Deep Eutectic Solvents for Biodiesel Purification in a Microextractor: Solvent Preparation, Selection and Process Optimization.

The most important and commonly used process for biodiesel synthesis is transesterification. The main by-product of biodiesel synthesis by transesterification is glycerol, which must be removed from the final product. Recently, deep eutectic solvent (DES) assisted extraction has been shown to be an effective and sustainable method for biodiesel purification. In this study, biodiesel was produced by lipase-catalysed transesterification from sunflower oil and methanol. A total of 12 different eutectic solvents were prepared and their physical properties were determined. Mathematical models were used to define which physical and chemical properties of DES and to what extent affect the efficiency of extraction of glycerol from the biodiesel. After initial screening, cholinium-based DES with ethylene glycol as hydrogen bond donor was selected and used for optimization of extraction process conditions performed in a microsystem. To determine the optimal process conditions (temperature, biodiesel:DES volume ratio, residence time), the experimental three-level-three-factor Box-Behnken experimental design was used. In the end, a combination of a mathematical model and experimental results was used to estimate how many micro-extractors are necessary for the complete removal of glycerol.

Development of NIR-Based ANN Models for On-Line Monitoring of Glycerol Concentration during Biodiesel Production in a Microreactor.

During the production process, a whole range of analytical methods must be developed to monitor the quality of production and the desired product(s). Most of those methods belong to the group of off-line monitoring methods and are usually recognized as costly and long-term. In contrast, on-line monitoring methods are fast, reliable, simple, and repeatable. The main objective of this study was to compare different methods for monitoring total glycerol concentration as one of the indicators of process efficiency during biodiesel production in a batch reactor and in a microreactor. During the biodiesel production process, the glycerol concentration was measured off-line using standard methods based on UV-VIS spectrophotometry and gas chromatography. Neither method provided satisfactory results, namely, both analyses showed significant deviations from the theoretical value of glycerol concentration. Therefore, near infrared spectroscopy (NIR) analysis was performed as an alternative analytical method. The analysis using NIR spectroscopy was performed in two ways: off-line, using a sample collected during the transesterification process, and on-line by the continuous measurement of glycerol concentration in a rector. Obtained results showed a great NIR application potential not only for off-line but also for on-line monitoring of the biodiesel production process.

An enhanced composting process with bioaugmentation: Mathematical modelling and process optimization.

In this paper, two different types of biowaste composting processes were carried out - composting without and with bioaugmentation. All experiments were performed in an adiabatic reactor for 14 days. Composting enhanced with bioaugmentation was the better choice because the thermophilic phase was achieved earlier, making the composting time shorter. Additionally, a higher conversion of substrate (amount of substrate consumed) was also noticed in the process enhanced by bioaugmentation. A mathematical model was developed and process parameters were estimated in order to optimize the composting process. Based on good agreement between experimental data and the mathematical model simulation results, a three-level-four-factor Box-Behnken experimental design was employed to define the optimal process conditions for further studies. It was found that the air flow rate and the mass fraction of the substrate have the most significant effect on the composting process. An improvement of the composting process was achieved after altering the mentioned variables, resulting in shorter composting time and higher conversion of the substrate.

Transesterification in Microreactors-Overstepping Obstacles and Shifting Towards Biodiesel Production on a Microscale.

Biodiesel, which was earlier used only as an alternative fuel, is now an indispensable component of commercial diesel. Conventional production processes are unable to cope with the increasing demand for biodiesel, and therefore more and more work is being done to intensify the existing processes. The intensification of the biodiesel production process, taking into account the environmental and economic factors, is based on increasing productivity. One way to achieve that is by reducing the volume of production units. The application of the enzymatic reaction path, while reducing the volume of process equipment to the micro-level, has significantly magnified the productivity of the biodiesel production process, which is primarily due to better mass transfer in microsystems. Additional breakthrough is the use of deep eutectic solvents (DES) instead of buffers for enzyme stabilization. In this study, a lipase from Thermomyces lanuginosus (TlL) (both commercial and produced by solid-state fermentation) was used as a catalyst for biodiesel production. Edible and waste sunflower oil, as well as methanol, were used as substrates. The reaction mediums were buffer and DES. The transesterification reaction was carried out in a batch reactor and the emphasis was made on different microreactor configurations. The highest yield of 32% for residence time of only τ = 30 min was obtained in the microreactor system with an emulsion of waste oil and a commercial enzyme suspended in a buffer. This indicates that enzymatic transesterification could be a valuable reaction path for dealing with waste oils. Furthermore, biodiesel synthesis in DES showed somewhat lower yields, but by increasing the water content in the system, the reaction could prove much better results. In the end, the effects of reaction conditions on the volumetric productivity of the process were analyzed.

