Results:
Stefanova K., Tomova I., Tomova A., Radchenkova N., Atanassov I., Kambourova M. Archaeal and bacterial diversity in two hot springs from geothermal regions in Bulgaria as demostrated by 16S rRNA and GH-57 genes. International Microbiology (in press), IF 1.326.
Margarita Stoilova-Disheva, Dimitrina Lyutskanova, Ivanka Boyadzhieva, Nadja Radchenkova, Margarita Kambourova. Unusually high archaeal diversity in Vlasa hot spring, Velingrad, Bulgaria, revealed by phylogenetic analysis. Participation in Seminar of Ecology with International Participation – 2016, Sofia, 21-22 April, 2016.
I N T E R M E D I A T E R E P O R T 1
On the project D03-100 / 05.06.2015
Programme of the Financial Mechanism of the European Economic Area (EEA grants), measure “Projects for inter-institutional cooperation”
DIVERSITY AND BIOTECHNOLOGICAL POTENTIAL OF ARCHAEA FROM BULGARIAN HOT SPRINGS
For the period 04.09.2015-31.12.2015
1. Billboard
In implementation of the Plan for publicity a billboard indicating the financial support of EEA program was installed at the entrance of the Institute.
2. Website
A page devoted to EEA program was created on the website of the Institute of Microbiology, BAS (www.microbio.bas.bg/wordpress/index.php/en/projects/project-d03-100-05-06-2015/, which includes information on:
(A) the program operator
(B) the beneficiaries of the grant and the contact person
(C) the funding with clear indication of the mechanism of sponsorship
(D) planned activities in the project by EEA program
(E) cooperation with staff from the donor country, information on the work of young Bulgarian scientist at Norwegian team
(F) the role of the donor country in project implementation
3. Project Management
This activity involved the following activities:
Corr. Memb. Hristo Naydenski, Director – Meeting with the EEA governing body, organization of procurement for supply of chemicals and consumables, a proposal for modification of the budget.
Antoaneta Tsareva, Ch. Accounter – Processing of financial documents, preparation of lists procurement, preparation of financial statements and contacts with the governing body.
4. Research activity
Activity 2, Sample collection
Springs with different geothermal origin and different properties of water, suggesting divergence of their inhabitants in them were selected for analysis of the archaeal biodiversity, namely Mizinka and Vlasa, Velingrad, and Sapareva banja. The trip to Velingrad was carried out on 5-6.10.2015 and to Sapareva banja – 31.10-1.11.2015. One liter of each sample water was analyzed for the chemical composition. The analysis was carried out in a trade company Dial LTD.
Activity 3, Isolation of DNA
The successful detection and characterization of microbial 16S rDNA in natural samples require extraction of high molecular weight DNA and its proper disposal of pollutants such as certain organic acids and ions, which inhibit the next stage of amplification.
The procedure for the isolation of total DNA included filtration of water, treatment of the filter with a lysis solution, RNase, protease, and DNA purification. As a result of the implementation of this phase high molecular total DNA was successfully isolated.
Activity 4, Аmplification of 16S rRNA gene and initialization the construction of clone libraries
A, Amplification of 16S rRNA gene
Using the 16S rRNA gene as a marker gene allows characterization the structure of microbial communities in nature and reaching the real microbial diversity. Archaeal DNAs were amplified with universal primers for archaea 21F and 958R. PCR products were observed into two samples,Vlasa hot spring and Sapareva banja. A product was not observed in the sample from Mizinka.
B, Ligation of the archaeal 16S rRNA into a plasmid and transformation into E. coli cells
The amplified 16S rRNA genes were ligated into a vector and the plasmid was transformed into competent cells. More than 200 clones were selected and tested for correctness of the insert.
C, Grouping of clones according to their restriction profiles
The diversity in the clone library was preliminary assessed by ARDRA analysis (Amplified Ribosomal DNA Restriction Analysis). To establish the correct polymorphism between cloned 16S rRNA genes, they were subjected to restriction with two 4-basic enzyme – MspI and HaeIII. Based on the diversity in the profiles obtained after restriction of 16S rDNA, 28 correct clones were allocated to 14 groups.
D, Sequencing
Amplified PCR product of one representative from each restriction group was sent for sequencing, a total of 14 PCR product. Additionally, six arhaeal clones from hot spring Levunovo were sent for sequencing. Sequencing was conducted in Macrogen, South Korea. An analysis of the results and further work with the rest clones from the library is forthcoming. The genes of the remaining clonesof Vlasa library will be amplified in the next stage, they will be grouped according to their restriction profiles, sequenced and analysed. Based on the results received the structure of archaeal community will be revealed.
