intensive and unsuitable to analyze in parallel a large number of samples. Based on an extensive literature review and on experimental results, this work compares reference and microplate adapted methods to define the most adequate filter paper, carboxymethylcellulase, ß-glucosidase and xylanase activity tests. In the adapted methods, the total reaction volume was reduced from 2.2-24.5 mL to 0.21-0.24 mL. Statistical analysis of the activities measured on enzyme mixtures by applying the 96-well plate reduced methods showed that they were not significantly different to the activities obtained with reference tests.

Fostering the action of versatile peroxidase as a highly efficient biocatalyst for the removal of endocrine disrupting compounds.

Response surface methodology (RSM) was used to optimize the removal of five endocrine disrupting compounds (EDCs) by the enzyme versatile peroxidase (VP): bisphenol A (BPA), triclosan (TCS), estrone (E1), 17ß-estradiol (E2) and 17α-ethinylestradiol (EE2). The optimal variables of enzyme activity (90-100 U L(-1)), sodium malonate (29-43 mM) and MnSO4 (0.8-1 mM) led to very high removal rates of the five pollutants (2.5-5.0 mg L(-1) min(-1)). The structural elucidation of transformation products arising from the enzymatic catalysis of the EDCs was investigated by Gas Chromatography coupled to Mass Spectrometry (GC-MS) and Liquid Chromatography Electrospray Time-of-Flight Mass Spectrometry (LC-ESI-TOF-MS). The presence of dimers and trimers, indicative of oxidative coupling, was demonstrated.

Continuous removal of nonylphenol by versatile peroxidase in a two-stage membrane bioreactor.

The ligninolytic enzymes versatile peroxidase (VP) and manganese peroxidase (MnP) have been previously described as efficient oxidizers of the endocrine disrupting chemical (EDC) nonylphenol at high concentrations of the pollutant. Envisaging the application of an enzymatic technology as a tertiary treatment in wastewater treatment plants, it is important to design a continuous reactor that performs the efficient removal of nonylphenol under environmental conditions. In the present research, a two-stage membrane bioreactor based on the production and use of Mn(3+)-malonate (chemical oxidant) was applied. The bioreactor consisted of an enzymatic reactor (R1) for the production of Mn(3+)-malonate by VP, coupled to an oxidation reactor (R2), where the oxidation of nonylphenol by Mn(3+)-malonate took place. The production of Mn(3+)-malonate in R1 was maintained constant: 500-700 µM with minimal deactivation of the enzyme. The oxidation reactor attained nearly complete removal of nonylphenol, even at a hydraulic retention time (HRT) shorter than 20 min. The operation with real wastewater containing nonylphenol at environmental concentrations (454 nM) was also successful, with a nonylphenol removal of 99.5% at a rate of 0.73 µM h(-1). Moreover, when the HRT of R2 was sharply reduced to 6.8 and 3.6 min, the removal of nonylphenol was maintained beyond 99%, which proves the feasibility of the system to remove the target compound present in a real effluent, even at very short HRTs.

Solvent screening methodology for in situ ABE extractive fermentation.

Solvent screening for in situ liquid extraction of products from acetone-butanol-ethanol (ABE) fermentation was carried out, taking into account biological parameters (biocompatibility, bioavailability, and product yield) and extraction performance (partition coefficient and selectivity) determined in real fermentation broth. On the basis of different solvent characteristics obtained from literature, 16 compounds from different chemical families were selected and experimentally evaluated for their extraction capabilities in a real ABE fermentation broth system. From these compounds, nine potential solvents were also tested for their biocompatibility towards Clostridium acetobutylicum. Moreover, bioavailability and differences in substrate consumption and total n-butanol production with respect to solvent-free fermentations were quantified for each biocompatible solvent. Product yield was enhanced in the presence of organic solvents having higher affinity for butanol and butyric acid. Applying this methodology, it was found that the Guerbet alcohol 2-butyl-1-octanol presented the best extracting characteristics (the highest partition coefficient (6.76) and the third highest selectivity (644)), the highest butanol yield (27.4 %), and maintained biocompatibility with C. acetobutylicum.

Operational strategies for producing bioethanol in a continuous single-stage reactor.

Novel strategies to facilitate the transition from batch to continuous simultaneous saccharification and fermentation were studied in this work. Implementing these strategies in bioethanol production plants to change production to a continuous mode will avoid large modifications in the process configuration. Therefore, experiments were carried out in a single-stage reactor applying strategies that favour a priori viability of yeast and stability of the process. The effects of (a) hydraulic residence time (HRT), (b) anaerobic and microaerobic operation, (c) inoculation strategy and (d) growth inhibition due to high ethanol concentrations were evaluated. The highest ethanol concentration (6.3 % w/w) was achieved during anaerobic operation, with reinoculations every 3-4 days and an HRT of 60 h; however, the processes suffered severe instability under these conditions. The greatest productivity and stability of the process was achieved using periodic microaeration and an HRT of 36 h (0.169 % ethanol weight/h), overcoming the result obtained during batch operation (0.128 % ethanol weight/h).

