Scientific activity of a laboratory of Extremophilic
Bacteria
1. Objects of scientific investigations:
The research activity of the Laboratory of Extremophilic Bactetria is oriented to the next
scientific problems:
1. Biodiversity of extremophilic bacteria. The application of the molecular approach
for characterization of the biodiversity in microbial communities has lead to basic
changes in our opinion about the extent of our knowledge on the organism world.
Bulgaria is rich in thermal springs, representing the natural ecosystem for
thermophilic microorganisms. New, cultivation-independent methodologies as well
as classical microbiological and biochemical approaches are used for searching of
unknown microorganisms which could hardly be assigned to the same phylogenetic
units with the previously isolated by cultivation techniques organisms.
2. Biosynthetic capacity of extremophilic microorganisms to synthesize
biotechnologically valuable enzymes. These organisms have been identified as
industrially important due to the production of thermostable enzymes. Regulation of
enzyme synthesis is investigated in batch and continuous cultures. Producers of
different hydrolases were isolated: α- and β-amylases, cyclo dextrin gluacno
transferase, glucosidase, pullulanase, inulinase, pectinase, xylanase, lipase, protease,
gellan-lyase et al.
3. Purification and characterization of thermostable enzymes. Optimal schemes for
purification of the enzymes with maximum yield were created. Substrate specificities
of enzymes, physico-chemical parameters of action, kinetic constants of enzyme
reactions, influence of different inhibitors, detergents and organic solvents on enzyme
activities were investigated. Data obtained were compared with the reported in
literature enzymes and considered their biotechnological potential.
4. Biosynthesis of exopolysaccharides from extremophilic microorganisms –
thermophiles and halophiles
The unusual metabolic pathways revealed in some extremophiles raised interest in
extremophilic microorganisms as potential producers of EPSs with novel and unusual
characteristics and functional activities under extreme conditions. Thermophilic
microorganisms and their EPSs suggest several biotechnological advantages, like
short fermentation processes and unusual thermostability of the polysaccharides. A
polysaccharide with very high biological activity was isiolated. It increased the lethal
dose of avarol almost 20 fold. EPS from halophiles easily formed stable emulsions.
Unlike mesophilic producers of EPSs, many of them being pathogenic, extremophilic
microorganisms provide non-pathogenic products, appropriate for applications in the
food, pharmaceutical and cosmetics industries as emulsifiers, stabilizers, gel agents,
coagulants, thickeners and suspending agents.
2. Last ten years projects:
I.
International projects
1. Magnetic immobilization of alkalophilic bacilli for cyclodextrin
glucanotransferase production.
Project leader: Assoc Prof. A. Tonkova
Joint research project between the Bulgarian and Czech Academies of
Sciences (2005-2007).
2. Purification, characterization and crystalogtaphic analysis of a
thermostable gellan lyase, synthesized by originally isolated
thermophilic aerobic bacterial strain
Project supervisor: Assoc. Prof. P. Hristova, Project leader: M.
Kambourova
Grant BG 16/2005. Joint research project between NFSR, Bulgaria
and Greece (2005-2007).
3. Advanced Fluorescence Sensors for Detection of Hazardous Agents.
Project supervisor: S. D’Auria (Italy),
Project leader: M. Kambourova.
NATO Collaborative Linkage Grant N.CBP.EAP.CLG.98243720062008.
4. Production of exopolysaccharides from thermophilic bacteria,
isolated from hot springs in Bulgaria and Italy.
Bilateral agreement between Bulgarian Academy of Sciences and
CNR (2007-2009).
Project leader Assoc. Prof. M. Kambourova.
II.
Bulgarian projects
5. Mixed bacterial populations degrading chelating agents and their metal complexes –
isolation from Bulgarian sources and identification.
Project supervisor: V. Ivanova, Project leader: A. Tonkova
Grant B-1205/02, NFSR, Bulgaria (2002-2006).
