Mesocellular Silica Foam as Immobilization Carrier for Production of Statin Precursors.

The employment of 2-deoxyribose-5-phosphate aldolase (DERA) stands as a prevalent biocatalytic route for synthesizing statin side chains. The main problem with this pathway is the low stability of the enzyme. In this study, mesocellular silica foam (MCF) with different pore sizes was used as a carrier for the covalent immobilization of DERA. Different functionalizing and activating agents were tested and kinetic modeling was subsequently performed. The use of succinic anhydride as an activating agent resulted in an enzyme hyperactivation of approx. 140%, and the stability almost doubled compared to that of the free enzyme. It was also shown that the pore size of MCF has a decisive influence on the stability of the DERA enzyme.

Heterotrophic Cultivation of Euglena gracilis in Stirred Tank Bioreactor: A Promising Bioprocess for Sustainable Paramylon Production.

Paramylon is a valuable intracellular product of the microalgae Euglena gracilis, and it can accumulate in Euglena cells according to the cultivation conditions. For the sustainable production of paramylon and appropriate cell growth, different bioreactor processes and industrial byproducts can be considered as substrates. In this study, a complex medium with corn steep solid (CSS) was used, and various bioreactor processes (batch, fed batch, semicontinuous and continuous) were performed in order to maximize paramylon production in the microalgae Euglena gracilis. Compared to the batch, fed batch and repeated batch bioprocesses, during the continuous bioprocess in a stirred tank bioreactor (STR) with a complex medium containing 20 g/L of glucose and 25 g/L of CSS, E. gracilis accumulated a competitive paramylon content (67.0%), and the highest paramylon productivity of 0.189 g/Lh was observed. This demonstrated that the application of a continuous bioprocess, with corn steep solid as an industrial byproduct, can be a successful strategy for efficient and economical paramylon production.

Application of Causality Modelling for Prediction of Molecular Properties for Textile Dyes Degradation by LPMO.

The textile industry is one of the largest water-polluting industries in the world. Due to an increased application of chromophores and a more frequent presence in wastewaters, the need for an ecologically favorable dye degradation process emerged. To predict the decolorization rate of textile dyes with Lytic polysaccharide monooxygenase (LPMO), we developed, validated, and utilized the molecular descriptor structural causality model (SCM) based on the decision tree algorithm (DTM). Combining mathematical models and theories with decolorization experiments, we have elucidated the most important molecular properties of the dyes and confirm the accuracy of SCM model results. Besides the potential utilization of the developed model in the treatment of textile dye-containing wastewater, the model is a good base for the prediction of the molecular properties of the molecule. This is important for selecting chromophores as the reagents in determining LPMO activities. Dyes with azo- or triarylmethane groups are good candidates for colorimetric LPMO assays and the determination of LPMO activity. An adequate methodology for the LPMO activity determination is an important step in the characterization of LPMO properties. Therefore, the SCM/DTM model validated with the 59 dyes molecules is a powerful tool in the selection of adequate chromophores as reagents in the LPMO activity determination and it could reduce experimentation in the screening experiments.

Biogas Production Systems and Upgrading Technologies: A Review.

The underutilized biomass and different organic waste streams are nowadays in the focus of research for renewable energy production due to the effusive use of fossil fuels and greenhouse gas emission. In addition, one of the major environmental problems is also a constant increase of the number of organic waste streams. In a lot of countries, sustainable waste management, including waste prevention and reduction, has become a priority as a means to reduce pollution and greenhouse gas emission. Application of biogas technology is one of the promising methods to provide solutions for both actual energy-related and environmental problems. This review aims to present conventional and novel biogas production systems, as well as purification and upgrading technologies, nowadays applicable on a large scale, with a special focus on the CO2 and H2S removal. It also gives an overview of feedstock and the parameters important for biogas production, together with digestate utilization and application of molecular biology in order to improve the biogas production.

Effect of Enzymatic, Ultrasound, and Reflux Extraction Pretreatments on the Chemical Composition of Essential Oils.

