Changes of cell size distribution maximum, cell relative content with size distribution maximum and half-width of curves of cell size distribution of
The need for reducing dependence on fossil fuels and the promoting the use of renewable fuels requires the development of alternative sources such as waste biomass for environmental benefits and alternative global energy supplies . Microbial fuel cells (MFCs) provide new opportunities for the sustainable production of energy from biodegradable and reduced compounds, and thus, have attracted substantial research efforts to develop various devices for generating electricity and removing wastes [1, 2]. The development of processes that can use bacteria to produce electricity represents a highly effective method for bioenergy production as the bacteria are self-replicating, and thus the catalysts of organic matter oxidation are self-sustaining . The substrates used in MFCs range from carbohydrates (e.g. glucose, sucrose, cellulose, starch), volatile fatty acids (e.g. formate, acetate, butyrate), alcohols (e.g. ethanol, methanol), amino acids, proteins and even inorganic components such as sulfides or acid mine drainages [2, 3-9]. The type of substrate fed to a MFC potentially has an impact on the structure and composition of the microbial community. Untill now, no clear image of the effect of the type of substrate on electricity generation by the microbial fuel cells is available.
Analysis of external resistances, electron donor concentrations, cell densities, rates of electron transfer to electrodes at various voltages, and anode potentials can aid in understanding the power production capabilities using microorganisms . A simplified model for the conversion of complex organic fuels to electricity is shown in figure 1 .
Complex organic matter is hydrolyzed to constituents that in the most cases are primarily fermented, but there are microorganisms that can completely oxidize such compounds with an electrode serving as the sole electron acceptor or incompletely oxidize these substrates with electron transfer to an electrode . Acetate and some other minor fermentation acids can be completely oxidized to carbon dioxide and it is typically the primary source of electrons for current production .
MFC is considered to be used also for hydrogen production from the generated potential of the organic matter electrolysis by bacteria .
Microbial fuel cell technologies also are a promising and yet completely distinct approach to wastewater treatment as the treatment process can become a method of capturing energy in the form of electricity or hydrogen gas, rather than a drain on electrical energy . Wastewater treatment processes currently employ the biological activities of complex microbial biofilms to remove organic pollutants . The most significant energy savings associated with the use of MFCs for wastewater treatment, besides electricity generation, result from savings in expenses for aeration and solids handling, because the major operating costs for wastewater treatment are wastewater aeration, sludge treatment, and wastewater pumping. The MFC process is inherently an anaerobic process, although, oxygen can diffuse into the system resulting in some aerobic organic matter removal .
At the same time, wastewater contains high concentrations of xenobiotics, such as heavy metal ions that have an overwhelming harmful effect towards all living organisms. These substances even in small concentration in the environment cause the increasing inhibition of physiological and biochemical properties of the most bacteria. Despite that, some genera of bacteria possess high resistance according to toxic heavy metals influence because of functioning of highly-efficient mechanisms of antioxidant defense system, ion efflux transport enzyme complexes etc. Examples of these genuses are
The variety of ion transport systems in gram-negative bacteria represents sophisticated mechanisms of bacterial cell metal homeostasis regulation. This is possible because of the formation of specific protein-metal coordination complexes used to effect uptake, efflux, intracellular trafficking within compartments, and storage .
Previous research has shown that gram-negative bacterium
Several redox-proteins have been elucidated of the cells of
This is the first investigated microorganism, which obtains energy by the complete acetate oxidation under the anaerobic conditions. It was shown that only 4% of consumed acetate by bacteria was assimilated into the cell material .