6. Isolation of alkalophilic bacilli from Bulgarian thermal springs and soils for
thermostable β-cyclodextrin glucanotransferase production
Project leader: A. Tonkova
Grant B-1521/05, NFSR, Bulgaria (2005-2008).
7. Molecular characterization of phylogenetic diversity of the bacteria in hot springs
located in South-West Bulgaria by 16S rDNA analysis
Project leader: M. Kambourova
Grant B 1511/05, NFSR, Bulgaria (2005-2008).
8. Regiospecific synthesis of structured lipids for food and medicine catalysed by
immobilized lipase from B. stearothermophilus MC7
Project supervisor: M. Guncheva, Institute of Organic Chemistry,
BAS; Project leader: A. Derekova, MSc
Grant C-МU, NFSR 1501/05, Bulgaria (2005-2007).
9. Molecular characterization (by 16S rDNA) of phylogenetic diversity of the
microorganisms in Bulgarian hot springs in the region of Velingrad (Central Bulgaria).
Isolation of novel microbial enzymes with biotechnological importance.
Project supervisor: Assoc. Prof. M. Kambourova
National Programme Genomics, NSFR, Bulgaria (2005-2008).
10. Metagenomic approach for identification and biosynthesis of an acidic thermostable αamylase from thermophilic microorganisms Grant DID 02/24, FNI (2009-2013).
Project supervisor: Assoc. Prof. Ivan Atanassov, AgroBioInstitute
Project leader for IMicB: Assoc. Prof. M. Kambourova
11. Biosynthesis of exopolysaccharides from extremophilic microorganisms. Grant DTK
02/46, FNI (2009-2013).
Project supervisor:: Prof. S. Vlaev, IEC, BAS.
Project leader for IMicB: Assoc. Prof. M. Kambourova
12. Biodiversity of halophilic microorganisms, isolated from Bulgarian salt niches and
their potential for exopolysaccharide synthesis. Grant GFNI 2-02/26 (2014-2016).
Project supervisor: Assoc. Prof. M. Kambourova
3. Selected publications for last ten years:
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Vassileva, A., Beschkov, V., Ivanova, V., Tonkova, A. (2005). Continuous
cyclodextrin glucanoransferase production by free and immobilized cells of
Bacillus circulans ATCC 21783 in bioreactors. Process Biochem., 40, 3290-3295.
Chernev, G., Samuneva, B., Djambaski, P., Salvado I.M., Fernandes, M.H.,
Kabaivanova, L. (2005). Silica Nanocomposites containing Polyacrylamide gel.
Glass Technol. 46/2 p.175-178.
Derekova, A., Sjoholm, C., Mandeva, R., Michailova, L., Kambourova, M.
(2006). Biosynthesis of a thermostable gellan lyase by newly isolated and
characterized strain of Geobacillus stearothermophilus 98. Extremophiles v. 10,
No 4, 321-326.
Kambourova, M., Mandeva, R., Fiume, I., Maurelli, L., Rossi, M., Morana, A.
(2007). Hydrolysis of xylan at high temperature by co-action of the xylanase from
Anoxybacillus flavithermus BC and the beta-xylosidase/alpha-arabinosidase from
Sulfolobus solfataricus Oalpha. J. Appl. Microbiol. Vol. 102, issue 6, 102(6).
Tonkova, A. (2006). Microbial starch converting enzymes of the α-amylase
family. In: Microbial Biotechnology in Horticulture (Ray, R.C. and Ward, O.P.,
eds.), v. 1, pp.421-472. Sci Publ. Inc., Enfield, NH, USA.
Kabaivanova, L., Dobreva, E., Emanuilova, E., Chernev, E., Samuneva, B.,
Solvado, I. (2006). Synthesis, structure and application of new hybrid
nanomaterials for cell immobilization. Minerva Biotechnologica 18, 23-29.