Kinetic Parameter Estimation and Mathematical Modelling of Lipase Catalysed Biodiesel Synthesis in a Microreactor.

Development of green, clean, and sustainable processes presents new challenges in today's science. Production of fuel is no exception. Considering the utilisation of various renewable sources, the synthesis of biodiesel, characterised as more environmentally-friendly then fossil fuel, has drawn significant attention. Even though the process based on chemical transesterification in a batch reactor still presents the most used method for its production, enzyme catalysed synthesis of biodiesel in a microreactor could be a new approach for going green. In this research, edible sunflower oil and methanol were used as substrates and lipase from Thermomyces lanuginosus (Lipolase L100) was used as catalyst for biodiesel synthesis. Experiments were performed in a polytetrafluoroethylene (PTFE) microreactor with three inlets and in glass microreactors with two and three inlets. For a residence time of 32 min, the fatty acids methyl esters (FAME) yield was 30% higher than the yield obtained for the glass microreactor with three inlets. In comparison, when the reaction was performed in a batch reactor (V = 500 mL), the same FAME yield was achieved after 1.5 h. In order to enhance the productivity of the process, we used proposed reaction kinetics, estimated kinetic parameters, and a mathematical model we developed. After validation using independent experimental data, a proposed model was used for process optimization in order to obtain the highest FAME yield for the shortest residence time.

Lipase catalysed biodiesel synthesis with integrated glycerol separation in continuously operated microchips connected in series.

Although the application of microreactors in different processes has been extensively explored in recent decades, microreactors continue to be underexplored in the context of the enzyme-catalysed process for biodiesel production. Due to their numerous advantages, microreactors could become the next step in the development of a biodiesel production process characterised by sustainability, cost-effectiveness and energy efficiency. In this investigation, biodiesel production was catalysed by lipase from Thermomyces lanuginosus (Lipolase L100). Edible sunflower oil was used as a model substrate in order to investigate the process. After optimal process conditions had been determined, waste-cooking oil was used for biodiesel production to make the production process more sustainable. Three different substrate-feeding strategies were investigated and finally an optimal strategy was proposed. In all the investigated systems, fatty acids methyl esters (FAME) content was higher than 95% and obtained in a significantly shorter time (less than 2 h) compared to the batch process in which biodiesel production was catalysed by lipase (C = 95%, t = 96 h). After the optimal biodiesel production system had been proposed, an integrated system with two microchips connected in series was developed. The first microchip was used for biodiesel production and the second for simultaneous purification i.e. glycerol separation. Finally, purified biodiesel was produced with glycerol content below the detection limit.

Corn silage fungal-based solid-state pretreatment for enhanced biogas production in anaerobic co-digestion with cow manure.

The objective of this research was to use white-rot fungus Trametes versicolor for corn silage pretreatment and to investigate the effect of pretreatment on biogas productivity. Semi-continuous pilot-scale experiment, comprised of two experimental phases, was carried out. In the first phase, operational conditions of the full-scale biogas plant were reproduced at pilot-scale. In that phase, the reactor was daily fed with the mixture of cow manure, digestate from industrial postfermentor, corn grits and ensiled corn silage, and the average methane generation rate was 0.167 m3CH4 kgVS-1. In the second phase, corn grits and ensiled corn silage were replaced with corn silage pretreated with T. versicolor, and the average methane generation rate increased up to 0.236 m3CH4 kgVS-1. The results of this study suggest that application of fungal-based solid-state pretreated corn silage has positive effect on pH stability and increase the biogas productivity.

Proximate analysis of cold-press oil cakes after biological treatment with Trametes versicolor and Humicola grisea.

In order to increase the current knowledge on cold-press oil cakes composition, the present study aims to determine the chemical composition of oil cakes from hull-less pumpkin (Cucurbita pepo L.), flax (Linum usitatissimum L.), and hemp (Canabis sativa L.) before and after the biological treatment with Trametes versicolor and Humicola grisea using fungal-based solid-state technology. After 10 days of treatment, the content of ash, total nitrogen, total proteins, and total organic carbon increased in all the three oil cakes, while the content of ether extracts decreased. After treatment, the concentration of soluble carbohydrates decreased in pumpkin and hemp seed oil cakes, whereas it increased in flaxseed oil cake. During treatment with T. versicolor, the content of fructose significantly increased in hull-less pumpkin seed oil cake. Fiber content decreased in pumpkin and flaxseed oil cakes after treatment with both of the fungi, whereas it increased in flaxseed oil cake.

