Bruno Zelić

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. Biotechnol. Bioeng. 2009; 104: 251–260

Modeling and kinetic parameter estimation of alcohol dehydrogenase-catalyzed hexanol oxidation in a microreactor

A mathematical model for hexanol oxidation catalyzed by NAD+‐dependent alcohol dehydrogenase from bakers yeast in a microreactor was developed and compared with the model when the reaction takes place in a macroscopic reactor. The enzyme kinetics was modeled as a pseudo‐homogeneous process with the double substrate Michaelis–Menten rate expression. In comparison with the kinetic parameters estimated in the cuvette, a 30‐fold higher maximum reaction rate and a relatively small change in the saturation constants are observed for the kinetic parameters estimated in the continuously operated tubular microreactor (Vm1=197.275 U/mg, Kmhexanol=9.420 mmol/L, and Km1NAD+=0.187 mmol/L). Kinetic measurements performed in the microreactor, estimated from the initial reaction rate experiments at the residence time of 36 s, showed no product inhibition, which could be explained by hydrodynamic effects and the continuous removal of inhibiting products. The Fourier amplitude sensitivity test method was applied for global kinetic parameter analysis, which shows a significant increase in the sensitivity of Km1NAD+ in the microreactor. Independent experiments performed in the microreactor were used to validate and to verify the developed mathematical model.

Enhancement of phenolic compounds oxidation using laccase from Trametes versicolor in a microreactor

Laccases catalyse the oxidation of a wide range of substrates by a radical-catalyzed reaction mechanism, with a corresponding reduction of oxygen to water in a four-electron transfer process. Due to that, laccases are considered environmentally friendly enzymes, and lately there has been great interest in their use for the transformation and degradation of phenolic compounds. In this work, enzymatic oxidation of catechol and L-DOPA using commercial laccase from Trametes versicolor was performed, in continuously operated microreactors. The main focus of this investigation was to develop a new process for phenolic compounds oxidation, by application of microreactors. For a residence time of 72 s and an inlet oxygen concentration of 0.271 mmol/dm3, catechol conversion of 41.3% was achieved, while approximately the same conversion of L-DOPA (45.0%) was achieved for an inlet oxygen concentration of 0.544 mmol/dm3. The efficiency of microreactor usage for phenolic compounds oxidation was confirmed by calculating the oxidation rates; in the case of catechol oxidation, oxidation rates were in the range from 76.101 to 703.935 g/dm3/d (18–167 fold higher, compared to the case in a macroreactor). To better describe the proposed process, kinetic parameters of catechol oxidation were estimated, using data collected from experiments performed in a microreactor. The maximum reaction rate estimated in microreactor experiments was two times higher than one estimated using the initial reaction rate method from experiments performed in a cuvette. A mathematical model of the process was developed, and validated, using data from independent experiments.

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.

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.

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.

Enzyme-catalysed Biodiesel Production from Edible and Waste Cooking Oils

Biodiesel synthesis was performed as transesterification of edible and waste cooking sunflower oil catalysed by free lipase from Thermomyces lanuginosus (Lipolase 100L). Experiments were performed at three different temperatures (T = 40, 50 and 60 °C) as one-step and four-step reactions with methanol. The highest fatty acids methyl esters (FAME) content (C = 95 %) was achieved in the one-step transesterification reaction of edible sunflower oil performed at 40 °C.

Optimization of biogas production from co-digestion of whey and cow manure

Anaerobic co-digestion is effective and environmentally attractive technology for energy recovery from organic waste. Organic, agricultural and industrial wastes are good substrates for anaerobic co-digestion because they contain high levels of easily biodegradable materials. In this paper enhancement of biogas production from codigestion of whey and cow manure was investigated in a series of batch experiments. The influence of whey ratio on specific biogas production in a mixture with cow manure was analyzed at 35 and 55°C, for different initial pH values and for different concentrations of supplemental bicarbonate in experiments carried out over 12 days. Good biogas production (6.6 dm3/dm3), methane content (79.4%) in a biogas mixture and removal efficiencies for total solids (16%) were achieved at optimum process conditions (temperature of 55°C, 10% v/v of whey and 5 g/dm3 NaHCO3 in the initial mixture). In order to validate optimized conditions for co-digestion of whey and cow manure in the one-stage batch process, the experiments were performed within 45 days. The high biogas production (21.8 dm3/dm3), a good methane content (78.7%) in a biogas mixture as well as maximum removal efficiencies for total solids (32.3%), and chemical oxygen demand (56.3%), respectively indicate that whey could be efficiently degraded to biogas in a onestage batch process when co-digested with cow manure.

Decolorization of dyes by Aspergillus ochraceus cultivated under solid state fermentation on sugar beet waste

Solid state fermentation (SSF) is defined as fermentation process performed in the absence of free water on non-soluble materials which can act as physical support and source of nutrient to microorganisms. In this paper, Aspergillus ochraceus was cultivated in solid state fermentation using sugar industry waste. In order to decolorize dyes, two different methods were applied using sugar beet waste a) as a substrate for A. ochraceus cultivation and as dyes adsorbent ; and b) as a substrate for A. ochraceus cultivation with the aim of extracellular enzymes production that can be used for dyes decolorization in batch experiments. 100 % conversion of textile violet dye decolorization, 57 % of textile green dye decolorization, 41.1 % of congo red and 51.9 % conversion of metylene blue was reached in batch experiments using produced extracellular enzymes.

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.