The effect of different hydrodistillation pretreatments, namely, reflux extraction, reflux extraction with the addition of cell wall-degrading enzymes, and ultrasound, on the yield and chemical composition of essential oils of sage, bay laurel, and rosemary was examined. All pretreatments improved essential oil yield compared to no-pretreatment control (40-64% yield increase), while the oil quality remained mostly unchanged (as shown by statistical analysis of GC-MS results). However, enzyme-assisted reflux extraction pretreatment did not significantly outperform reflux extraction (no-enzyme control), suggesting that the observed yield increase was mostly a consequence of reflux extraction and enzymatic activity had only a minute effect. Thus, we show that ultrasound and reflux extraction pretreatments are beneficial in the production of essential oils of selected Mediterranean plants, but the application of enzymes has to be carefully re-evaluated.

Microreactor Production by PolyJet Matrix 3D-Printing Technology: Hydrodynamic Characterization§.

This work investigates the methodology of producing a 3D-printed microreactor from the acrylic resin by PolyJet Matrix process. The PolyJet Matrix technology employs different materials or their combinations to generate 3D-printed structures, from small ones to complex geometries, with different material properties. Experimental and numerical methods served for the evaluation of the geometry and production of the microreactor and its hydrodynamic characterization. The operational limits of the single-phase flow in the microchannels, further improvements and possible applications of the microreactor were assessed based on the hydrodynamic characterization.

Bioethanol production from raw sugar beet cossettes in horizontal rotating tubular bioreactor.

Fossil fuels are still major energy sources, but the search for renewable energy sources has been encouraged. Bioethanol has been recognized as an alternative to fossil fuels and nowadays it represents more than 90% of the global biofuel production. Bioethanol production from raw sugar beet cossettes as a semi-solid substrate was studied. The study was carried out in the horizontal rotating tubular bioreactor (HRTB) with Saccharomyces cerevisiae as a microbial production strain. The impact of different combinations of HRTB operational parameters such as, rotation speed (5-15 min-1), rotation type [constant or interval (3-15 min h-1)] and working volume (ratio V W/V T = 0.2-0.7) on the bioethanol production was examined. In this study, the highest bioprocess efficiency parameters ([Formula: see text] = 0.47 g g-1, E = 87.36% and Pr = 0.618 g L-1 h-1) were observed at 0.20 V W/V T, interval rotation of 12 min h-1 and rotation speed of 15 min-1. It has to be pointed out that bioethanol production efficiency in the HRTB was on the similar level as observed by bioethanol production from the raw sugar beet juice. Naturally present microorganisms of sugar beet could have a significant impact on bioethanol production. Higher yeast inoculation rate could reduce contaminant activities and, consequently, the bioethanol production efficiency would be improved.

Removal of Cr, Mn, and Co from textile wastewater by horizontal rotating tubular bioreactor.

Environmental pollution by industrial wastewaters polluted with toxic heavy metals is of great concern. Various guidelines regulate the quality of water released from industrial plants and of surface waters. In wastewater treatment, bioreactors with microbial biofilms are widely used. A horizontal rotating tubular bioreactor (HRTB) is a combination of a thin layer and a biodisc reactor with an interior divided by O-ring shaped partition walls as carriers for microbial biomass. Using a biofilm of heavy metal resistant bacteria in combination with this special design provides various advantages for wastewater treatment proven in a pilot study. In the presented study, the applicability of HRTB for removing metals commonly present in textile wastewaters (chromium, manganese, cobalt) was investigated. Artificial wastewaters with a load of 125 mg/L of each metal underwent the bioreactor treatment. Different process parameters (inflow rate, rotation speed) were applied for optimizing the removal efficiency. Samples were drawn along the bioreactor length for monitoring the metal contents on site by UV-vis spectrometry. The metal uptake of the biomass was determined by ICP-MS after acidic microwave assisted digestion. The maximum removal rates obtained for chromium, manganese, and cobalt were: 100%, 94%, and 69%, respectively.

Mathematical modeling of Fe(II), Cu(II), Ni(II) and Zn(II) removal in a horizontal rotating tubular bioreactor.