Synergy of Microtechnology and Biotechnology: Microreactors as an Effective Toolfor Biotransformation Processes§: §The paper was presented at European Biotechnology Congress, 25-27 May 2017, Dubrovnik, Croatia.

Despite the fact that microreactors have been present for more than 40 years now and that their potential has been extensively exploited in chemical synthesis, analytics and screening, to date very few biocatalytic processes have been explored in microreactors. It is claimed that enzymatic microreactor technology is exactly in the same place where chemical microreactors were 15 years ago. However, general opinion is that the efforts devoted to the research of micro-enzymatic reactors will inaugurate a new breakthrough in bio-based processing. The aim of this review is to explore the synergy between microtechnology, mainly microreactors, and biotechnology, and to assess its potential, opportunities, challenges and future application in biotechnology.

Biogas Production from Brewer's Yeast Using an Anaerobic Sequencing Batch Reactor.

Renewable energy sources are becoming increasingly important in the beverage and food industries. In the brewing industry, a significant percentage of the used raw materials finishes the process as secondary resource or waste. The research on the anaerobic digestion of brewer's yeast has been scarce until recent years. One of the reasons for this is its use as a secondary resource in the food industry and as cattle feed. Additionally, market value of brewer's yeast is higher than its energy value. Due to the increase of energy prices, brewer's yeast has become of interest as energy substrate despite its difficult degradability in anaerobic conditions. The anaerobic co-digestion of brewer's yeast and anaerobically treated brewery wastewater was studied using a pilot-scale anaerobic sequencing batch reactor (ASBR) seeded with granular biomass. The experiments showed very good and stable operation with an organic loading rate of up to 8.0 kg/(m3·day), and with a maximum achieved organic loading rate of 13.6 kg/(m3·day) in a single cycle. A specific biogas productivity of over 0.430 m3/kg of the total chemical oxygen demand (COD) inserted, and total COD removal efficiencies of over 90% were achieved. This study suggests that the brewer's yeast can be successfully digested in an ASBR without adverse effects on the biogas production from brewer's yeast/wastewater mixtures of up to 8% (by volume). By using the brewer's yeast in the ASBR process, the biogas production from brewery wastewater could be increased by 50%.

Catechol Removal from Aqueous Media Using Laccase Immobilized in Different Macro- and Microreactor Systems.

Laccase belongs to the group of enzymes that are capable to catalyze the oxidation of phenols. Since the water is only by-product in laccase-catalyzed phenol oxidations, it is ideally "green" enzyme with many possible applications in different industrial processes. To make the oxidation process more sustainable in terms of biocatalyst consumption, immobilization of the enzyme is implemented in to the processes. Additionally, when developing a process, choice of a reactor type plays a significant role in the total outcome.In this study, the use of immobilized laccase from Trametes versicolor for biocatalytic catechol oxidation was explored. Two different methods of immobilization were performed and compared using five different reactor types. In order to compare different systems used for catechol oxidation, biocatalyst turnover number and turnover frequency were calculated. With low consumption of the enzyme and good efficiency, obtained results go in favor of microreactors with enzyme covalently immobilized on the microchannel surface.

Recovery of Phenolic Acid and Enzyme Production from Corn Silage Biologically Treated by Trametes versicolor.

Corn silage is used as high-energy forage for dairy cows and more recently for biogas production in a process of anaerobic co-digestion with cow manure. In this work, fresh corn silage after the harvest was used as a substrate in solid-state fermentations with T. versicolor with the aim of phenolic acid recovery and enzyme (laccase and manganese peroxidase) production. During 20 days of fermentation, 10.4-, 3.4-, 3.0-, and 1.8-fold increments in extraction yield of syringic acid, vanillic acid, p-hydroxybenzoic acid, and caffeic acid, respectively, were reached when compared to biologically untreated corn silage. Maximal laccase activity was gained on the 4th day of fermentation (V.A. = 180.2 U/dm3), and manganese peroxidase activity was obtained after the 3rd day of fermentation (V.A. = 30.1 U/dm3). The addition of copper(II) sulfate as inducer during solid state fermentation resulted in 8.5- and 7-fold enhancement of laccase and manganese peroxidase activities, respectively. Furthermore, the influence of pH and temperature on enzyme activities was investigated. Maximal activity of laccase was obtained at T = 50 °C and pH = 3.0, while manganese peroxidase is active at temperature range T = 45-70 °C with the maximal activity at pH = 4.5.