2.1. Microbial strains, medium and cultivation
2.2. Cell size distribution and relative content measurement
Bacterial growth commonly can be investigated by the registration of bacterial suspension turbidity or by the methods of dynamic or static light dispersion. The new method of rapid measurement of cell size distribution and their relative content, which is based on cell light scattering changes [31, 32] is proposed in this study. It includes the sounding of flow suspended bacterial cells by monochromatic coherent light, the registration of cooperative signals of sounding radiation and the explored microbiological objects by detecting of amplitudes and duration of scattered light impulses. The distribution of particles in size is determined on the basis of the measured functional dependence of the number of registered particles upon the amplitude and duration of corresponding electric impulses on the photoreceiver output by solving integral equation of Fredholm of the first kind (1):
where rmin and rmax – upper and lower limits of particle size distribution, which is registered;
However, presence of bacterial metabolism products in the growth medium could lead to errors in cell size distribution measurement because of additional light scattering. These errors were eliminated in the next way. Bacteria were cultivated in the liquid growth medium. Dependence between quantities of microbial cells and background particles in the growth medium had been determined during the time of bacterial cultivation. Liquid growth medium with and without bacterial cells was diluted in the same proportions by using highly purified liquid (deionized water). Then were registered separately the total quantity of cells and background particles in the highly purified liquid, which contains cells, and the total size distribution of background particles in the growth medium without cells. Then, relative content of bacterial cells was determined in the chosen interval of sizes, which equaled 0.3-1.9 μm. The cells relative content was measured by the calculation of quantity ratio of the set size cells to their total quantity. Specimens for determination of cell size distribution were prepared by dilution of 1 ml of bacterial suspension in 100 ml of deionized water [31, 32]. Measurements have been carried out by using the equipment PRM-6M, which was constructed at the Laboratory of Optical-Electronic Device of Faculty of Electronics of Ivan Franko National University of Lviv. The errors of cell size distribution measurement of constructed equipment are 5%.
2.3. Electron microscopy
After the third day of
2.4. Measurement of catalase, superoxide dismutase activity and intracellular reduced glutathione content
Antioxidant defense system activity has been measured in the cell-free extract after the second, third and fourth day of bacterial growth. Cells were washed by 0.9% NaCl solution and disintegrated on the ultrasonic homogenizer at 22 kHz at 0ºC during five minutes. Cell debris were sedimented by centrifugation at 5640-8800g at 4 ºC during 30 minutes. Catalase activity was measured spectrophotometrically at λ=410 nm by the degree of breakdown of hydrogen peroxide in the cell-free extract . Superoxide dismutase activity was measured by the level of inhibition of 2,3,5-triphenyltetrazolium chloride reduction that follows formazan formation (absorbance maximum λ=405 nm) [35, 36]. Reduced glutathione content has been measured by the degree of dithionitrobenzoic acid reduction in cell-free extract (absorbance maximum λ=412 nm) .
2.5. Microbial fuel cell construction and maintenance
In this study two chamber microbial fuel cell has been constructed, in which
2.6. Power output measurements of the microbial fuel cell
Electric current and voltage generation in constructed MFC were determined from the measured voltage drop across the resistor by multimeter DT-830C. The external load resistor with value 2.2 kΩ, which was shown to be the most optimal in constructed microbial fuel cell, was applied. The power output of an MFC was calculated from the measured voltage, EMFC, across the load and the current as (2):
Power generation by
3. Results and discussion
3.1. Particularities of
D. acetoxidans IMV B-7384 growth physiology
Biomass accumulation of
Electron micrographs of
Bacterial growth usually is characterized by increasing of cell quantity or cell size. Thus, analyses of cell size distribution and their relative content allow to obtain more detailed data of cell growth and division processes under various cultivation conditions in comparison with standard turbidimetric method of biomass measurement. Proposed method of cell light scattering determination allows to calculate possible changes of cell division on the basis of cumulative analyses of three histogram parameters: cell size distribution maximum, cell relative content and half-width of cell size distribution. It can be the basis for inventing of new methodologies for obtaining of synchronous cell cultures and also for development of new effective cytometers with low self-cost and high productivity.
Cell size distribution maximum equaled 0.55±0.01 μm on the third day of
From the first to the fifth day of
It indicated the decrease of cell size variations with the increase of cultivation time. Obviously, it is caused by intensive bacterial division on the third-fourth days of their cultivation. As a result cell’s relative content with lower size distribution maximum (0.49±0.01 μm) increased in comparison with its initial value with higher maximum (0.55±0.01 μm). Possible inhibition of cell division on the fifth day of bacterial growth caused increase of cell relative content with higher size distribution maximum, which equaled 0.55±0.02 μm.
