ИСТИНА

Microbial fuel cells (MFC) – are electrochemical devices, which are capable of converting chemical energy into electricity by the metabolic activity of microorganisms, since the electron transport chains (ETC) of these microorganisms can carry out an electron transfer to external insoluble acceptors. The possibility of using microbial fuel cell for purposes of electricity generation was demonstrated first time in 1912t, but about 90% of published works in this area is related to the last 10-15 years. The international scientific journal ChemSusChem (impact factor 6.827) devoted the special issue to MFC technology in June 2012 (ChemSusChem, 2012, V. 5, I. 6). This is mainly caused by the prospect of using MFC in wastewater treatment and processing of various types of waste from the relatively safe food wastes to toxic and even radioactive wastes. At the same time, the study of bioelectrochemical reduction of mineral compounds has been carried out for more than two decades, but the molecular mechanisms of this process remain widely unknown. The fact that this is a complex problem and its solution requires cooperation of specific methodological approaches (microbiological, molecular biological, ecological, geological and etc.). Moreover, there is a little understanding how the process works at the level of the genome of microorganisms which are capable to implement this type of anaerobic respiration. Now scientists have fully sequenced genomes of only two most studied microorganisms which are capable of carrying out the process of mineral compounds reduction (Shewanella oneidensis MR-1 and Geobacter sulfurreducens). But we should not forget that the mechanism of anaerobic respiration is specific to each individual mineral compound, the electron acceptor. In this regard, the study of the process of bioelectrochemical reduction of mineral compounds is very important from both the point of view fundamental problems and from the point of view of technology MFC development. In the framework of the project that I perform in Moscow State University it was suggested to focus on research of system that includes microorganism interacting with mineral compound. For this purpose, it was necessary to isolate microorganisms from natural habitat where they normally exist in the form of multicomponent syntrophic microbial communities. The communities will be more stable to the introduction of contaminated raw material in to anode part of MFC compare with pure culture of electrochemical active microorganisms (Shewanella oneidensis, Geobacter sulfurreducens and etc.). However, the main criteria for the selection of these communities is its electrochemical activity and ability to process complex organic substrates. Therefore, the selected communities of microorganisms were studied in a specially designed electrochemical bioreactor cells. As the main substrate for microorganisms we used distillery grains. Some of the most effective communities were selected. Now I am interested in a detailed study of selected syntrophic communities. In the nature, the mineral reduction process usually carried out by different types of microorganisms that are only small part of microbial communities. These communities can include dozens of species among of which only a few are able to reduce the mineral compounds. The ‘electrogenic” microorganisms are components of such communities. As substrates “electrogenic” microorganisms can utilize glucose, pyruvate, fumarate, acetate, and other easily accessible compounds. In natural anaerobic niches these organisms can compete with acetoclastic methanogens for the available acetate. Homoacetogens reduce CO2 with hydrogen to form acetate, at the same time in the presence of acetate-consuming microorganisms they oxidize sugars and lactate to acetate. Thus the second process coupled to hydrogen formation. Therefore during consumption of complex substrates in anaerobic environments, key organisms can use or form hydrogen depending on the prevailing conditions. Syntrophic communities are known to be involved in the degradation of substrates that cannot be fermented by individual species alone, but the importance of syntrophy is less apparent for substrates that are easily fermented. Therefore for the processing of distillers grains using the microbial community is necessary. Further we should consider the metabolic flexibility of the microorganisms involved in syntrophic communities. The availability of genome sequences of key microorganisms will allow us to explore this flexibility in detail. The physiological studies will give us more information about occurrence and significance of less obvious metabolic interactions between microorganisms. For these purposes, syntrophic communities are ideal models. So, selected electrochemically active communities are of considerable interest for the understanding of the processes occurring in anaerobic niches where the final electron acceptor is an insoluble compound.