Derekova, A., Sjøholm, C., Mandeva, R., Kambourova, M. (2007). Anoxybacillus
rupiensis sp. nov., a novel thermophilic bacterium isolated from Rupi basin
(Bulgaria). Extremophiles 11(4), 577-583.
Chernev, G., Samuneva, B., Djambaski, P., Kabaivanova, L., Emanuilova, E.,
Miranda Salvado I. M., Fernandes, M.H.V., Wu, A. Synthesis and structure of
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new biomaterials containing chitosan. Physics and Chemistry of Glasses:
European J. Glass Science and Technology, 2007, B48/3, 147-150.
Kambourova, M., Mandeva, R., Fiume, I., Maurelli, L., Rossi, M., Morana, A.
(2007). Hydrolysis of xylan at high temperature by co-action of the xylanase from
Anoxybacillus flavithermus BC and the beta-xylosidase/alpha-arabinosidase from
Sulfolobus solfataricus Oalpha. J. Appl. Microbiol. Vol. 102, issue 6, 1586 - 1593.
Safarikova M., Atanasova N., Ivanova V., Weyda F., Tonkova A. (2007)
Cyclodextrin glucanotransferase synthesis by semicontinuous cultivation of
magnetic biocatalysts fron cells of Bacillus circulans ATCC 21783. Process
Biochemistry, 42, 1454-1459.
Guncheva, M., Zhiryakova, D., Radchenkova, N., Kambourova, M. (2007). Effect
of nonionic detergents on the activity of a thermostable lipase from Bacillus
stearothermophilus MC7. Journal of Molecular Catalysis B: Enzymatic 49 (1-4),
pp. 88-91.
Atanassova, M., Derekova, A., Mandeva, R., Sjøholm, C., Kambourova, M.
(2008). Anoxybacllus bogrovensis sp. nov., a novel thermophilic bacterium
isolated from Dolni Bogrov’s hot spring, Bulgaria. Int. J. Syst. Evol. Microb. 58,
2330 – 2335.
Atanasova, N., Petrova, P., Ivanova, V., Yankov, D., Vassileva, A., Tonkova, A.
(2008). Isolation of novel alkaliphilic Bacillus strains for cyclodextrin
glucanotransferase production. Applied Biochemistry and Biotechnology 149,
155-167.
Derekova, A., Mandeva, R., Kambourova, M. (2008) Phylogenetic diversity of
thermophilic carbohydrate degrading bacilli from Bulgarian hot springs. World J.
Microbiol. Biotechnol. 24, 1697-1702.
Kabaivanova, L., Emanuilova, E., Dimitrov, P., Engibarov, S., Boyadzhieva, I.,
Dobreva, E. (2008). Nitrile degradation by free and immobilized cells of the
thermophile Bacillus sp. UG-5B, isolated from polluted industrial waters. World
J. Microbiol. Biotechnol. 2, 2383–2388.
Chernev, G., Samuneva, B., Djambaski, P., Kabaivanova, L., Emanuilova, E.,
Miranda Salvado, I.M., Wu, A. (2008). Synthesis and selected properties of
silicate hybrids containing sepharose Advanced Materials Research 39-40, 53-56.
Samuneva, B., Kabaivanova, L., Chernev, G., Djambaski, P., Kashchieva, E.,
Emanuilova, E., Miranda Salvado, I.M., (...), Wu, A. (2008). Sol-gel synthesis
and structure of silica hybrid materials Journal of Sol-Gel Science and
Technology 48, 73-79.
Guncheva, M., Zhiryakova, D., Radchenkova, N., Kambourova, M. (2008).
Acidolysis of Tripalmitin with Oleic Acid Catalyzed by a Newly Isolated
Thermostable Lipase. J. Am. Oil Chem. Soc., 85, 129-132.