Corn forage biological pretreatment by Trametes versicolor in a tray bioreactor.

Trametes versicolor is a white-rot fungus known to be efficient in lignin removal due to its complex extracellular lignocellulolytic enzymatic system. Therefore, it can be used in the treatment of lignocellulose waste from agro, food, and wood industries. In a first experiment, corn forage treatment with T. versicolor was investigated in laboratory jars. In a second experiment, the process was scaled up to a tray bioreactor. In the tray bioreactor, the process of lignin degradation was improved, resulting in an increase in lignin conversion of up to 71% during seven days' treatment.

Anaerobic Biodegradation of Raw and Pre-treated Brewery Spent Grain Utilizing Solid State Anaerobic Digestion.

The brewery spent grain (BSG) represents approximately 85% of the total quantity of by-products from the brewing industry. The biogas production from the BSG has been the subject of several studies in recent years, due to relatively high energy consumption in the brewing process and due to the increasing energy costs. The biodegradability of raw and pre-treated BSG in a single-stage and two-stage solid-state anaerobic digestion (SS-AD) system was determined in this study. The results show that the BSG have a biogas potential of 120 L/kg(-1). In the single-stage system, the biogas yield obtained from raw BSG (87.4 L/kg(-1)) was almost equal to the yield obtained from the pre-treated BSG (89.1 L/kg(-1)), while the methane yield was 51.9 and 55.3 L/kg(-1) and the biodegradation was 62.0% and 62.2% for raw and pre-treated BSG, respectively. In two-stage SS-AD the pre-treated BSG showed better results, with the biogas yield of 103.2 L/kg(-1) and the biodegradation of 73.6%, while the biogas yield obtained from raw BSG was 89.1 L/kg(-1), with the biodegradation of 63.5%. In two-stage process the obtained methane yields from raw and pre-treated BSG were identical (58.7 L/kg(-1)).

Bioproduction of food additives hexanal and hexanoic acid in a microreactor.

Hexanal and hexanoic acid have number of applications in food and cosmetic industry because of their organoleptic characteristics. Problems like low yields, formation of unwanted by-products, and large quantities of waste in their traditional production processes are the reasons for developing new production methods. Biotransformation in a microreactor, as an alternative to classical synthesis processes, is being investigated. Because conditions in microreactors can be precisely controlled, the quality of the product and its purity can also be improved. Biocatalytic oxidation of hexanol to hexanal and hexanoic acid using suspended and immobilized permeabilized whole baker's yeast cells and suspended and immobilized purified alcohol dehydrogenase (ADH) was investigated in this study. Three different methods for covalent immobilization of biocatalyst were analyzed, and the best method for biocatalyst attachment on microchannel wall was used in the production of hexanal and hexanoic acid.

Modelling of the whey and cow manure co-digestion process.

The production of renewable energy, a reduction of waste and prevention of environmental pollution promote the industrial application of anaerobic co-digestion for the treatment of agro-industrial organic waste. In this paper production of biogas/methane was studied by performing a series of laboratory batch experiments using whey and cow manure as substrates. The influence of substrate concentration, temperature and pH on biogas production was analysed. A mathematical model has been developed that describes the co-digestion process. The hydrolysis of proteins, lipids and cellulose has been modelled using first-order kinetics. Fermentation of sugars and amino acids, anaerobic oxidation of long chain fatty acids (LCFA), acetogenesis and methanogenesis have been described using an unstructured model based on Monod kinetic equations taking into account different inhibitory effects. Model applicability was demonstrated by comparing experimental results with the model simulation results.

Optimization of laccase production by Trametes versicolor cultivated on industrial waste.

Laccases are very interesting biocatalysts for several industrial applications. Its production by different white-rot fungi can be stimulated by a variety of inducing substrates, and the use of lignocellulosic wastes or industrial by-products is one of the possible approaches to reduce production costs. In this work, various industrial wastes were tested for laccase production by Trametes versicolor MZKI G-99. Solid waste from chemomechanical treatment facility of a paper manufacturing plant showed the highest potential for laccase production. Enzyme production during submerged cultivation of T. versicolor on the chosen industrial waste has been further improved by medium optimization using genetic algorithm. Concentrations of five components in the medium were optimized within 60 shake-flasks experiments, where the highest laccase activity of 2,378 U dm(-3) was achieved. Waste from the paper industry containing microparticles of CaCO(3) was found to stimulate the formation of freely dispersed mycelium and laccase production during submerged cultivation of T. versicolor. It was proven to be a safe and inexpensive substrate for commercial production of laccase and might be more widely applicable for metabolite production by filamentous fungi.