The following team members have participated in the implementation of activities 2, 3 and 4:
1. Assoc. Prof. Margarita Kambourova
2. Assis.Prof. Nadia Radchenkova
3. Assis. Prof. Ivanka Boyadjieva
4. Assis.Prof. Margarita Stoilova-Disheva
5. Assis. Prof. Dimitrina Lyutskanova
13/01/2016 Project leader:
Assoc. Prof. M.Kambourova
Director IMicB:
Cor. Mem. H. Naydenski
I N T E R M E D I A T E R E P O R T 2
For the project D03-100 / 05.06.2015
Programme of the Financial Mechanism of the European Economic Area (EEA grants), measure “Projects for inter-institutional cooperation”
DIVERSITY AND BIOTECHNOLOGICAL POTENTIAL OF ARCHAEA FROM BULGARIAN HOT SPRINGS
For the period 01.01.2016-30.04.2016
Website
The page on the website of the Institute of Microbiology, BAS, dedicated to the project has been updated (www.microbio.bas.bg),http://microbio.bas.bg/IMB%20web%20page/microbio/Norwegia_Priekt_Kamburova_bg.html, in which the next information was included:
(A) The publication activity of the group and participation in a conference
(B) First and second 4-month reports
(C) Gallery of photos demonstrating the research activity of the team
Activity 1, Project Management
Corr. Mem. H. Naydenski, Director – Control of the project implementation.
Antoaneta Tsareva, Ch. Accountant – Financial records, preparation of financial statements, modification of the budget.
Assoc. Prof. DSc M. Kambourova, Project Manager:
– Meetings of the commission for consideration, evaluation and ranking of participants in the procurement for delivery of materials, chemicals and consumables were organized.
– Modification of the project budget
– Procedure for granting a loan from BAS to cover expenses until the last installment of the contract.
– Participation in the organized on 30.03.2016 meeting with the management of the EEA Program operator BG09, in which discussion on the progress on the projects implementation was performed.
Activity 2, Experimental work
– Members of the team together with Norwegian partner Prof. Birkeland visited hot springs in southwestern Bulgaria and in the region of Velingrad. Samples for isolation of culturable archaea were taken.
– PhD student Nicoleta Boteva (team member) has achieved growth of mixed cultures from 9 samples in Norwegian laboratory. Their ability to synthesize biotechnologically valuable enzymes (xylanase, cellulase, gellan lyase) was established. She is working on the isolation of pure strains that will be sent to the Bulgarian laboratory for characterization.
– The work on the construction of clone library with DNA isolated from a hot spring Vlasa, Velingrad continued in Bulgarian laboratory. The selected clones were splited into 57 groups according to the identity of their restriction profiles. One representative from each group was sent for sequencing in a Korean company Macrogen. The team successfully works with chemicals and consumables borrowed from colleagues from the institute and other scientific organizations.
– A contract for suppling a system for anaerobic cultivation of archaeal strains has been signed. Incoming system components are assembled.
Activity 3, Exchange of visits
– The Norwegian partner Nils-Kare Birkeland visited Bulgaria in the period 3-7.01.2016. He participated in trips for collecting samples from hor springs in southwestern Bulgaria and the region of Velingrad. He met the working team and an interesting discussion on the specifics in archaeal cultivation took place.
– The team member PhD student Nicoleta Boteva left on 10.01.2016 for 6 months work on the project in the laboratory of Prof. Nils-Kare Birkeland, Faculty of Biology, centered on Geobiology, University of Bergen, Bergen, Norway.
Activity 4, Information and Publicity
– Results obtained from clone libraries of 16S rRNA gene and amylase gene from Levunovo and Vetren dol were summarized in a publication submitted and accepted for print in the journal International Microbiology.
Stefanova K., Tomova I., Tomova A., Radchenkova N., Atanassov I., Kambourova M. Archaeal and bacterial diversity in two hot springs from geothermal regions in Bulgaria as demostrated by 16S rRNA and GH-57 genes. International Microbiology (in press), IF 1.326.
– The team participated in the “Seminar on Environment – 2016” with international participation with poster:
Margarita Stoilova-Disheva, Dimitrina Lyutskanova, Ivanka Boyadzhieva, Nadja Radchenkova, Nicoleta Boteva, Margarita Kambourova. Unusually high archaeal diversity in Vlasa hot spring, Velingrad, Bulgaria, revealed by phylogenetic analysis. Seminar on ecology with international participation – 2016, Sofia, 21-22.04.2016.
30/04/2016 Project leader:
Assoc. Prof. M. Kambourova
Director IMicB:
Cor. Mem. H. Naydenski