Biotechnologia Acta

5-13

Authors: D. Koltysheva, K. Shchurska, Y. Kuzminskyi

A biofuel cell (BFС) is a bioelectrochemical device that can directly produce electricity or biohydrogen as a result of highly efficient “cold” fuel combustion. Nowadays there is no unified definitive classification and terminology, because BFCs are complex devices and they are still at research stage. Ukrainian scientists have proposed a classification of BFCsbased on nature of the biological component in the anode chamber, type of enzymes, presence of mediators, etc. Such classification is still relevant today, but due to the expansion of research areas and promising fields of BFC implementation and creation of hybrid and integrated systems there is a need to expand the review of existing BFC. The aim of the work was to study the current state of development of different BFC types and prospects for their implementation. Results of the analysis of modern publications in the field of BFC research have revealed a wide range of variations and possible promising fields of BFC application. Further research and implementation of these devices as environmentally friendly fuel for autonomous operation of robots, in biosensors and for wastewater treatment etc. should be based on the study of biotechnological parameters of biofilm formation and operation of BFC.

26-38

Authors: O. Mezhenska, A. Rebriev, O. Kobzar, N. Zlatoust, A. Vovk, Yu. Parkhomenko

The aim of this work was evaluation of binding affinity of thiamine to malate dehydrogenase isoforms. The methods of affinity chromatography, SDS PAGE electrophoresis, and MALDI-TOF mass spectroscopy, as well as molecular modeling in silico were used in the study. Affinity sorbent (T-AS) contained C2-conjugated thiamine fragment as an anchor which was bound to activated Sepharose 4B via a spacer composed of N-4-azobenzoylcaproic acid hydrazide. A commercial preparation of MDH from the porcine heart was chosen for the experimental study. Analysis of the protein content in fractions performed by the Bradford method showed that three separate protein peaks with malate dehydrogenase activity were obtained after elution with a thiamine solution. The results of one-dimensional electrophoresis of the initial MDH preparation and pooled fraction of proteins which were eluted from the affinity sorbent with a thiamine solution demonstrated that almost all protein fractions detected in the commercial MDH preparation were also present in eluates obtained by T-AS affinity chromatography. Four isoforms of MDH, including cytoplasmic malate dehydrogenase (MDH1), mitochondrial malate dehydrogenase (MDH2) and its isoform, and malate dehydrogenase 1B (MDH1B) were specifically bound to the affinity sorbent. According to the molecular docking results, the most preferred for both monomeric and homodimeric MDH1 and MDH2 could be ligand position at the NAD (NADH) binding site. Additional binding site could be located between two subunits of the homodimeric form of enzyme. Our results confirm the previously obtained data and expand an idea of ability of MDH isoforms to interact with the thiamine molecule in vivo. These data can also be useful for identification of thiamine binding protein (ThBP) which was previously isolated from rat brain, taking into account the possible partial homology of this protein with proteins that show MDH activity.

49-59

Authors: V .M. Hovorukha, O. A. Havryliuk, G. V. Gladka, O. B. Tashyrev,

Chromium(VI) is one of the most hazardous contaminant of the water and soil environment. Thermodynamic calculations enable to determine the optimal metabolic pathways for microbial extraction of toxic soluble hexavalent chromium compounds from contaminated sewage. The purpose of this research was to predict theoretically and confirm experimentally the possibility of hazardous chromium(VI) removal by hydrogen producing microbiome with simultaneous destruction of multicomponent organic waste and hydrogen synthesis. Gas composition was determined by the standard gas chromatography method. The redox potential (Eh) and рН of the medium were measured potentiometrically by EZODO MP-103 universal ionomer. The Cr(VI) concentration was determined by a photocolorimetric method with 1,5-diphenylcarbazide (DFK). Multicomponent organic waste was effectively destroyed by hydrogen producing microbiome in control conditions (at the absence of chromium). Maximum concentration of H2 was 36% and biohydrogen yield was 81 l/kg of absolutely dry weight of waste. The weight of organic waste was 92 times decreased (Kd=92) during only 82 hours. The hydrogen fermentation cycle was not significantly inhibited by addition of Cr(VI). Thus, the efficiency of waste destruction and biohydrogen yield decreased consequently on 10% (Kd=83) and 7% (75 l/kg of waste) after addition of 50 ppm Cr(VI) to the culture liquid at the beginning of the final phase (50 hours) of the fermentation. As it was expected, the addition of 100 ppm Cr(VI) into the liquid phase during the final fermentation phase (80 hours) caused rapid increase of the redox potential and short-term inhibition of the fermentation process. However, after complete reduction of soluble CrO42- to insoluble Cr(OH)3∙nH2O↓ by hydrogen producing microorganisms, H2 synthesis resumed and metabolic parameters returned to initial values (the Eh was -185 mV and -99 mV after complete extraction of 50 ppm and 100 ppm Cr(VI) consequently). The optimal pathway of microbial detoxification of toxic chromium(VI) compounds was thermodynamically predicted and experimentally confirmed. High efficiency of CrO42- removal by strict anaerobic hydrogen producing microbiome via dark hydrogen fermentation of multicomponent organic waste was demonstrated. The obtained results could be used as the basis for development of novel environmental biotechnology of chromium-containing sewage purification and simultaneous destruction of environmentally hazardous organic waste as well as obtaining of eco-friendly energy carrier – biohydrogen.

71-80

Authors: Pirog Т. P., Voronenko А. А., Yarosh М. B.

The aim of the work was to establish the cultivation conditions of Acinetobacter sp. IMV B-7005 for providing the maximum indicators of the exopolysaccharide ethapolan synthesis on the mixture of acetate and sunflower oil, as well as to study the possibility of replacing the refined oil in the mixture with acetate on a waste one. The optimal molar ratio of concentrations for refined sunflower oil and acetate in the mixture was calculated theoretically according to Babel’s concept of “auxiliary substrate”. The EPS concentration was determined gravimetrically after precipitation with isopropanol. The EPS-synthesizing ability was calculated as the ratio of the EPS concentration to the concentration of biomass and expressed in g EPS/g biomass. Based on theoretical calculations of energy requirements for EPS synthesis and biomass of Acinetobacter sp. B-7005 on energy-deficient substrate (acetate) it was found that molar ratio for the concentrations of sodium acetate and oil in the mixture, at which the maximum EPS synthesis was achieved, should be 1:0.13. It was experimentally confirmed that at this ratio of monosubstrate concentrations and the use of the inoculum grown on refined oil, the synthesis rates of ethapolan were higher than at the other ratios of acetate and oil concentrations in the mixture. However, the assimilation of sodium acetate through the symport with proton led to an increase in pH of the culture liquid to 9.0–9.3, which is not optimal for EPS synthesis. Decrease of the medium alkaline component and fractional introduction of substrates enabled not only to stabilize pH at the level of 7.8–7.9, but to increase the amount of synthesized ethapolan to 16–17 g/l, which was achieved regardless of the type of used oil (refined or mixed waste) in the mixture with acetate.