Nándor Nemestóthy

Enzymatic esterification in ionic liquids integrated with pervaporation for water removal

The application of ionic liquids as solvents for the enantioselective esterification of (R,S)-2-chloropropanoic acid with butan-1-ol using Candida rugosa lipase is reported. The role of water produced during the reaction and controlling of the water activity with pervaporation was studied. The enantioselective esterification of (R,S)-2-chloropropanoic acid with butan-1-ol in different organic solvents and ionic liquids was studied. The reaction was effectively catalysed by Candida rugosa lipase. From the ionic solvents investigated, the best results were achieved in [bmim]PF6. The presence of water had a strong effect on the activity of the lipase. Since in esterification reactions with acid and alcohol equimolar amounts of water are formed, the excess of water was removed and the water activity was kept constant using a pervaporation system without any additives. At the optimum water activity Candida rugosa lipase showed high thermal stability in [bmim]PF6 and it could be reused for at least five recycles with only a small lost of activity.

Improved microbial conversion of de-oiled Jatropha waste into biohydrogen via inoculum pretreatment: process optimization by experimental design approach

In this study various pretreatment methods of sewage sludge inoculum and the statistical process optimization of de-oiled jatropha waste have been reported. Peak hydrogen production rate (HPR) and hydrogen yield (HY) of 0.36 L H2/L-d and 20 mL H2/g Volatile Solid (VS) were obtained when heat shock pretreatment (95 oC, 30 min) was employed. Afterwards, an experimental design was applied to find the optimal conditions for H2 production using heat-pretreated seed culture. The optimal substrate concentration, pH and temperature were determined by using response surface methodology as 205 g/L, 6.53 and 55.1 oC, respectively. Under these circumstances, the highest HPR of 1.36 L H2/L-d was predicted. Verification tests proved the reliability of the statistical approach. As a result of the heat pretreatment and fermentation optimization, a significant (~ 4 folds) increase in HPR was achieved. PCR-DGGE results revealed that Clostridium sp. were majorly present under the optimal conditions.

Influence of pervaporation process parameters on enzymatic catalyst deactivation

Abstract This paper presents a study of enzymatic esterification of oleic-acid and i-amyl alcohol. The product of this reaction, i-amyl-oleate, is widely used as a bio-lubricant. During the esterification reaction, water is produced as a by-product, which has a disadvantageous effect on the reaction rate and enzyme activity. To enhance the effectiveness of the process, water should be removed. One of the most promising techniques for realizing this goal is pervaporation, which can be integrated on-line with the reaction system. Such integration can be realized in the form of a membrane reactor as was studied in this work. An immobilised lipase enzyme, Novozym 435 (Novo Nordisk, Denmark), which was taken as a catalyst in the experiments, proved to be very sensitive for the presence of water and alcohol in the reaction mixture. Below a certain level of water concentration, enzymatic catalyst activity is very low. From the other side, high initial concentrations of alcohol deactivate the catalyst. Both of these influences were taken into consideration and introduced into the mathematical model of an integrated esterification-pervaporation process. Computer calculations performed with the use of this model showed that there exists a range of pervaporation process parameters which prohibits the reaction from proceeding.

Biogenic H2 production from mixed microalgae biomass: impact of pH control and methanogenic inhibitor (BESA) addition

Hydrogen production from mixed microalgae biomass, predominantly containing Scendesmus and chlorella species, was investigated with a focus on enhancement strategies, in particular (i) pH control (at 5.5) and (ii) methanogenic inhibitor (BESA) addition along with pH control at 5.5. The results obtained showed that the later condition remarkably increased the performances. This was mainly ascribed to the occurrence of a suitable environment for the hydrogen producers to perform actively. Hydrogen production under these conditions (i.e., both pH 5.5 and pH5.5+BESA) was significantly higher than that of the control experiment. Using the pH control at 5.5 and BESA addition, peak hydrogen production rate (HPR) and hydrogen yield (HY) were attained as 210 mL/L/d and 29.5 mL/g VSadded, respectively. This improvement was nearly 3-folds higher compared with the control experiment with an HPR of 62 mL/L/d and an HY of 9.5 mL/g VSadded.

