SPECTROPHOTOMETRY AS A METHOD FOR MICROALGAE CULTIVATION ANALYSIS. Correspondence address:

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1 SPECTROPHOTOMETRY AS A METHOD FOR MICROALGAE CULTIVATION ANALYSIS Carolina Ribero Cereijo 1 ;Hugo Santana 2 ; Félix Gonçalves de Siqueira 3 ;Bruno Brasil Federal University of Tocantins, Gurupi - TO Brazil 2. Federal Universityof Bahia, Vitoria Da Conquista - BA Brazil 3. Embrapa Agroenergy, Brasília - DF Brazil Correspondence address: carolina.cereijo@colaborador.embrapa.br KEYWORDS Chlamydomonas, Chlorella, biomass, productivity, cultivation

2 SPECTROPHOTOMETRY AS A METHOD FOR MICROALGAE CULTIVATION ANALYSIS Carolina Ribero Cereijo 1 ;Hugo Santana 2 ; Félix Gonçalves de Siqueira 3 ;Bruno Brasil Federal University of Tocantins, Gurupi - TO Brazil 2. Federal Universityof Bahia, Vitoria Da Conquista - BA Brazil 3. Embrapa Agroenergy, Brasília - DF Brazil Correspondence address: carolina.cereijo@colaborador.embrapa.br KEYWORDS Chlamydomonas, Chlorella, biomass, productivity, cultivation INTRODUCTION Microalgae are photosynthetic unicellular microorganisms found in seawater and fresh water.this group of microorganisms shows high plasticity in terms of adaptation to various environments due its high genetic diversity and low requirement of nutrients. The basic nutrients required for microalgae growth is the presence of nitrate, phosphate, carbon dioxide, water and light, but other nutrients are also required(li et al., 2008).Due to this characteristic, microalgae have been studied as a potential production source of many products, such as pigments, fatty acids and proteins, which make this microorganism suitable for use as animal fed, fertilizers and biofuel production(gouveia et al., 2005; Gouveia e Oliveira, 2009). For industrial application of microalgae, growth phases, growth rate and biomass accumulation of the microorganismsmust be known. The classic methodologies applied for these analyses are cell counting and dry weight, however alternative methodologies can be used to estimate the growth rate and biomass accumulation in this group of microorganisms. The spectrophotometry is an alternative methodology that can be used to estimate these growth parameters due the fact that microalgae cells have chloroplasts rich in chlorophyll a, a molecule that have an well-defined absorbance peak at 680nm that have less interference of other pigments produced by this group of microorganisms. The presence of this pigment make the estimation of these growth parameters by spectrophotometry relatively easy, once the amount of this pigment in the growth medium is the most significant and increases with the cell proliferation (Bidigare et al., 1990; Bricaud et al., 2004). The objective of this study was to evaluate the applicability of in vivo spectrophotometry for determination of cell counting and biomass accumulation in microalgae strains. MATERIALS AND METHODS For comparative analysis, four microalgae strains (two Chlamydomonas sp.and two Chlorella sp.) of EMBRAPA Agroenergymicroorganisms collection (Brasília DF Brazil) were cultivated in 500 mlof Bold s Basal Medium (BBM) ph 7.1during 8 days

3 in aerated flasks(5 L/h of atmospheric air).the samples were incubated at 26 C with 12h/12h light/dark cycle at8.000 Lux. To perform the analysis, samples were collected every day in sterile flow cabinet. For spectrophotometry analysis, samples were analyzed in spectrophotometer at 680nm. For cell counting, samples were analyzed using Neubauer chamber. For dry weight estimation, 10 ml of the samples were washed 3 times using refrigerated centrifuge (4 C, 10000g, 15 minutes) and the re-suspended precipitate were dried overnight using drying oven (105 C). All cultivation were performed in triplicate. RESULTS AND DISCUSSION As can be seen in the Figure 1a, the optical density of the culture stabilizes at the sixth day in all strains. This observation also can be seen when the cell number was accessed (Figure 1 b). However, when the analysis of the dry weight was performed (Figure 1c), was possible to observe that continues to increase until the end of the cultivation, showing that the analyzed strains show high potential of biomass accumulation Optical density (680nm) a cell number.ml b

4 Biomass yield (g.l -1 ) c LBA8 LBA32 LBA39 LBA40 Figure 2.Microalgae growth curve.the strains LBA8, LBA32, LBA39 and LBA40 were grow in triplicate using aerated flasks with Bold s Basal Medium (BBM), ph 7.1 during 8 days in 12h/12h light/dark cycles with Lux at 26 C. The optical density at 680nm (a) was accessed by spectrophotometry. The cell number of the cultivated strains was accessed by cell counting in Neubauer chamber. Data are represented as cell number per milliliter (cell number.ml -1 ). The biomass yeld (c) was accessed by dry weight analysis. Productivity is represented as g per liter (g.l -1 ). Table 1. Correlation (R 2 ) between cell number and optical density at 680nm and between dry weight and optical density at 680nm. Correlation (R 2 ) Strain Optical density x Cell number Optical density x Dry Weight LBA LBA LBA LBA The data showed that is possible to correlate the dry weight and the cell counting with the spectrophotometric analysis. When the correlation between the data was accessed (Table 1), was observed strong correlation (R 2 >0.93)between the cell number and optical density in all strains analyzed. This strong correlation also was observed with the dry weight, however the strain LBA40 showed relatively weak correlation (0.89) in comparison to the others strains. This may be due the reproduction characteristics of Chlamydomonas sp., which also contributed to the lack of precision when the cell number was accessed (Figure 1b). CONCLUSIONS The data obtained in this study showed that spectrophotometry is a suitable methodology for the estimation of growth parameters of microalgae strains. This technique can be used to simplify the acquisition of the data used to prepare inoculum for a start culture or estimate the biomass productivity before the collection of the microalgal biomass. However, more analysis are required to determinate the applicability of spectrophotometry at an industrial level. ACKNOWLEDGEMENTS The authors thank the financial and structure support from EMBRAPA and CNPq.

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