Industrial wastewaters polluted with toxic heavy metals are serious ecological and environmental problem. Therefore, in this study multi-heavy metals (Fe(2+), Cu(2+), Ni(2+) and Zn(2+)) removal process with mixed microbial culture was examined in the horizontal rotating tubular bioreactor (HRTB) by different combinations of process parameters. Hydrodynamic conditions and biomass sorption capacity have main impact on the removal efficiency of heavy metals: Fe(2+) 95.5-79.0%, Ni(2+) 92.7-54.8%, Cu(2+) 87.7-54.9% and Zn(2+) 81.8-38.1%, respectively. On the basis of experimental results, integral mathematical model of removal heavy metals in the HRTB was established. It combines hydrodynamics (mixing), mass transfer and kinetics to define bioprocess conduction in the HRTB. Mixing in the HRTB was described by structured cascade model and metal ion removal by two combined diffusion-adsorption models, respectively. For Langmuir model, average variances between experimental and simulated concentrations of metal ions were in the range of 1.22-10.99 × 10(-3) and for the Freundlich model 0.12-3.98 × 10(-3), respectively. On the basis of previous facts, it is clear that developed integral bioprocess model with Freundlich model is more efficient in the prediction of concentration of metal ions in the HRTB. Furthermore, the results obtained also pointed out that the established model is at the same time accurate and robust and therefore it has great potential for use in the scale-up procedure.

New approach to the evaluation of lignocellulose derived by-products impact on lytic-polysaccharide monooxygenase activity by using molecular descriptor structural causality model.

Lytic-polysaccharide monooxygenase (LPMO) is one of the most important enzyme involved in biocatalytic lignocellulose degradation, and therefore inhibition of LPMO has significant effects on all related processes. Structural causality model (SCM) were established to evaluate impact of phenolic by-products in lignocellulose hydrolysates on LPMO activity. The molecular descriptors GATS4c, ATS2m, BIC3 and VR2_Dzs were found to be significant in describing inhibition. The causalities of the molecular descriptors and LPMO activity are determined by evaluating the directed acyclic graph (DAG) and the d-separation algorithm. The maximum causality for LPMO activation is ß = 0.79 by BIC3 and the maximum causality of inhibition is ß = -0.56 for the GATS4c descriptor. The model has the potential to predict the inhibition of LPMO and its application could be useful in selecting an appropriate lignocellulose pretreatment method to minimise the production of a potent inhibitor. This will subsequently lead to more efficient lignocellulose degradation process.

Natural deep eutectic solvent as a unique solvent for valorisation of orange peel waste by the integrated biorefinery approach.

This research investigates the use of seven natural deep eutectic solvents (NADESs) for valorisation of orange peel waste, with the final goal to propose a unique NADES for integrated biorefinery. Initial screening of NADESs revealed the excellent ability of cholinium-based NADES with ethylene glycol as hydrogen bond donor (ChEg50) to serve as a medium for orange peel-catalysed kinetic resolution (hydrolysis) of (R,S)-1-phenylethyl acetate with high enantioselectivity (ee = 83.2%, X  = 35%), as well as it's stabilizing effect on the hydrolytic enzymes (hydrolytic enzymes within ChEg50 peel extract were stabile during 20 days at 4 °C). The ChEg50 also showed a satisfactory capacity to extract D-limonene (0.5 mg gFW-1), and excellent capacity to extract polyphenols (45.7 mg gFW-1), and proteins (7.7 mg gFW-1) from the peel. Based on the obtained results, the integrated biorefinery of orange peel waste using ChEg50 in a multistep process was performed. Firstly, enantioselective kinetic resolution was performed (step I; ee = 83.2%, X  = 35%), followed by isolation of the product 1-phenylethanol (step II; h = 82.2%) and extraction of polyphenols (step III; h = 86.8%) from impoverished medium. Finally, the residual orange peel was analysed for sugar and lignin content, and results revealed the potential of waste peel for the anaerobic co-digestion process. The main bottlenecks and futures perspective of NADES-assisted integrated biorefinery of orange peel waste were outlined through SWOT analysis.