Thus, it was shown that the maximal biomass accumulation is observed during third-fourth day of bacterial growth. High value of cell relative content with size distribution maximum and intensive decrease of half-width of cell size distribution indicates on significant increase of cell quantity with lower size distribution maximum. It is a possible result of intensive cell division during this time. Overall analysis of mentioned above parameters allow to assume that
3.2. Response of
D. acetoxidans IMV B-7384 cells to the influence of heavy metals
The activity of antioxidant defense system of
biosynthesis of reduced glutathione (GSH), a tripeptide that serves as universal electron donor detoxifying reactive oxygen species ;
activity of catalase and superoxide dismutase, a basic enzymes of antioxidant defense system [36, 39].
Reactive oxygen species (ROS) such as superoxide radical, hydrogen peroxide, hydroxyl radical etc are produced as a result of prolonged influence of oxygen or toxic xenobiotics, such as heavy metal ions, on the living cell. Glutathione (γ-L-glutamyl-L-cysteinylglycine) is the most abundant intracellular thiol-dependent antioxidant, which protects living cells against oxidative stress. It has low redox-potential (Е'0=-240 mV under pH=7.0) and it is constantly maintained in reduced state because of NADF-glutathione reductase functioning. Therefore it serves as cellular redox-buffer . Superoxide dismutase catalyzes disproportionation of superoxides with oxygen and hydrogen peroxide formation. Catalase causes decomposition of produced H2O2 with neutralization of its toxicity. Н2О and О2 are final products of this reaction . Thus, aforementioned components of antioxidant defense system protect the cell against oxidative stress, which may be caused by reactive oxygen species.
It was revealed that under the influence of various concentrations of FeSO4, FeCl3, MnCl2, NiCl2, CoCl2 and CuCl2 on the cells of
Obtained results show that investigated bacteria possess specific mechanisms of rapid defense against toxic influence of external factors, such as high concentrations of various metal ions. It allows to assume their tolerance according to detrimental xenobiotics, which wastewaters are enriched with. This shows the prospect of
3.3.Power generation by
D. acetoxidans IMV B-7384 in constructed microbial-anode fuel cell
3.3.1. The influence of external load resistance on power generation by
D. acetoxidans IMV B-7384 as the anode biocatalyst in constructed MFC
External load resistance in microbial fuel cell is one of the crucial factors, which influence the electricity generation. Correlation between value of external load resistance and power generation in constructed MFC was determined. The influence of changes of external load resistance on volt-ampere characteristic of constructed MFC was investigated during
The maximal value of power density equaled 4.7 mW/m2 on 84 hour of bacterial cultivation with addition of lactate (6 g/l) as electron donor and elemental sulfur (10 g/l) as electron acceptor (normal growth conditions), and application of an external load resistor of 0.2 kΩ. With the increase of cultivation time till 192 hour (eighth day of growth) generated power decreased by 42% in comparison with its maximal value.
With the aim to determine the most optimal load in term of electricity generation by
Therefore, all further experiments on MFC development were carried out with application of such external load resistance (2.2 kΩ), which was shown to be the most effective in term of electric power generation by
Accurate selection of external load resistance plays an important role in effective MFC development, because it significantly influences the value of generated power by bacteria, which are cultivated under different conditions in constructed MFC.
3.3.2. Power output in MFC under usage of various electron donors by
D. acetoxidans IMV B-7384 in MFC
184.108.40.206. Lactic acid and power output in MFC
In previous researches it was shown that maximal power output equaled 4.3 mW/m2 under addition the external electron acceptors, such as sulfur and Ferric iron in concentrations, which are favorable for
Lactic acid was applied as the sole organic electron donor in concentrations 6 and 9 g/l during
Thus, gradual lactate oxidation and the following diminishing of its quantity in the anode chamber because of bacterial growth in MFC caused gradual decrease of produced power with the increase of duration of cultivation time. Thus, application of lactic acid as the single electron donor in the aforementioned concentration in constructed MFC can’t be used for sustainable long-term electricity generation.
