EEEP 1/ 2023


Editorial comment

H. Najdenski                  3-4


Biokinetic characterization of Ferroplasma acidiphilum

J. Daoud, D. Karamanev                     5-16

Mineral waters in Bulgaria

A. Sadovski                   17-26


Microbial bioremoval of divalent toxic metals

I. Bida, O. Havryliuk, V. Hovorukha, G. Gladka, O. Tashyrev                    27-35


Cellulolytic microorganisms: aerobic, microaerophilic, anaerobic bacteria and microbial consortia (Part II)

Y. Gocheva, L. Dimitrova, V. Hubenov, L. Kabaivanova, P. Angelov, I, Simeonov, H. Najdenski                     36-53


Methodology for a kinematic model of seismic early warning system about critical infrastructure

B. Ranguelov, G. Mardirosian                     54-61


Carbon materials from waste biomass as anti-bacterial air filters

G. Georgiev, V. Hubenov, I. Stoycheva, R. Eneva, B. Petrova, U. Szeluga, S. Pusz, M. Belcheva, B. Tsyntsarski                        62-67


Biocidal action of anolyte

S. Karadzhov, T. Popova, I. Ignatov, L. Dimitrova, I. Nikolova, V. Kussovski,  H. Naidenski, G. Gluhchev                         68-76


Gross alpha and beta activity in mineral water from Southwest Bulgaria

E. Geleva, S. Dimitrov, N. Goutev, V. Variyska, D. Tonev                            77-82





Jamal Daoud, Dimitre Karamanev

Western University, London, Ontario, Canada, N6G 5B9

Abstract. Recently a mixed culture dominated by the iron-oxidizing microorganisms Leptospirillum and Ferroplasma has been used in a large-scale microbial fuel cell for electrical power generation. There are many factors that affect the kinetics of iron oxidation by the mixotroph Ferroplasma acidiphilum. This study investigated the effects of pH, temperature, and substrate and yeast extract concentrations in order to arrive at kinetically favorable operating conditions with minimal jarosite precipitation. Furthermore, F. acidiphilum was cultured with Leptospirillum sp. in order to determine its viability as a species capable of limiting the organic by-products of chemolithotrophic microbial growth. Bacterial characterization in the culture was accomplished using fluorescent in situ hybridization (FISH). It was found that the ferrous iron oxidation was most favorable at yeast extract concentration of 0.02% (w/v), pH of 1.6, temperature of 35 oC and an initial ferrous iron concentration of 1 g/L yielding a maximum specific growth rate of 0.0351-0.042 h-1. Moreover, F. acidiphilum displayed a symbiotic relationship with its chemolithotrophic counterpart, Leptospirillum sp. in that they were able to utilize the metabolic organic products of the chemolithotroph and limit the organic concentration to ~20 ppm total organic carbon (TOC), well below the threshold concentration of 250 ppm for chemolithotroph activity.

Keywords: Ferroplasma acidiphilum; Bioelectrochemical system; Mixotroph; Ferrous iron oxidation; Biokinetics; Total organic carbon.



Alexander Sadovski

: The present study aims to review the sources of mineral water in Bulgaria, as well as the various aspects of their use as an energy source, benefit for human health and the national economy. Several literary sources on the subject are cited. Statistical analysis was performed and data were summarized about their physical and chemical characteristics, and especially to find distribution by temperature. The production of cheap thermal energy from the geothermal plants contribute to the reduction of the environment pollution, which is of great importance for the country. The extremely good bio-climatic resources in the country provide a base for the prospective development of the balneological branch. The barriers to geothermal development in the country are shown. It is concluded that the importance of geothermal energy from mineral waters in Bulgaria is underestimated.

Keywords: mineral waters, energy, temperature, human health.


Iryna Bida, Olesia Havryliuk, Vira Hovorukha, Galyna Gladka, Oleksandr Tashyrev

Zabolotny Institute of Microbiology and Virology of the NAS of Ukraine, Kyiv, Ukraine

