Construction of Local Isolates of Cyanobacteria for Ethanol Production.

Payam Bayazeed Hasan and Dlnya Asad Mohamad

Faculty of Science and Science Education- Sulaimani University- Qliasan- Sulaimani- Iraq


Cyanobacteria can use solar energy and convert carbon dioxide into biofuel molecules in
one single biological system. In this research, Synechococcus sp. was isolated from
Saray Subhan Agha fresh water, a pure culture of Synechococcus was obtained by
several subculturing on BG11 media. For the production of ethanol by Synechococcus
sp. pyruvate decarboxylase (PDC) and alcohol dehydrogenseII (ADH II), genes from
Zymomonas mobilis ATCC (29191), were amplified by PCR and cloned into the
pSyn_1⁄D-TOPO® Vector. The Synechococcus and Synechococcus elongates
transformed with constructed vector (pSyn_1⁄D-TOPO®) that harboring the two ethanol
fermenting genes. The transformation was performed using a double homologous
recombination system to integrate the PDC and ADHII genes into the local isolates of
Synechococcus sp. and Synechococcus elongatus chromosome under the control
cyanobacterial weak constitutive nickel inducible promoter. The recombinant
Synechococcus cells grow in different concentrations of NiSO4 (1, 2.5, 5, 7.5, 10) μM in
BG11 media, under different temperature (15, 30, 45)  ̊C and different light intensity (10,
50, 150) μE. The enzymatic ethanol assay kit was used to determine ethanol
concentration produced by both recombinant Synechococcus sp. and recombinant
Synechococcus elongatus. Highest ethanol concentration obtained by those cultures
containing five μl NiSO4, which incubated under continues light of 50μE at 30 ̊C
(Optimum um condition for ethanol production by recombinant Synechococcus cells).
The amount of ethanol produced by local isolates of Synechococcus sp. was 0.00103 g/l,
whereas for Synechococcus elongatus was 0.0138 g/l. The amount of ethanol produced
by those Synechococcus cultures containing different concentrations of NiSO4 were
incubated under continuous light of (10 and 150) μE and temperature of (15 and 45)  ̊C
was less than those cultures were incubated under light of 50 μE and temperature of
30 ̊C.

Key Words:
Ethanol, Sulaimani.


[1] Almarsdóttir, A. R. (2011). Thermophilic Ethanol and Hydrogen Production from Lignocellulosic Biomass. MSc. Thesis, University of Akureyri. Akureyri-Iceland.assignments, strains histories and properties of pure cultures of cyanobacteria.

[2] Nagy, M. (2009). Biofuels from Lignin and Novel Biodiesel Analysis. Ph. D. Thesis. Georgia Institute of Technology, Atlanta- Georgia.

[3] Quintana, N., Van der Kooy, F., Van de Rhee, M.D., Voshol, G. P., and Verpoorte R. (2011). Renewable Energy from Cyanobacteria: Energy Production Optimization by Metabolic Pathway Engineering. PubMed Central®,91(3): 471–490.

[4] Lu, X. (2010). A perspective: Photosynthetic production of fatty acid-based biofuels in genetically engineered cyanobacteria. Biotechnology advances,28(6), 742-746.

[5] Nogales, J., Gudmundsson, S., and Thiele, I. (2013). Toward Systems Metabolic Engineering in Cyanobacteria: Opportunities and Bottlenecks. Bioengineered, 4(3): 158 – 163.

[6] Callieri, C., Corno, G., Caravati, E., Galafassi, S., Bottinelli, M., and Bertoni, R. (2007). Photosynthetic Characteristics and Diversity of Freshwater Synechococcus at two Depths during Different Mixing Conditions in a Deep Oligotrophic Lake. Journal of Limnology, 66(2): 81-89.
[7] Perkins, F.O., Haas, L.W., Phillips, D.E., and Webb, K.L. ( 1981). Ultrastructure of a Marine Synechococcus Possessing Spinae. Canadian Journal of Microbiology, 27(3):318-29.

[8] Deng, M. D., and Coleman, J. R. (1999). Ethanol Synthesis by Genetic Engineering in Cyanobacteria. Applied and Environmental Microbiology, 65(2): 523-528.

[9] Talarico, L. A., Gil, M. A., Yomano, L. P., Ingram, L. O., and Maupin-Furlow, J. A. (2005). Construction and Expression of an Ethanol Production Operon in Gram-positive Bacteria. Microbiology, 151(12), 4023-4031.

[10] Wang, B., Wang, J., Zhang, W., and Meldrum, D. R. (2012). Application of Synthetic Biology in Cyanobacteria and Algae. Frontiers in Microbiology, 3,1-15.

[11] Dexter, J., and Fu, P. (2009). Metabolic Engineering of Cyanobacteria for Ethanol production. Energy & Environmental Science, 2(8): 857-864.

[12] Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. 2nd edition. Cold spring harbor laboratory press, NY.

[13] Hardter, U., Luzhetska, M., Ebeling, S., nd Bechthold, A. (2012). Ethanol Production in Actinomycetes after Expression of Synthetic adhB and pdc. Open Biotechnology Journal, 6, 13-16.

[14] Bookless, N., Hartley, B., Baghaei-yazdi, N., and Javed, M. (2007). Fermentation Process for the Production of Ethanol.,: World Intellectual Property Organization. WIPO Patent No. 2007110592

[15] Gold, R. S., Meagher, M. M., Tong, S., Hutkins, R. W., and Conway, T. (1996). Cloning and Expression of the Zymomonas mobilis “production of ethanol” Genes in Lactobacillus casei. Current Microbiology, 33(4): 256-260.

[16] Casali, N., and Preston, A. (2003). E. coli Plasmid Vectors: Methods and Applications. Human Press; 235.

[17] Daniell, H., and McFadden, B. A. (1986). Characterization of DNA Uptake by the Cyanobacterium Anacystis nidulans. Molecular and General Genetics MGG, 204(2): 243-248.

[18] Lightfoot, D. A., Walters, D. E., and Wootton, J. C. (1988). Transformation of the Cyanobacterium Synechococcus PCC 6301 Using Cloned DNA. Journal of General Microbiology, 134(6): 1509-1514.

[19] Golden, S. S., and Sherman, L. A. (1984). Optimal Conditions for Genetic Transformation of the Cyanobacterium Anacystis nidulans R2. Journal of Bacteriology, 158(1): 36-42.

[20] Chauvat, F., Astier, C., Vedel, F., and Joset-Espardellier, F. (1983). Transformation in the Cyanobacterium Synechococcus R2: improvement of efficiency; Role of the pUH24 plasmid. Molecular and General Genetics MGG,191 (1): 39-45.

[21] Shestakove, S. V., Karbysheva , E. A. and Elanskaya, I. V. (1982). The nature of damage in mutants of Anacystis nidulans deficient in genetic transformation. Genetika 18, 1271-1275.

[22] Los, D. A., Zorina, A., Sinetova, M., Kryazhov, S., Mironov, K., and Zinchenko, V. V. (2010). Stress Sensors and Signal Transducers in Cyanobacteria. Sensors,10 (3): 2386-2415.

[23] Thiel, T. (1995). Genetic Analysis of Cyanobacteria, p. 581–611. In D. A. Bryant (ed.), The molecular biology of cyanobacteria. Kluwer Academic Press, Dordrecht, The Netherlands.