Optimization of algal-bacterial co-cultures for semi-continuous hydrogen and biomass production Gergely Lakatos1, Daniella Balogh1, Gergő Balogh1, Vince Ördög2, and Gergely Maróti1 1 Institute of Biochemistry, Biological Research Centre, Szeged, Hungary 2 Institute of Plant Biology, Faculty of Agricultural and Food Sciences, University of West Hungary, Mosonmagyaróvár, Hungary E-mail: lakatos.gergely@brc.mta.hu Introduction It is well known that some of the green algae can produce hydrogen by their FeFe-hydorgenases under anaerobic and illuminated circumstances. Most of the methods are based on nutrient deprivations like sulfur- (Melis et. al, 2000), phosphorus- (Batyrova et. al, 2012), nitrogen- (Philipps et. al, 2012) and magnesium-deprivation (Volgusheva et. al, 2015). Algal-bacterial co-culture doesn’t require nutrient deprivations, which allows the parallel production of biohydrogen and mixed algal-bacterial biomass. Our previous studies resulted in lower hydrogen production rates compared to nutrient deprived cultures and short, approximately one day long hydrogen production period (Lakatos et. al, 2014, Wirth et. al, 2015). Here we demonstrate the first steps of the culture condition optimization for a semi-continuous hydrogen production, focusing on selection of algae strains with high hydrogen productivity, optimization of culture densities/cell numbers, and fine adjustments of liquid and gas phase ratios. Basic conception Optimization of algal-bacterial hydrogen production O2 CO2 H2 1. Three different algae strains with signifficantly different cell sizes were chosen for hydrogen production rate comparison. Chlorella sp. MACC 360 Average area per cell: 14,46 +- 3,92 mm2 Chlamydomonas reinhardtii cc124 Average area per cell: 51,07 +- 12,86 mm2 Chlamydomonas sp. MACC 549 Average area per cell: 105,97 +- 42,72 mm2 2. Six gas-to-liquid phase ratios were tested. Gas-to-liquid ratios: 5 ml/35 ml, 10 ml/30 ml, 15 ml/25 ml, 20 ml/20 ml, 25 ml/15 ml, 30 ml/10 ml. Algal-bacterial culture is incubated in Tris-Acetate-Phosphate (TAP) media under 50 mmol m-2 s-1 light intensity at 25°C in 40 ml serum bottles. Bacterial partner consumes the evolved oxygen, evolves carbon dioxde and enhances the establishment of the anaerobic environment. Acetic acid (1 ml l-1 culture) accelerates the algal cell respiration, thus reduces the net oxygen emission of the algae and enhances the establishment of the anaerobic environment for hydrogen production. Algae uses acetic acid, carbon dioxide and light energy to produce biohydrogen and biomass. 3. Different optical densities/cell numbers were tested with each gas-to-liquid phase ratio. Optical densities of the Chlorella sp. MACC 360 were set to 0.7 (2.77*108 cells ml-1), 1 (3.96*108 cells ml-1), 2 (7.93*108 cells ml-1), 3 (11.89*108 cells ml-1), 4 (15.86*108 cells ml-1), 5 (19.83*108 cells ml-1) at 750nm. Results: highest accumulated hydrogen yields after 24 hour long incubation. 124: Chlamydomonas reinhardtii cc 124: 18.66 ml l-1 d-1 (algae OD750: 0.7; 25 ml/15 ml gas/liquid phase; 1.03*108 cells ml-1) 360: Chlorella sp. MACC 360: 98.98 ml l-1 d-1 (OD750: 1; 20 ml/20 ml gas/liquid phase; 3.96*108 cells ml-1) 549: Chlamydomonas sp. MACC 549: 6.84 ml l-1 d-1 (OD750: 1; 20 ml/20 ml gas/liquid phase; 0.38*108 cells ml-1 ) Semi-continuous hydrogen production with the Chlorella strain Conclusion The cell sizes of the algal strains indirectly effected the accumulated hydrogen yields of the co-cultures. The algae cell numbers in the algal-bacterial co-cultures showed strong correlation with the accumulated hydrogen yields and also with the relative hydrogen level. The Chlorella sp. MACC 360 - E. coli DhypF co-cultures had a much higher algae cell number at the same optical density - therefore produced more hydrogen - compared to the Chlamydomonas reinhardtii cc124 - E. coli DhypF and Chlamydomonas sp. MACC 549 - E. coli DhypF co-cultures. The algae cell numbers showed reciprocal proportionality with the cell size of the specific algae strains, thus, the size of the specific algae cells is one of the most important factor in hydrogen productivity of the algal-bacterial co-cultures. The lower oxygen production rate and the unaltered respiration (algal and bacterial) establish anaerobic environment earlier in the more concentrated cultures permitting the initiation of algal hydrogen evolution. On the other hand higher initial algae concentration affects not only oxygen consumption, but results also in faster acetic acid consumption leading to hydrogen saturation at lower level. The depletion of acetic acid allowed the re-accumulation of photosynthetic oxygen in the headspace of the algal-bacterial cultures. Thus, co-cultures containing more algae cells consume acetic acid faster from the TAP medium leading to the elevation of oxygen level and the inhibition of hydrogen evolution. 1152.49 ml H2 was produced with 1 l mixed culture containing 10 ml l-1 acetic acid in 12 days by using the optimized culture setup, daily releasing of accumulated hydrogen with gas mixture and re-adjusting of culture density with biomass harvesting. The average daily hydrogen production was 96.04 ml H2 l-1 culture. Culture: Chlorella sp. MACC 360 – E. coli DhypF (JW5433) Gas phase was pured with gas mixture once per day (CO2 2.29 n/n%, O2 4.33 n/n%, N2 100 %). Optical density was reset daily by the partial harvesting of mixed biomass. Total TAP media was changed on every third day (marked with red arrows). 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Volgusheva, A., Kukarskikh, G., Krendeleva, T., Rubin, A., & Mamedov, F. (2015). Hydrogen photoproduction in green algae Chlamydomonas reinhardtii under magnesium deprivation. RSC Advances, 5(8), 5633-5637. Wirth, R., Lakatos, G., Maróti, G., Bagi, Z., Minárovics, J., Nagy, K., & Kovács, K. L. (2015). Exploitation of algal-bacterial associations in a two-stage biohydrogen and biogas generation process. Biotechnology for biofuels, 8(1), 1.a Acknowledgement: This work was supported by HU09-0091-A1-2016 Norway Grant and the GINOP-2.3.2-15-2016-00011 grant supported by the European Union and the State of Hungary.