Mathematical model for Trametes versicolor growth in submerged cultivation.

Trametes versicolor is a white-rot fungus known as a producer of extracellular enzymes such as laccase, manganese-peroxidase, and lignin-peroxidase. The production of these enzymes requires detailed knowledge of the growth characteristics and physiology of the fungus. Submerged cultivations of T. versicolor on glucose, fructose, and sucrose as sole carbon sources were performed in shake flasks. Sucrose hydrolysis catalyzed by the whole cells of T. versicolor was considered as one-step enzymatic reaction described with Michaelis-Menten kinetics. Kinetic parameters of invertase-catalyzed sucrose hydrolysis were estimated (K (m) = 7.99 g dm(-3) and V (m) = 0.304 h(-1)). Monod model was used for description of kinetics of T. versicolor growth on glucose and fructose as sole carbon sources. Growth associated model parameters were estimated from the experimental results obtained by independent experiments (mu(G)(max) = 0.14 h(-1), K(G)(S) = 8.06 g dm(-3), mu(F)(max) = 0.37 h(-1) and K(F)(S) = 54.8 g dm(-3)). Developed mathematical model is in good agreement with the experimental results.

Metabolomics for biotransformations: Intracellular redox cofactor analysis and enzyme kinetics offer insight into whole cell processes.

For redox reactions catalyzed by microbial cells the analysis of involved cofactors is of special interest since the availability of cofactors such as NADH or NADPH is often limiting and crucial for the biotransformation efficiency. The measurement of these cofactors has usually been carried out using spectrophotometric cycling assays. Today LC-MS/MS methods have become a valuable tool for the identification and quantification of intracellular metabolites. This technology has been adapted to measure all four nicotinamide cofactors (NAD, NADP, NADH, and NADPH) during a whole cell biotransformation process catalyzed by recombinant Escherichia coli cells. The cells overexpressing an alcohol dehydrogenase from Lactobacillus brevis were used for the reduction of methyl acetoacetate (MAA) with substrate-coupled cofactor regeneration by oxidation of 2-propanol. To test the reliability of the measurement the data were evaluated using a process model. This model was derived using the measured concentrations of reactants and cofactors for initiation as well as the kinetic constants from in vitro measurements of the isolated enzyme. This model proves to be highly effective in the process development for a whole cell redox biotransformation in predicting both the right concentrations of cofactors and reactants in a batch and in a CSTR process as well as the right in vivo expression level of the enzyme. Moreover, a sensitivity analysis identifies the cofactor regeneration reaction as the limiting step in case for the reduction of MAA to the corresponding product (R)-methyl 3-hydroxybutyrate. Using the combination of in vitro enzyme kinetic measurements, measurements of cofactors and reactants and an adequate model initiated by intracellular concentrations of all involved reactants and cofactors the whole cell biotransformation process can be understood quantitatively.

Process strategies to enhance pyruvate production with recombinant Escherichia coli: from repetitive fed-batch to in situ product recovery with fully integrated electrodialysis.

Using the pyruvate production strain Escherichia coli YYC202 ldhA::Kan different process alternatives are studied with the aim of preventing potential product inhibition by appropriate product separation. This strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate, resulting in acetate auxotrophy during growth in glucose minimal medium. Continuous experiments with cell retention, repetitive fed-batch, and an in situ product recovery (ISPR) process with fully integrated electrodialysis were tested. Although the continuous approach achieved a high volumetric productivity (QP) of 110 g L(-1) d(-1), this approach was not pursued because of long-term production strain instabilities. The highest pyruvate/glucose molar yield of up to 1.78 mol mol(-1) together with high QP 145 g L(-1) d(-1) and high pyruvate titers was achieved by the repetitive fed-batch approach. To separate pyruvate from fermentation broth a fully integrated continuous process was developed. In this process electrodialysis was used as a separation unit. Under optimum conditions a (calculated) final pyruvate titer of >900 mmol L(-1) (79 g L(-1)) was achieved.