Abstract. The problems of polymetallic wastewater treatment from mining enterprises as well as the accumulation of organic waste are acute worldwide. The application of any existing methods of wastewater purification is ineffective and impossible due to the huge volumes and high concentrations of metals. Similarly, modern methods are ineffective for the treatment of huge amounts of organic waste. Therefore, there is a necessity to develop novel environmental biotechnologies providing the simultaneous degradation of organic waste and detoxification of toxic metals. The purpose of the work was to theoretically substantiate and experimentally confirm the possibility of toxic divalent cations removal using dissimilatory sulfate reduction via anaerobic fermentation of ecologically hazardous model organic waste. Colorimetric and potentiometric methods were used for pH and redox potential measurement; volumetric and chromatographic methods – to control volume and composition of synthesized gas; permanganate method – to determine the concentration of dissolved organic carbon (DOC); photocolorimetric method via the qualitative reaction with Nessler’s reagent was used to determine the concentration of ammonium ions. The Co2+ and Ni2+ content in medium was determined by a colorimetric method with 4-(2-pyridylazo)resorcinol (PAR). Fermentation parameters were calculated with the use of mathematical and statistical ones. Modified Postgate B medium with different sources of carbon and energy (potatoes, alanine, and meat) was used for cultivation of dissimilatory sulfate reducing bacteria. The anaerobic microbiome obtained from the sludge of methane tanks showed high efficiency to remove Co2+ and Ni2+ from the liquid medium. The highest efficiency (100% in 9 days) was observed when alanine was used as a source of carbon and energy. The slowest metal precipitation process occurred using meat (20 days). Also, the use of a protein substrate did not provide the expected alkalinization of the medium, which could significantly accelerate the process of metal precipitation. The precipitation of cobalt and nickel cations during the hydrogen fermentation of potato starch was complicated by acidification of the medium, but it was equally effective when the pH was adjusted. The proposed approach, the slow dissimilatory sulfate reduction, due to the sparingly soluble calcium sulfate as electron acceptor, can be used as a basis for the development of new biotechnologies for the treatment of wastewater contaminated with divalent heavy metals with the simultaneous treatment of ecologically hazardous compounds.

Keywords: thermodynamic prediction, metal containing wastewater purification, environmental biotechnologies, metals detoxification, metal pollution, toxic metals, sulfate reduction, ammonification, environmentally hazardous compounds.


Yana Gocheva1, Lyudmila Dimitrova1, Venelin Hubenov1, Lyudmila Kabaivanova1,Plamen Algelov2, Ivan Simeonov, Hristo Najdenski1

1 The Stephan Angeloff Institute of Microbiology at the Bulgarian Academy of Sciences

2 Space Research and Technology Institute at the Bulgarian Academy of Sciences

Abstract. In nature, cellulose, lignocellulose and lignin are major sources of plant biomass therefore their recycling is indispensable for the carbon cycle. The synergistic action of a variety of microorganisms is needed for recycling lignocellulosic materials. The capacities of microorganisms to assimilate complex carbohydrates, such as cellulose, hemicellulose and lignin, depend on the ability to produce the enzymes that work synergically. Populations growing in compost piles consist mainly of bacteria (including actinobacteria) and fungi. Polymers such as hemicellulose, cellulose, and lignin are only degraded once the more easily degradable compounds have been consumed. Afterwards, the lignocellulosic materials are partly transformed into humus. In the present review, numerous studies on the isolation of cellulose-degrading bacteria and fungi, their identification, enzymatic activities, and their ability to grow in the presence of lignocellulose and components of these industrial waste streams (phenolic compounds, sulfides, and dyes are analyzed and discussed. This is of particular interest to design future studies to isolate those bacteria that can specifically degrade cellulose matrix and more recalcitrant components such as lignin and aromatic lignin degradation products. Cultivation and characterization of microorganisms alone is not adequate without preservation techniques that do not alter the morphology, physiology or genetics of pure strains. Careful preservation is imperative for future research, teaching and industrial applications.
cellulolytic microorganisms, bacteria, fungi, cellulose waste, biodegradation, culture preservation.
Boyko Ranguelov1, Garo Mardirosian2

Sofia University of Mining and Geology „St. Ivan Rilski”, Sofia, Bulgaria.
Space Research and Technology Institute at the Bulgarian Academy of Sciences, Sofia, Bulgaria.
: The destructive earthquakes generated huge losses and human victims. As for the moment the time of impending strong seismic event is unpredictable, during the last years – new global innovation is under development – the seismic early warning systems (SEWS). All of them signalize the population and/or governmental bodies and after the earthquake starts just providing information that the seismic event is fact, to expect stronger shakings and some very fast measures to be performed – for example NPP’s reactors stop, as well as superfast trains, chemical production, gas and oil pipeline disconnection, etc. In lucky cases some people can react (especially in the areas of low buildings) and to evacuate effective in the time domain of few seconds, thus saving their lives. Such system is in operation in Japan since 2007 and some other countries also develop their own SEWS. The present investigation is targeted to the methodology development of so called kinematic SEWS. They are using a fundamental property of the body seismic waves P and S. As it is well known from the theory, the P-waves are faster and less destructive than S-waves. This property gives the possibility to use the detection of the P waves as important signal that after a while (the time domain depends on the distance between the seismic source and the investigated site) the slower (1.41 times) more destructive S waves will arrive and struck the infrastructure. Our previous experience of the developed SEWS (for example for Baku and Venice) gives the possibility to upgrade the modeling methodology extending to the formalized algorithm and accuracy estimations, especially for the critical infrastructure (for example NPP).