Concentration of lactic acid in the anode chamber has been increased up to 9 g/l. Under these cultivation conditions the highest power output equaled 5.90±0.21 mW/m2 on the 64 hour (third day) of
Increase of lactic acid content in the anode chamber caused enhance of stability of power generation in constructed MFC, but such manipulation did not boost its value significantly in comparison with application of lower concentration of lactic acid.
Thus, lactic acid as the sole electron donor in high concentrations supports durability of constructed MFC with application of
220.127.116.11. Fumaric acid and power output in MFC
With the aim to determine the most optimal electron donor in term of electricity generation lactic acid has been substituted by fumaric acid in the anode chamber of constructed MFC. The highest value of generated power equaled 5.69±0.29 mW/m2 on the 56 hour (the third day) of bacterial cultivation under usage of 6 g/l of fumaric acid as the sole electron donor (fig. 11). It’s value decreased by 38% on the 250 hour (the tenth day) and by 43% on the 480 hour (the twentieth day) of
Thus, application of fumaric acid (6 g/l) causes higher stability of power generation by investigated bacteria in constructed MFC in comparison with the lactic acid in the same concentration. Therefore, fumaric acid is more preferable electron donor in term of power generation by
It was shown that increase of fumaric acid concentration up to 9 g/l caused maximal power generation (2.38±0.10 mW/m2) at 56 hour (fig. 12).
It was less by 2.4 times in comparison with the highest power density value, which was observed under bacterial cultivation with application of less concentration of fumaric acid (6 g/l). The minimal observed power density value equaled 1.95±0.047 mW/m2 on the 160 hour of bacterial cultivation under these conditions. Increasing of cultivation time caused insignificant enhance of power production. On the 480 hour it value equaled 1.97±0.07 mW/m2, which was less by 17% in comparison with its maximal measured value in this investigation.
Thus, increase of fumaric acid concentration from 6 to 9 g/l in the anode chamber of constructed MFC reduced its productivity but enhanced its stability in comparison to the application of lower concentration of investigated electron donor.
18.104.22.168. Acetic acid and power output in MFC
Acetic acid was applied as the separate electron donor in constructed MFC. It was shown that the maximal power value equaled 5.78±0.24 mW/m2 on the 40 hour (second day) of bacterial cultivation under addition of 6 g/l of CH3COOH (fig. 13).
Its value decreased by 42% on the 232 hour (10 day) of
Thus, increase of acetic acid concentration as electron donor in the anode chamber of MFC caused partial inhibition of electricity generation in comparison with application of its lower content.
It can be summarized that low concentrations of investigated organic acids caused higher stability of power generation in constructed microbial fuel cell apart from their higher concentrations.
Possibly, it may be explained because of raising of by-products concentrations in the growth medium under increasing of organic source concentration. It may cause negative influence according to
The optimal external resistance in constructed MFC in term of power generation was determined to be 2.2 kΩ. Separate application of lactic, fumaric, and acetic acids caused differences in power generation by investigated bacterium. It was shown that addition of fumaric and acetic acids in concentration 6 g/l improved stability of generated power in constructed microbial fuel cell in comparison with application of lactic acid in the same concentration. Increase of concentration of investigated organic electron donors up to 9 g/l reduced generated electric power .
4.1. Possible directions of further research on application of
D. acetoxidans IMV B-7384 as the anode biocatalyst in MFC
Exploration of the utility of
We are highly grateful to Dr. Neelkanth G. Dhere, Jaroslav Ferensovych, Dr. Vasyl Getman, Dr. Dariya Fedorovych, Dr. Yurij Boretsky, Dr. Oleksandr Kulachkovskyj, and all other our coworkers for their support provided in carrying out of investigations and book chapter preparation.
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