Chernev, G., Samuneva, B., Djambaski, P., Kabaivanova, L., Emanuilova, E.,
Salvado, I.M.M., Fernandes, M.H.V., Wu, A. (2008). Synthesis and structure of
new biomaterials containing silica and chitosan Physics and Chemistry of
Glasses: European Journal of Glass Science and Technology Part B 49 , 11-14,.
Samuneva, B., Djambaski, P., Kashchieva, E., Chernev, G., Kabaivanova, L.,
Emanuilova, E., Salvado, I.M.M., (...), Wu, A. (2008). Sol-gel synthesis and
structure of silica hybrid biomaterials Journal of Non-Crystalline Solids 354, 733740.
Kambourova, M., Mandeva, R., Dimova, D., Poli, A., Nicolaus, B., Tommonaro,
G. (2009) Production and characterization of a microbial glucan, synthesized by
Geobacillus tepidamans V264 isolated from Bulgarian hot spring. Carbohydrate
Polymers, 77(2), 338-343.
22. Atanasova N., Kitayska Ts., Yankov D., Safarikova M., Tonkova A. (2009).
Cyclodextrin glucanotransferase production by cell biocatalysts of alkaliphilic
bacilli. Biochem. Eng. J. 46, 278-285.
23. Guncheva, M., Zhiryakova, D., Radchenkova, N., Kambourova, M. (2009).
Properties of immobilized lipase from Bacillus stearothermophilus MC7.
Acidolysis of triolein with caprylic acid. World Journal of Microbiology and
Biotechnology, 25(4), 727-731.
24. Ivanova I, Atanassov I, Lyutskanova D, Stoilova-Disheva M, Dimitrova D,
Tomova I, Derekova A, Radeva G, Buchvarova V, Kambourova M. (2011). High
Archaea diversity in Varvara hot spring, Bulgaria. Journal of Basic Microbiology
51:163-172.
25. Atanasova N., Kitayska Ts., Boyadzhieva I., Yankov D., Tonkova A. (2011). A
novel cyclodextrin glucanotransferase from alkaliphilic Bacillus pseudalcaliphilus
20RF: purification and properties. Process Biochemistry 46:116-122
26. Kitayska Ts., Petrova P., Ivanova V., Tonkova A. (2011). Purification and
properties of a new thermostable cyclodextrin glucanotransferase from Bacillus
pseudalcaliphilus 8SB. Applied Biochemistry and Biotechnology 165:1285-1295.
27. Ivanova, I., Atanassov, I., Lyutskanova, D., Stoilova-Disheva, M., Dimitrova, D.,
Tomova, I., Derekova, A., Radeva, G., Buchvarova, V., Kambourova, M. (2011).
High Archaea diversity in Varvara hot spring, Bulgaria. Journal of Basic
Microbiology 51, 163-172.
28. Tomova I., Dimitrova D., Stoilova-Disheva M., Lyutskanova D., Kambourova M.
(2011). Archaeal diversity at two hot springs, Rupi basin, Bulgaria.
Biotechnology&Biotechnological Equipment 25(SUPPL. 4), 105-113.
29. Radchenkova N., Tomova, A., Kambourova M. (2011). Biosynthesis of an
exopolysaccharide, produced by Brevibacillus thermoruber 438. Biotechnology
and Biotechnological Equipment, 25(SUPPL. 4), 77-79.
30. Petrova P., Tonkova A., Petrov K. (2012). Sequence analysis, cloning and
extracellular expression of cyclodextrin glucanotransferase gene from the
alkaliphilic Bacillus pseudalcaliphilus 8 SB in Escherichia coli. Process
Biochemistry 47, 2139-2145.
31. Rezanka, T., Kambourova, M., Derekova, A., Kolouchova, I., Sigler, K. (2012).
LC-ESI-MS/MS identification of polar lipids of two thermophilic Anoxybacillus
bacteria containing a unique lipid pattern. Lipids, 47, 729-739.