Activation of Kraft Lignin by an enzymatic treatment with a versatile peroxidase from Bjerkandera sp. R1.

Enzymatic lignin activation may be an environmentally friendly alternative to the use of chemicals in the production of wood fibers composites. Most studies on enzymatic activation of lignin for improving the adhesion of lignocellulosic products have been carried out using laccases. In this work, the use of a versatile peroxidase (VP) from the white-rot fungus Bjerkandera sp. (anamorph R1) for activating Kraft lignin was studied. The effect of enzyme dosage, incubation time, and H(2)O(2) addition profile on lignin activation was evaluated by quantifying the phenoxy radicals formed using electron paramagnetic resonance (EPR) spectroscopy. Two alternative enzymatic systems based on the use of VP (a two-stage and an enzymatic cascade system) were also assayed. At optimal conditions (dose of 15 U g(-1) and continuous addition of H(2)O(2) (5.24 µmol h(-1)) during 1 h) the content of phenoxy radicals was doubled as compared with an untreated control. Moreover, using the two-stage VP system, a lignin activation similar to that found at optimal conditions could be reached in a shorter time.

Enhanced saccharification of biologically pretreated wheat straw for ethanol production.

The biological pretreatment of lignocellulosic biomass with white-rot fungi for the production of bioethanol is an alternative to the most used physico-chemical processes. After biological treatment, a solid composed of cellulose, hemicellulose, and lignin-this latter is with a composition lower than that found in the initial substrate-is obtained. On the contrary, after applying physico-chemical methods, most of the hemicellulose fraction is solubilized, while cellulose and lignin fractions remain in the solid. The optimization of the combination of cellulases and hemicellulases required to saccharify wheat straw pretreated with the white-rot fungus Irpex lacteus was carried out in this work. The application of the optimal dosage made possible the increase of the sugar yield from 33 to 54 %, and at the same time the reduction of the quantity of enzymatic mixture in 40 %, with respect to the initial dosage. The application of a pre-hydrolysis step with xylanases was also studied.

Optimisation of the biological pretreatment of wheat straw with white-rot fungi for ethanol production.

The biological pretreatment of lignocellulosic biomass for the production of bioethanol is an environmentally friendly alternative to the most frequently used process, steam explosion (SE). However, this pretreatment can still not be industrially implemented due to long incubation times. The main objective of this work was to test the viability of and optimise the biological pretreatment of lignocellulosic biomass, which uses ligninolytic fungi (Pleurotus eryngii and Irpex lacteus) in a solid-state fermentation of sterilised wheat straw complemented with a mild alkali treatment. In this study, the most important parameters of the mechanical and thermal substrate conditioning processes and the most important parameters of the fungal fermentation process were optimised to improve sugar recovery. The largest digestibilities were achieved with fermentation with I. lacteus under optimised conditions, under which cellulose and hemicellulose digestibility increased after 21 days of pretreatment from 16 to 100 % and 12 to 87 %, respectively. The maximum glucose yield (84 %) of cellulose available in raw material was obtained after only 14 days of pretreatment with an overall ethanol yield of 74 % of the theoretical value, which is similar to that reached with SE.

Reductive dechlorination of α-, ß-, γ-, and δ-hexachlorocyclohexane isomers with hydroxocobalamin, in soil slurry systems.

The present study was carried out to test the viability of a method of reductive dehalogenation of α-, ß-, γ-, and δ-hexachlorocyclohexane (HCH) in soil slurry systems. The soil slurries were maintained under anaerobic conditions, with titanium(III) citrate as a reducing agent and hydroxocobalamin (vitamin B(12a)) as a catalyzing agent. Experiments were carried out with two soil samples with markedly different characteristics (particularly regarding organic matter content), at a small scale and larger reactor scale. HCH concentration was monitored throughout the 24 h duration of the tests. In the low organic matter soil HCH isomers degraded rapidly, in both the small scale and reactor systems, and undetectable levels (<0.5%) were reached within 5 h. However, complete degradation of HCH isomers was not achieved in soil with high organic matter content, and there were differences between the results obtained in the small scale and reactor systems. In the small scale system, the levels of degradation reached 93, 88, 94, and 91%, for α-, ß-, γ-, and δ-HCH, respectively, and the nondegraded HCH was sorbed in the soil. In the reactor system, the reaction stopped after two hours (no more than 65% of any of the isomers was degraded).