Keywords: kinematic model, early warning system, critical infrastructure.



Georgi Georgiev1, Venelin Hubenov2, Ivanka Stoycheva1, Rumyana Eneva2, Bilyana Petrova1, Urszula Szeluga3, Slawomira Pusz3, Marieta Belcheva2, Boyko Tsyntsarski1

1 Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria

2 The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria

3 Centre of Polymer and Carbon Materials, Polish Academy of Sciences, Poland

Abstract. Carbon materials from almond shells were developed for the adsorption of microorganisms from air. Samples were processed by means of a one-step process – high-temperature hydro-pyrolysis. Studies were carried out in a wide temperature range of 600-900 °C. As a result, carbonates with predominant micro- and mesopores were obtained. The samples were impregnated with zinc, silver, iron and copper 5% by weight. The obtained samples were characterized by XDR, BET, scanning electron spectroscopy and elemental analysis. The final products are distinguished by a moderate surface and the presence of nanosized metal particles. The antibacterial properties of the activated carbon composites were examined using standard methodology under dynamic contact conditions and Escherichia coli K12 as test microorganism. All tested composite materials exhibit strong antibacterial properties after 48 h of contact with microbial cells. Thus the application of these materials in filtering system will be possible solution for successful reduction of microbial cell number. It is assumed that a similar effect can be achieved in an air environment.
Keywords: activated carbon composites, antibacterial activity, waste almond shells.

S. Karadzhov1, T. Popova2, I. Ignatov3, L. Dimitrova4, I. Nikolova4, V. Kussovski4,  H. Naidenski4, G. Gluhchev5
1 Bulgarian Association of Activated Water, 39 Kutuzov blvd, Sofia 1619, Bulgaria
2 Institute of Forestry at BAS, 10 Kl. Ohridsky blvd, 1756, Sofia, Bulgaria
3 Scientific Research Center of Medical Biophysics, 32, N. Kopernik Street, Sofia 1111, Bulgaria
4 Institute of Microbiology at BAS, 25 Acad. G. Bonchev, Sofia 1113, Bulgaria

5 Institute of Information and Communication Technologies at BAS,  2, Acad. G. Bonchev, Sofia 1113, Bulgaria

Abstract. The anolyte, obtained during electrochemical activation of low mineralized water solutions possesses a strong biocidal action due to its physical-chemical properties. This study presents a brief report for the effect of anolyte application to samples of different kind of microorganisms. An idea of the physical-chemical processes taking place in the anode chamber of an electrolyzer is present. The changes in the parameters pH, ORP and the presence of active chlorine during the time and at different storage conditions of the anolyte were examined. The experiments showed their stability and preservation of biocidal activity for a period longer than 9 months. Experimental results from investigations of the biocidal effect of anolyte carried out in different laboratories are present.

Keywords: electrochemical activation, anolyte, pH, ORP (Oxidation Reduction Potential), biocidal effect, pathogens


Elena Geleva, Svetoslav Dimitrov, Nikolay Goutev, Verzhinia Variyska, Dimitar Tonev

Institute for Nuclear Research and Nuclear Energy, Bulgarian Academy of Sciences,Sofia, Bulgaria

Abstract. The natural radioactivity levels of mineral water from certain most frequently used sources in Southwest Bulgaria were determined. The mineral water was investigated with regard to gross alpha and beta activity to determine whether the activity concentrations are below levels at which no further action is required. The measurements were made by Liquid Scintillation Spectrometer. The gross alpha and beta activity varied from 0.003 Bq/L to 0.671 Bq/L and from 0.054 and to 0.375 Bq/L, respectively. The values obtained in this study were compared with data reported by other authors in different countries and with reference values accepted for drinking water.
The annual effective doses were calculated for all investigated waters for adult inhabitants assuming yearly consumption of 730 litres.
The results show that the annual effective dose of ingestion of these water samples excluding one, are below the individual dose criterion of 100 µSv/y according to the World Health Organization recommendations.
Keywords: gross alpha activity, gross beta activity, mineral water, annual effective dose.