Phenotypic analysis of different strains of Synechocystis sp Phenotypic analysis of different strains of Synechocystis sp. PCC 6803 for determination of the optimal strain for ethanol production Dawn Carey1, Patricia Armshaw1, Lorraine Quinn1, Con Sheahan2, J. Tony Pembroke1 1Molecular Biochemistry Laboratory, Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland 2Department of Design and Manufacturing Technology, University of Limerick, Limerick, Ireland Abstract Global energy demands are at an all-time high, and due to the depletion of fossil fuels, sustainable energy sources are being looked upon as a substitute. Cyanobacteria have been utilised for the production of biofuels, including the model microorganism Synechocystis sp. PCC 6803. These organisms have minimal requirements, needing only solar energy, carbon dioxide and minimal amounts of trace elements for growth, and have been broadly used to produce a range of biotechnological products such as ethanol [1], isobutanol [2] and lactic acid [3]. Within the DEMA project [Direct Ethanol from MicroAlgae]; the utility of Synechocystis PCC 6803 for ethanol production at a commercial level is being investigated [4]. A non-native ethanol biosynthesis pathway has been integrated into a strain of Synechocystis PCC 6803 [termed “Berkeley”], to generate an ethanol-producing strain. We have determined that current ethanol yields from this strain are still below what would be required at a commercial scale. In addition, the growth rate of this strain is significantly lower when the ethanol-producing genes are present within the chromosome. Recently, two phenotypically different strains or “wild types” of Synechocystis PCC 6803 were examined and found to differ by only six point mutations. Yet these strains were found to significantly differ in terms of growth rate [5]. We have obtained six different “wild type” strains of Synechocystis PCC 6803 and are currently evaluating these strains phenotypic characteristics such as growth rate, settleability and transformation efficiency to determine the optimal “wild type” strain for maximising ethanol production yields. DEMA concept WT Synechocystis Phenotype Differences Photoautotrophic ethanol biosynthesis pathway Table 1: Transformation Efficiency of various Synechocystis PCC 6803 strains A B C D E F A B C D E F Settling rate of various Synechocystis PCC 6803 sub-strains (72 h) Settling rate of various Synechocystis PCC 6803 sub-strains (168 h) Synechocystis PCC 6803 Transformation rates were determined for each of the six wild type Synechocystis PCC 6803 strains being investigated, as shown in Table 1. Sub-strain B was deemed the only strain to be untransformable, while E had the highest transformation efficiency rate, indicating this strain takes up extracelllular DNA most readily. Settleability of each wild type showed D and C to settle most rapidly, with C producing the tightest pellet and clearest supernatant. Sub-strains A and F behaved similarly, being slowest to settle and displaying a gradient of cell density throughout the supernatant. DEMA Synechocystis PCC 6803 Photobioreactor Within the DEMA project, ethanol is produced via heterologous expression of the Zymomonas mobilis pyruvate decarboxylase (pdc) along with overexpression of the endogenous Zn2+ dependent alcohol dehydrogenase (adhII) [4, 6]. Within DEMA, the performance of Synechocystis PCC 6803 is being substantially enhanced by a series of metabolic engineering strategies with the aim of achieving a concentration level of > 10 g/L (v/v). The modified cyanobacteria strains are cultivated in autotrophic culture medium using advanced closed photobioreactors and the bioethanol is continuously extracted from the culture media via a membrane technology process exploiting existing EU expertise and technology. Figure 1: Wild Type growth rates over time, 2% CO2, 150 µE light Figure 2: WT with integrated EtOH cassette growth rates over time with 2% CO2, 150 µE light Conclusions Over the course of this investigation, various phenotypic discrepancies were seen between all six wild type Synechocystis PCC 6803 strains. Sub-strain B was the only untransformable strain, while A was shown to be incapable of ethanol production. The optimum performer for the majority of investigations was C , while F, the strain utilised in the DEMA project, showed comparable ethanol production but had poor settleability. Additional investigations are currently underway to establish optimal wild type strain for maximising ethanol production. Figure 3: Ethanol production (g/L) of sub-strains with integrated EtOH cassette over time, 2% CO2, 150 µE light Figure 4: Ethanol production (g/L/OD1) of substrains with integrated EtOH cassette over time, 2% CO2, 150 µE light Three of the five ethanol producers grew at a slightly slower rate than their native wild types over a period of two weeks (Figures 1 and 2). Ethanol production is shown in Figures 3 and 4 (each being the average of at least 3 replicates). Sub-strain A is unable to produce ethanol although the ethanol-producing cassette is integrated in all strains and verified to have no mutations by sequencing. C produces the highest volume of ethanol both in g/L and g/L/OD1. References [1] J. Dexter, P. Fu, Energy Environ Sci 65 (2009) 857 [2] A.M. Varman, Y. Xiao, H.B. Pakrasi, Y.J. Tang, Appl Environ Microbiol 79 (2013) 908 [3] S.A. Angermayr, M. Paszota, K.J. Hellingwerf, Appl Environ Microbiol 78 (2012) 7098 [4] dema-etoh.eu. DEMA – Direct Ethanol from MicroAlgae; 2015 [cited 18 Feb 2015]. Available from http://www.dema-etoh.eu/ [5] J.N. Morris, T.S. Crawford, A. Jeffs, P.A. Stockwell, J.J. Eaton-Rye, T.C. Summerfield, New Zeal J Bot 10 (2014) 1080 [6] J. Dexter, P. Armshaw, C. Sheahan, J. T. Pembroke, J Appl Microbiol (2015) DOI: 10.1111/jam.12821 Acknowledgements This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no 309086.