Biolubricant production in ionic liquids by enzymatic esterification

Esterification of oleic acid with short carbon chain alcohols gives biofuels, with long chain alcohols biolubricants can be produced. Previously biolubricants was successfully produced from fusel oil and isoamyl-alcohol substrates by enzyme catalised esterification, but solution of polar substrates was problematic, that was solved by using ionic liquids. The optimal reaction parameters such as substrate molar ratios, initial water content, amount of enzyme and type of ionic liquid were determined by experimental design using Statistica software. Some of the ionic liquids have catalytic effect by itself, therefore control reactions were necessary to determine which ionic liquid has negligible catalytic effect. In five of the six investigated ionic liquid the yield was the same without enzyme, while in case of B[mim]PF6, the yield was negligible. In shorter time higher yield was achieved in presence of ionic liquid against other methods. Increasing the temperature results higher yield till a limit, higher temperature does not make moreproduct, but thermal desactivation of the enzyme was not occur.

Asymmetric lactic acid esterification with biocatalysts in ionic liquid

Biodegradability and environmentally friendly technologies recently came into prominence; this is the reason why we assayed to develop a new “green” technology for L-lactic-acid (LLA) production. Racemic lactic acid (rLA) mixture produced by chemical industry is difficult to handle. The product of esterification with low carbon chain alcohols has higher volatility than lactic acid (LA) itself, therefore it can be more effectively separated. Our reactions were carried out with biocatalysts (enzymes) — some of them prefer reactionswith L-enantiomer — result in enantioselective esterification. After LLA ester production the hydrolysis leads to separated LLA, which is the starting material of a biodegradable plastic. Our aim was to achieve enantioselectivity in phosphonium-type ionic liquid solvents by the optimization of several parameters, such as temperature, substrate molar ratio, amount of IL, water content. Reasonable results were achieved with three types (Candida antarctica, Candida rugosa, AMANO PS-IM) of lipases. The use of enzymes and ionic liquids can make the technology “greener”, where an ingredient of a biodegradable plastic can be produced. The toxic heavy metals or hazardous acids can be replaced by biocatalyst (enzymes). These intermediates are re-usable, and they work at lower temperature, than conventional catalysts, thus the operational costs can be reduced. Ionic liquids — compared with conventional organic solvents — have insignificant vapour pressure, they are non-flammable and re-usable after a purification process, furthermore they can betailor made for a certain application. It is not negligible that the structure affects the environmental features like biodegradability or toxicity. The high lactic acid dissolving capacity is the reason why phosphonium-type ionic liquids were used. There are research teams, apply them for lactic acid extraction from fermentation broth.

Kinetic Study on Hydrolysis of Various Pectins by Aspergillus niger Polygalacturonase

Hydrolysis of pectins from various sources by a polygalacturonase (PG) enzyme was studied from a kinetic point of view. Pectin substrates – which are commercially not available – were extracted from sugar beet pulp, apple, red currant and black currant. Strong product inhibition was found in each pectin preparations that could be described by a competitive mechanism. The kinetic parameters (Michaelis-Menten constants, maximal reaction rates and inhibition constants) were determined and compared. Differences in the parameters imply distinctions in structure of the pectins studied.