Monitoring of Cu, Fe, Ni, and Zn in wastewater during treatment in a horizontal rotating tubular bioreactor.

The most appropriate systems for treatment of metal-contaminated waters are bioreactors with microbial biofilms. A horizontal rotating tubular bioreactor (HRTB) was studied for its applicability for removing copper, iron, nickel, and zinc (Cu, Fe, Ni, and Zn) from wastewater. Monitoring of the concentrations of Cu, Fe, Ni, and Zn by a fast, simple, onsite method was needed to make decisions for further optimization. The UV-VIS spectrophotometric quantification of Cu, Fe, Ni, and Zn using sodium diethyldithiocarbamate, 1,10-phenathroline, dimethylglyoxime, and 2-{[alpha-(2-Hydroxy-5-sulfophenylazo)-benzylidene]-hydrazino}-benzoic acid monosodium salt (=zincon monosodium salt) as reagents, respectively, was optimized and validated. The limits of quantification were 0.14, 0.12, 0.21, and 0.03 mg/L for Cu, Fe, Ni and Zn, respectively. The recovery for all elements was between 98 and 104%, the uncertainty of measurement was less than 6%. Depending on the reactor parameters applied, metal removals from 40 to more than 90% could be obtained.

Improved spectrophotometric assay for lytic polysaccharide monooxygenase.

BACKGROUND: The availability of a sensitive and robust activity assay is a prerequisite for efficient enzyme production, purification, and characterization. Here we report on a spectrophotometric assay for lytic polysaccharide monooxygenase (LPMO), which is an advancement of the previously published 2,6-dimethoxyphenol (2,6-DMP)-based LPMO assay. The new assay is based on hydrocoerulignone as substrate and hydrogen peroxide as cosubstrate and aims toward a higher sensitivity at acidic pH and a more reliable detection of LPMO in complex matrices like culture media. RESULTS: An LPMO activity assay following the colorimetric oxidation of hydrocoerulignone to coerulignone was developed. This peroxidase activity of LPMO in the presence of hydrogen peroxide can be detected in various buffers between pH 4-8. By reducing the substrate and cosubstrate concentration, the assay has been optimized for minimal autoxidation and enzyme deactivation while maintaining sensitivity. Finally, the optimized and validated LPMO assay was used to follow the recombinant expression of an LPMO in Pichia pastoris and to screen for interfering substances in fermentation media suppressing the assayed reaction. CONCLUSIONS: The biphenol hydrocoerulignone is a better substrate for LPMO than the monophenol 2,6-DMP, because of a ~ 30 times lower apparent K M value and a 160 mV lower oxidation potential. This greatly increases the measured LPMO activity when using hydrocoerulignone instead of 2,6-DMP under otherwise similar assay conditions. The improved activity allows the adaptation of the LPMO assay toward a higher sensitivity, different buffers and pH values, more stable assay conditions or to overcome low concentrations of inhibiting substances. The developed assay protocol and optimization guidelines increase the adaptability and applicability of the hydrocoerulignone assay for the production, purification, and characterization of LPMOs.

Comparing soluble and co-immobilized catalysts for 2-ketoaldose production by pyranose 2-oxidase and auxiliary enzymes.

The tri-enzyme system pyranose 2-oxidase (P2O), laccase, and catalase was used to study major parameters in the homogeneous and heterogeneous application of a multi-component enzymatic machinery. P2O oxidizes aldoses to 2-ketosugars, which are interesting intermediates in carbohydrate chemistry, and concomitantly reduces oxygen or alternative electron acceptors. The enzyme was immobilized on eleven agarose or acrylic resins using various coupling methods. The binding capacity was determined and an acrylic carrier with the most suitable properties selected for detailed studies. As P2O shows higher turnover numbers with the electron acceptor 1,4-benzoquinone than with oxygen, the use of this alternative electron acceptor was enabled by employing laccase for the continuous reoxidation of hydroquinone. The laccase regeneration system was found to increase the specific productivity up to 3-fold. Catalase was used to disproportionate the formed hydrogen peroxide in close proximity to the oxygen consuming enzymes and applied in different amounts to adjust the hydrogen peroxide concentration, which was found to be the main reason for enzyme deactivation under turnover conditions. In contrast to homogeneous catalysis, the specific productivity of heterogeneous catalysts under the applied experimental conditions was limited primarily by oxygen transfer, an effect significantly reduced by the laccase regeneration system.