32. Safarikova M., Horska K., Maderova Z., Tonkova A., Ivanova V., Safarik I.
Magnitic porous corn starch for the affinity purification of cyclodextrin
glucanotransferase produced by Bacillus circulans. Biocatalysis and
Biotransformation, 30, 1, 2012, 96-101.
33. Ivanova V., Tonkova A., Petrov K., Petrova P., Gencheva P. (2012). Covalent
attachment of cyclodextrin glucanotransferase from genetically modified
Escherichia coli on surface functionalized silica coated carriers and magnetic
particles. J. BioSci. Biotech. SE/ONLINE: 7-13.
34. Tomova I., Lazarkevich I., Tomova A., Kambourova M. and Vasileva-Tonkova
E. (2013). Diversity and biosynthetic potential of culturable aerobic heterotrophic
bacteria isolated from Magura Cave, Bulgaria. International Journal of
Speleology, 42 (1), 65-76.
35. Ivanova, V., Tomova, I., Kamburov, A., Tomova, A., Vasileva-Tonkova, E.,
Kambourova, M. (2013). High phylogenetic diversity of bacteria in the area of
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prehistoric paintings in Magura Cave, Bulgaria. Journal of Cave and Karst
Studies, 75 (3), 218-228.
Atanassov I., Stefanova K., Tomova I., Kambourova M. (2013). Seamless GFP
and GFP-amylase cloning in Gateway shuttle vector, expression of the
recombinant proteins in E. coli and Bacillus megaterium and assessment the GFPamylase thermostability. Biotechnol. Biotechnol. Eq., 27, , 4172-4180.
Radchenkova N., Vassilev S., Panchev I., Anzelmo G., Tomova I., Nicolaus B.,
Kuncheva M., Petrov K., Kambourova M. (2013). Production and properties of
two novel exoplosaccharides synthesized by a thermophilic bacterium
Aeribacillus pallidus 418. Appl. Biochem. Biotechnol., 171, 31-43.
Tomova A., Tomova I., Vasileva-Tonkova E., Lazarkevich I., Stoilova-Disheva
M., Lyutskanova D., Kambourova M. (2013). Myroides guanonis sp. nov.,
isolated from prehistoric paintings. Int. J. Syst. Evol. Microbiol., 63, 4266–4270.
Kambourova M., Derekova A. (2013). Developments in thermostable gellan
lyase. In: Thermophilic Microbes in Environmental and Industrial Biotechnology
(T. Satyanarayana, J. Littlechild, Y. Kawarabayasi, eds). Springer, Second
Edition, рр. 711-730.
Radchenkova N., Vassilev S., Martinov M., Kuncheva M., Panchev I., Vlaev S.,
Kambourova M. Optimization of aeration and agitation speed on
exopolysaccharide production by Aeribacillus palidus 418 and emulsifying
properties of the product. Process Biochemistry, 49, 2014, 576-582.
Yasar Yildiz S., Anzelmo G., Ozer T., Radchenkova N., Genc S., Di Donato P.,
Nicolaus B., Toksoy O., Kambourova M. Brevibacillus themoruber: A promising
microbial cell factory for exopolysaccharide production. Journal of Applied
Microbiology, 116 (2), 2014, 314–324.
Yasar Yildiz, S., Kambourova, M., Yalcin Arga, K., Toksoy Oner E. (2013).
Draft Genome Sequence of Exopolysaccharide-Producing Thermophilic
Bacterium Brevibacillus thermoruber Strain 423. Genome announcements, 1(5), ,
e00774-13.
4. Current staff of the laboratory of Extremophilic Bacteria
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Assoc. Prof. Margarita Kambourova, DSc
Assist. Prof. Iva Tomova, Dr.
Assist. Prof. Nadja Radchenkova, Dr.
Assist. Prof. Ivanka Boyadzhieva, Dr.
Assist. Prof. Miroslava Atanassova, Dr.
Researcher Nikolina Atanassova