Enzymatic Esterification of Lactic Acid under Microwave Conditions in Ionic Liquids

Ethyl lactate is a natural flavouring compound and can be used as an environmentally friendly solvent, as well. Lactic acid production requires costly downstream processes, which increases the price of the products. One of the latest purification methods is the extraction of the lactic acid from the fermentation broth by phosphonium type ionic liquids. This method gives the possibility to synthetise lactates in the extracting agent avoiding an expensive separation process. Microwave heating is widely used in organic chemistry because it usually shortens the reaction time and enhances the reaction rate, but its effect on enzymatic esterification reactions in ionic liquid media was hardly investigated. For comparison of the ethyl lactate synthesis in different media two organic solvents and 20 ionic liquids were tested. Eight suitable media were found: toluene and 7 ionic liquids. The reaction conditions of the enzymatic synthesis were optimised in toluene and in Cyphos 104. Using toluene the highest yield (80%) was achieved in a reaction mixture consisting of 1 mmol lactic acid, 5 mmol ethanol and 4.5 w/w% initial water content diluted by organic solvent to 5 cm3. The enzyme amount needed was 250 mg. In Cyphos 104 medium 0.8 mmol ionic liquid, 2 mmol lactic acid, 7 times ethanol excess, 2 w% initial water content and 25 mg immobilised Candida antarctica lipase B was enough to carry out the reaction up to 95% yield in 24 hour on 40 °C. The obtained yields and reaction parameters were compared using the two previous media and enzyme reusability tests were done. This experiment gave the result that smaller enzyme amount is enough in ionic liquid than in toluene and the enzyme stability is also much better in it. The synthesis was studied under microwave conditions as well, and the following effects were observed: The optimal initial water content was shifted from 3.7 w/w% to 3 w/w% but the same yield was achieved. Microwave heating accelerated the hydrolysis of lactoyllactic acid providing the mixture with fresh lactic acid and enhancing the reaction rate.

Comparative study of various E. coli strains for biohydrogen production applying response surface methodology

The proper strategy to establish efficient hydrogen-producing biosystems is the biochemical, physiological characterization of hydrogen-producing microbes followed by metabolic engineering in order to give extraordinary properties to the strains and, finally, bioprocess optimization to realize enhanced hydrogen fermentation capability. In present paper, it was aimed to show the utility both of strain engineering and process optimization through a comparative study of wild-type and genetically modified E. coli strains, where the effect of two major operational factors (substrate concentration and pH) on bioH2 production was investigated by experimental design and response surface methodology (RSM) was used to determine the suitable conditions in order to obtain maximum yields. The results revealed that by employing the genetically engineered E. coli (DJT 135) strain under optimized conditions (pH: 6.5; Formate conc.: 1.25u2009g/L), 0.63u2009mol H2/molu2009formate could be attained, which was 1.5 times higher compared to the wild-type E. coli (XL1-BLUE) that produced 0.42u2009molu2009H2/molu2009formate (pH: 6.4; Formate conc.: 1.3u2009g/L).

Biocatalytic Hydrogen Sulphide Removal from Gaseous Streams

Hydrogen sulphide is one of the most important substances responsible for unpleasant odour emissions in gas phase. It is often formed in higher concentration beyond other sulphur containing volatile compounds like methane thiol (MT), dimethyl sulphide (DMS) and dimethyl disulphide (DMDS). Removal of hydrogen sulphide is usually carried out by physical-chemical methods (e.g. adsorption), but nowadays some bio-processes may be considered as promising alternatives. Certain sulphur oxidising thiobacteria can be successfully applied for hydrogen sulphide conversion from gaseous streams like biogas. Various strains have been applied so far for degradation of hydrogen sulphide, they belong mostly to the group of Thiobacillus, which are autotrophic microorganisms. These autotrophic bacteria have the drawback in application that they grow slower than the heterotrophic ones and it is more difficult to control their growth. A number of chemotrophs are suitable for the biodegradation of H2S. These bacteria grow and produce new cell material by using inorganic carbon (CO2) as a carbon source and chemical energy from the oxidation of reduced inorganic compounds such as H2S.The objective of the work described here was to study the ability of elimination of hydrogen sulphide by two chemotrophic microorganisms (Thiomonas intermedia, Thiobacillus thioparus) in a batch bioreactor. The other aim was the study of the immobilization of these bacteria to different supports.