Photo-mixotrophic Cultivation of Algae Euglena gracilis for Lipid ...

Photo-mixotrophic Cultivation of Algae Euglena gracilis for Lipid Production67centrifugation (4500 rpm for 20 min). Sample solutions werefiltrated through a 0.20 μm filter before HPLC determination.CO 2 concentrations were measured in the bioreactor air inflowand outflow. CO 2 concentration measurements were performedby infrared gas analyzers (Oxybaby ® V range, WITT-Gastechnik,Witten, Germany) every 12 hours.Cell number of E. gracilis in cultivation medium was determinedby counting under microscope (400 X magnification) inThoma chamber. Samples were continuously drawn from thestirred tank photo-bioreactor and after appropriate dilutionwith sterile water plated in the chamber.Results and discussionOptimization of medium composition for E. gracilisphoto-heterotrophic cultivationDuring this investigation photo-heterotrophic cultivation ofE. gracilis was performed in order to increase biomass and lipidyield. Therefore, different carbon sources (glucose, ethanol andacetic acid) and CSL were used for the modification of Hutnermedium composition. The CSL was used to replace glutamic,malic and succinic acids, glycine, asparagine and urea as wellas vitamins (B 1 and B 12 ) in order to simplify and reduce the costof cultivation medium. In this investigation E. gracilis cultivationwas also done on the original Hutner medium as control.Results of these photo-heterotrophic cultivations are presentedin Table 1. As it can be seen in Table 1, the highest biomass yieldand cell density were observed by the cultivation on the originaland modified Hutner medium with glucose and CSL. Thiseffect can be explained by the fact that glucose is more favorablecarbon source compared to the ethanol and acetic acid due toenzyme induction and synthesis for glucose transport and metabolismin the algae cells (Yamane et al., 2001). However, thehighest lipid yield was observed during cultivation on the modifiedHutner medium with glucose and CSL what clearly showsthat this medium composition is suitable for lipid accumulationin algae E. gracilis. On the basis of these results it is also clearthat CSL is adequate complex source of carbon, nitrogen andgrowth factors (amino acids and vitamins) for E. gracilis cultivationand lipid production.In order to examine the effect of CSL concentration on thelipid yield during photo-heterotrophic cultivation of E. gracilissecond set of experiments was carried out on modified Hutnermedium with glucose as a carbon source. In this research, CSLconcentration was changed in the range of 20 - 40 g/L and thereforelipid yield was reduced as a consequence of the increase ofeasy metabolized nitrogen content in the medium (Table 2).It is well known that nitrogen depletion from the medium induceslipid accumulation (Evans and Ratledge, 1984; Yoon andRhee, 1983a; Yoon and Rhee, 1983b). In the nitrogen exhaustedmedium, glucose conversion in lipids is occurred due to presenthigh ATP:AMP ratio (Sheehan et al., 1998; Chen et al., 2011). Inthis investigation, cell number was at approximately constantlevel (0.22 - 0.24 · 10 8 cell/mL), but biomass and lipid yield werereduced with increase of CSL concentration in the medium.Therefore, for further study modified Hutner medium with 20g/L of glucose and 20 g/L of CSL was selected (Table 2).Table 1. The effect of medium composition on the E. gracilisgrowth and lipid productionMediumCell number(10 8 cell/mL)Biomassyield (g/g)Lipid yield(g/g)Hutner medium 0.24 0.3 0.08MHM* (glucose and CSL) 0.22 0.24 0.20MHM* (ethanol and CSL) 0.15 0.09 0.10MHM* (acetic acid and CSL) 0.11 0.05 0.04*MHM - modified Hutner mediumTable 2. The effect of CSL concentration in the modifiedHutner medium on the E. gracilis growth and lipid productionCSL(g/L)Cell number(10 8 cell/mL)Biomass yield(g/g)Lipid yield(g/g)20 0.22 0.24 0.2030 0.23 0.15 0.1540 0.24 0.14 0.12Effect of bioreactor operational conditions on thephoto-heterotrophic cultivation of E. gracilis andlipid productionAt the start of research in the stirred tank photo-bioreactorthe effect of bioreactor operational parameters on the algaegrowth and lipid production was studied. Mixing intensity isimportant parameter of E. gracilis cultivation since it affects thecells and medium compounds distribution as well as mass andheat transfer. In algae large scale production optimal degree ofturbulence is required due to the circulation of cells from thedark to the light zone of the bioreactor (Mata et al., 2010). On theother hand, high liquid velocities and degrees of turbulence (dueto mechanical mixing or air bubbles mixing) can damage algaecells because of high shear stress (Eriksen, 2008). The maximallevel of turbulence (above which cell death occurs) is strain dependentand it has to be experimentally determined in order toavoid decline in the