Effect of culture temperature on the heterologous expression of Pleurotus eryngii versatile peroxidase in Aspergillus hosts.

Production of recombinant versatile peroxidase in Aspergillus hosts was optimized through the modification of temperature during bioreactor cultivations. To further this purpose, the cDNA encoding a versatile peroxidase of Pleurotus eryngii was expressed under control of the alcohol dehydrogenase (alcA) promoter of Aspergillus nidulans. A dependence of recombinant peroxidase production on cultivation temperature was found. Lowering the culture temperature from 28 to 19 degrees C enhanced the level of active peroxidase 5.8-fold and reduced the effective proteolytic activity twofold. Thus, a maximum peroxidase activity of 466 U L(-1) was reached. The same optimization scheme was applied to a recombinant Aspergillus niger that bore the alcohol dehydrogenase regulator (alcR), enabling transformation with the peroxidase cDNA under the same alcA promoter. However, with this strain, the peroxidase activity was not improved, while the effective proteolytic activity was increased between 3- and 11-fold compared to that obtained with A. nidulans.

Effect of pH on the stability of Pleurotus eryngii versatile peroxidase during heterologous production in Emericella nidulans.

Complementary DNA (cDNA) encoding the new versatile peroxidase from the ligninolytic basidiomycete Pleurotus eryngii has been expressed in the ascomycete Emericella nidulans. In recombinant E. nidulans cultures, the pH reached values as high as 8.3, correlating with a sharp decrease in peroxidase activity. Peroxidase was rapidly inactivated at alkaline pH, but was comparatively stable at acidic pH. The peroxidase inactivation in alkaline buffer could be reversed by adding Ca(2+) and lowering the pH. However, reactivation did not result after incubating the enzyme in non-buffered E. nidulans cultures that reached pH 7.5. To optimize recombinant peroxidase production, the effect of controlling the pH in E. nidulans bioreactor cultures was studied. An extended growth period, and a significant increase in the recombinant peroxidase level (5.3-fold higher activity than in the bioreactor without pH control) was obtained when the pH was maintained at 6.8, showing that culture pH is an important parameter for recombinant peroxidase production.

Anaerobic and aerobic continuous cultures of Saccharomyces cerevisiae: comparison of plasmid stability and EXG1 gene expression.

Two bioreactor continuous cultures, at anaerobic and aerobic conditions, were carried out using a recombinant Saccharomyces cerevisiae strain that over-expresses the homologous gene EXG1. This recombinant system was used to study the effect of dissolved oxygen concentration on plasmid stability and gene over-expression. Bioreactor cultures were operated at two dilution rates (0.14 and 0.03 h(-1)) to investigate the effect of other process parameters on EXG1 expression. Both cultures suffered severe plasmid instability during the first 16 generations. Segregational plasmid loss rate for the aerobic culture was two-fold that of the anaerobic operation. In spite of this fact, exo-beta-glucanase activity at aerobic conditions was 12-fold that of the anaerobic culture. This maximal activity (30 U ml(-1)) was attained at the lowest dilution rate when biomass reached its greatest value and glucose concentration was zero.

Population dynamics of a continuous fermentation of recombinant Saccharomyces cerevisiae using flow cytometry.

The plasmid instability of genetically modified microorganisms during prolonged bioreactor operations is one of the major problems to be overcome in the production of recombinant proteins. The use of flow cytometry to monitor a fermentation process with recombinant cells in a CSTR is reported here. This technique has been applied to determine the fraction of plasmid-bearing cells (P+) of a recombinant Saccharomyces cerevisiae strain harboring the EXG1 gene in a continuous stirred tank bioreactor with a working volume of 2 L. The different levels in the expression of the EXG1 gene, which encodes the enzyme exo-beta-glucanase, were used to determine the P+ fraction. Other parameters such as viability, cellular protein, cell size and structure were also monitored using flow cytometry. This technique has two main advantages over the conventional method of determining the P+ fraction (plating in selective and non-selective solid media): (a) it takes a very short period of time to obtain a measurement that provides multiple parametric information; and (b) it is more representative of the bioreactor cell population since it can analyze thousands of cells in the same sample. A continuous operation (432 h) with the recombinant strain in a CSTR was carried out to test the application of this technique. Measurements of cellular exo-beta-glucanase activity and cellular protein content closely correlates to the measured fraction of plasmid-containing cells in the population. Moreover, the standard deviation of the fraction of P+ cells determined using this technique was very low (about 2%). Recombinant protein production also increased the size of the yeast cells, whereas the recombinant cells also had a more complex internal structure than the non-recombinant host strain.