This research project focused on studying the system that includes a microorganism interacting with a mineral compound. For this purpose, the microorganisms were isolated from their natural habitat (mineral springs near the town of Kislovodsk, Russia), where they normally exist in the form of a multicomponent, syntrophic microbial consortia. One of the main criteria for selection of this consortia was electrochemical activity. Therefore, the selected consortia was studied in a specially designed electrochemical bioreactor cell. As the result of this test, it was shown that the microbial consortia was able to form a negative redox potential in the anode chamber of the electrochemical bioreactor cell and maintain it during the cultivation process. Microscopic examination of samples from the anodic electrode of the electrochemical bioreactor revealed cultures related to both Gram-positive and Gram-negative bacteria. The observation of biofilms on the electrode showed densely packed agglomerations of cells. Therefore, the selected electrochemically active consortia was interesting because it could be used in understanding the processes occurring in the anaerobic niches where the final electron acceptor is an insoluble compound. During the process of cultivation in liquid media with ferric iron oxide as an electron acceptor, the dispersed mineral phase was finally comprised in the biofilm structure. The fouling of flat surfaces of large crystalline particles deprived of structural defects occurred less effectively. The ferric iron oxide with a particle size less than 100 nm was also used as an electron acceptor. In this case, samples of cell surface “incrustation” by nanoscale iron oxide particles were observed. This strategy is typical for the microorganisms carrying out direct electron transfer from the terminal reductase of the membrane electron transport chain to the acceptor of electrons. The selected microbial consortia covered the surface of iron oxides by a multilevel, dense biofilm. In some parts of the biofilm, long filaments could be observed. Their diameter was approximately 0.2 microns and length more than 23 microns. The functional role of these filaments was unclear. They may be performing a structural function, helping a single cell firmly entrench in the biofilm. It is well known that some of the electrochemically active microorganisms, members of the Geobacter genus, synthesized conductive filaments through which the electrons are transferred to the mineral acceptors or to the surface of the anodic electrode in the microbial fuel cell [1]. Therefore, in future work we are going to clarify the functional role of the filaments found in the microbial consortia. Gas analysis indicated that 24 mM CO2, 8 mM CH4, and 2 mM H2 were produced during cultivation. Moreover, H2S was also detected in the gas phase. In syntrophic consortia, hydrogen-producing bacteria and hydrogen-utilizing methanogens sense redox conditions, influencing each other’s metabolism [2]. Low concentrations of hydrogen and the presence of methane suggest that consortium composition includes hydrogen-consuming methanogens. Thus, hydrogen can play role of the interspecies extracellular electron mediator. The amount of reduced iron was approximately 30% for 11 days of cultivation in the medium containing formate and acetate as a substrates (the initial concentration of ferric iron was 140 mM). Formate also could be used for interspecies electron transfer, but the relative contributions of formate and hydrogen transfer to consortia are difficult to determine. As substrates were also used—glucose, acetate, lactate, and the mixture of yeast extract with peptone. The iron reduction was observed in the medium with addition of the peptone and yeast extract mixture and in the medium with added lactate. The contribution of H2S in the iron-reduction process was also featured in experimentation, in which the culture medium and sterile agar with solution of ferric iron oxide were separated by the gas phase of the cultivation vessel. In this case, ferric iron oxide was also reduced due to the spread of H2S through the gas phase. In another experiment, 10 mM Na2MoO4 was added to the cultivation medium. This compound was able to specifically inhibit the growth of sulphate-reducing bacteria. After addition of Na2MoO4, the microbial consortium partly slowed the rate of ferric iron reduction. In the nature, H2S may be locally produced by sulphate-reducing components of microbial biofilms and instantly oxidized on the surface of ferric iron particles, thereby reducing it to FeS [3]. Genomic analysis of 16S rRNA gene clone libraries indicated a predominance of bacteria with sequences most similar to several species: Eubacterium aggregans, Advenella kashmirensis, Oscillibacter sp., and Lactococcus lactis. A number of less prevalent bacteria was also detected. Among them were Clostridium sp., Acetivibrio sp., and Chlorobi sp. This information did not allow us to make the overall conclusion about the distribution of functions within the consortium. We managed to identify mainly hydrolytic and fermenting components of microbial consortia. However, we cannot exclude that the identified microorganisms were also involved in the process of ferric iron reduction or transfer of electrons to the anodic electrode. Perhaps these microbes could interact directly (via contact) or indirectly (via excreted molecules) with insoluble acceptors (ferric iron or anodic electrodes) [4]. Because pelagic cells were also spread in the anodic chamber, extracellular electron mediators, such as H2 or H2S, might have served as electron shuttles. Although we could not identify the sulphate-reducing microorganisms, H2S can be released during the process of dissimilatory sulphate reduction. Soluble mediators might also be involved, since the peaks indicated by the cyclic voltammetry showed the presence of soluble redox metabolites in the cultivation medium. Finally, a few pure cultures were isolated from this consortium and their ability to reduce ferric iron using different substrates was tested.