bioprocess productivity (Mata et al., 2010).In order to increase the biomass and lipid production, E. gracilisphoto-heterotrophic cultivation in the stirred tank photobioreactorwas carried out at various combinations of bioreactorstirrer speed and aeration rate and only a few characteristic combinationsare presented in Table 3. The cell number and biomassyield were increased with increase of stirrer speed and aerationrate due to the fact that cell growth is affected by the substrate(carbon source and oxygen) and light distribution as well as bythe shear stress in the photo-bioreactor. However, the highestlipid yield (0.25 g lipid / g cell dry weight) was observed withthe stirrer speed of 200 min -1 and aeration rate of 0.4 v/v min.Further increase of both bioreactor operational parameters wasrelated to the slight decrease of lipid yield as a consequence ofshear stress enlargement in these conditions. However, in theseconditions biomass yield was also increased what resulted inapproximately similar lipid concentrations at the end of thesebioprocesses (Table 3). On the basis of these results it can be concludedthat for successful lipid production by E. gracilis aerationrate has to be in the range of 0.4 - 0.8 v/v min and stirrer speedAgric. conspec. sci. Vol. 78 (2013) No. 1

Photo-mixotrophic Cultivation of Algae Euglena gracilis for Lipid Production69Table 4. The effect of light source type on the E. gracilisgrowth and lipid production during photo-mixotrophiccultivation (8 days) in the stirred tank photo-bioreactorRunType of lightsourceCell number(10 8 cell/mL)Biomassyield (g/g)Lipid yield(g/g)1 aqua-glow lamps 0.28 0.25 0.172 sun-glo lamps 0.38 0.36 0.29concentration increases in the photo-bioreactor. Therefore, duringphoto-autotrophic phase biomass concentration was reducedfor 23.5%, but lipid concentration remains at approximatelyconstant level (3.70 - 4.03 g/L). At the same time, CO 2 in the airoutflow of photo-bioreactor was almost completely consumed,what clearly pointed out that it is used for biomass growth andlipid production (Figure 2.).Comparison between these two photo-mixotrophic cultivationson the basis of bioprocess efficiency parameters clearlyshows that considerably higher biomass and lipid yields as wellas cell number were observed during E. gracilis cultivation withsun-glo lumps (Table 4). This effect can be explained by thefact that during cultivation with sun-glo lumps CO 2 is moreefficiently used for biomass growth and lipid production (CO 2concentration in the air outflow was only 0.1%). Therefore, it isclear that for photo-autotrophic cultivation of algae E. gracilislight source has to reproduce sun light in order to avoid significantreduction of biomass and lipid yields. Results obtained inthis research clearly indicate E. gracilis potential for photo-mixotrophiccultivation and lipid production. However, reductionof biomass yield during photo-autotrophic cultivation gives additionalchallenge for the further research of E. gracilis growthin these conditions.ConclusionsResults of this research clearly show that the photo-mixotrophiccultivation of E. gracilis for lipid production can besuccessfully carried out during two stage cultivation inside onestirred tank photo-bioreactor. Modification of Hutner mediumwas successfully done by the replacement of organic acids,amino-acids and vitamins (B 1 and B 12 ) with corn steep liquor(CSL) as complex source of these compounds. Modified Hutnermedium with 20 g/L of glucose and 20 g/L of CSL provided thehighest lipid content in E. gracilis cells and confirmed potentialof CSL as alternative and low-cost medium compound forthe E. gracilis cultivation. The lipid production of E. gracilis dependedhighly on the type of light source and CO 2 level in thebioreactor air inflow. During photo-mixotrophic cultivation ofE. gracilis with sun-glo lamps and 2% CO 2 in the air inflow thelipid yield reached the highest level (0.29 g/g). However, in thisphoto-mixotrophic cultivation the reduction of biomass concentrationduring photo-autotrophic cultivation was also observedwhat requires further research in order to define optimal conditionsfor E. gracilis cultivation in these conditions.ReferencesBligh E. G., Dyer W. J. (1959). A rapid method of total lipid extractionand purification. Can J Biochem Physiol 37: 911-917Chen M., Tang H., Ma H., Holland T. C., Ng K. Y. S. Salley S. O.(2011). Effect of nutrients on growth and lipid accumulation inthe green algae Dunaliella tertiolecta. Bioresour Technol 102:1649-1655Chisti Y. (2007). Biodiesel from microalgae. Biotechnol Adv 25:294-306Demirbas A. (2009). Production of biodiesel from algae oils. EnergSources Part A 31: 163-168Eriksen N. T. (2008). The technology of microalgal culturing.Biotechnol Lett 30: 1525- 1536Evans C. 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