Lignocellulose degradation: An overview of fungi and fungal enzymes involved in lignocellulose degradation.

This review aims to present current knowledge of the fungi involved in lignocellulose degradation with an overview of the various classes of lignocellulose-acting enzymes engaged in the pretreatment and saccharification step. Fungi have numerous applications and biotechnological potential for various industries including chemicals, fuel, pulp, and paper. The capability of fungi to degrade lignocellulose containing raw materials is due to their highly effective enzymatic system. Along with the hydrolytic enzymes consisting of cellulases and hemicellulases, responsible for polysaccharide degradation, they have a unique nonenzymatic oxidative system which together with ligninolytic enzymes is responsible for lignin modification and degradation. An overview of the enzymes classification is given by the Carbohydrate-Active enZymes (CAZy) database as the major database for the identification of the lignocellulolytic enzymes by their amino acid sequence similarity. Finally, the recently discovered novel class of recalcitrant polysaccharide degraders-lytic polysaccharide monooxygenases (LPMOs) are presented, because of these enzymes importance in the cellulose degradation process.

Fungal secretomes enhance sugar beet pulp hydrolysis.

The recalcitrance of lignocellulose makes enzymatic hydrolysis of plant biomass for the production of second generation biofuels a major challenge. This work investigates an efficient and economic approach for the enzymatic hydrolysis of sugar beet pulp (SBP), which is a difficult to degrade, hemicellulose-rich by-product of the table sugar industry. Three fungal strains were grown on different substrates and the production of various extracellular hydrolytic and oxidative enzymes involved in pectin, hemicellulose, and cellulose breakdown were monitored. In a second step, the ability of the culture supernatants to hydrolyze thermally pretreated SBP was tested in batch experiments. The supernatant of Sclerotium rolfsii, a soil-borne facultative plant pathogen, was found to have the highest hydrolytic activity on SBP and was selected for further hydrolyzation experiments. A low enzyme load of 0.2 mg g(-1) protein from the culture supernatant was sufficient to hydrolyze a large fraction of the pectin and hemicelluloses present in SBP. The addition of Trichoderma reesei cellulase (1-17.5 mg g(-1) SBP) resulted in almost complete hydrolyzation of cellulose. It was found that the combination of pectinolytic, hemicellulolytic, and cellulolytic activities works synergistically on the complex SBP composite, and a combination of these hydrolytic enzymes is required to achieve a high degree of enzymatic SBP hydrolysis with a low enzyme load.

Integrated hydrolyzation and fermentation of sugar beet pulp to bioethanol.

Sugar beet pulp is an abundant industrial waste material that holds a great potential for bioethanol production owing to its high content of cellulose, hemicelluloses, and pectin. Its structural and chemical robustness limits the yield of fermentable sugars obtained by hydrolyzation and represents the main bottleneck for bioethanol production. Physical (ultrasound and thermal) pretreatment methods were tested and combined with enzymatic hydrolysis by cellulase and pectinase to evaluate the most efficient strategy. The optimized hydrolysis process was combined with a fermentation step using a Saccharomyces cerevisiae strain for ethanol production in a single-tank bioreactor. Optimal sugar beet pulp conversion was achieved at a concentration of 60 g/l (39% of dry weight) and a bioreactor stirrer speed of 960 rpm. The maximum ethanol yield was 0.1 g ethanol/g of dry weight (0.25 g ethanol/g total sugar content), the efficiency of ethanol production was 49%, and the productivity of the bioprocess was 0.29 g/l·h, respectively.