## 3 Results and Discussion
### Effect of Enzymatic Hydrolysate on Chlorella Biomass, Fatty Acid and Pigment Productivity  
Wood hydrolysates were reported to possess negative or positive effects on microalgae growth. Wood hydrolysate from pulp and paper mills [25] or wood hydrolysate from diluted acid treatment [13] inhibited _Chlorella_ growth, if applied at high loadings, and supported _Chlorella_ growth when applied at low loadings. In our previous study [13], neutralized beech wood diluted acid hydrolysate at a 12% loading partially inhibited _Chlorella_ growth, when compared to a synthetic organic carbon-based medium. In the literature, lignocellulosic hydrolysates contain a range of compounds that inhibit the metabolism of yeast [26; 27], and can also negatively affect microalgae growth [10]. In the present study, neutralized enzymatic beech wood hydrolysate at 10% loading (TGP-Enz10) also inhibited _Chlorella_ growth. However, inhibitory activity of this hydrolysate seems to be attributed solely to the presence of neutralized citrate buffer (TGP-Cit10). Our results show (Tables 1 and 2) that the inhibitory effect of enzymatic hydrolysate (TGP-Enz10) is not greater that the inhibitory effect of a synthetic medium containing glucose and citrate buffer (TGP-Cit10). This suggests that 10% enzymatic beech wood hydrolysate does not contain lignocellulosic-derived compounds that could inhibit microalgae growth, and that the only inhibitory substances are those added externally in order to produce wood hydrolysate. Moreover, inhibition in enzymatic hydrolysate containing neutralized citrate buffer (TGP-Enz10) seems to be partially alleviated when compared to TGP-Cit10. As a result, the following biomass productivities were achieved: 403 \(\pm\) 20 (mg L\({}^{-1}\) d\({}^{-1}\)) for TGP, 345 \(\pm\) 20 (mg L\({}^{-1}\) d\({}^{-1}\)) for TGP-Enz10 and 279 \(\pm\) 15 (mg L\({}^{-1}\) d\({}^{-1}\)) for TGP-Cit10 (Tables 1 and 2). Therefore, enzymatic wood hydrolysate shows the potential to be used as a feedstock for microalgal biomass production. Moreover, enzymatic wood hydrolysate should contain only glucose as a final product from cellulose hydrolysis. Cellobiose was tested in terms of its effect on _Chlorella_ growth, and was found to be neutral, as this sugar neither supported nor inhibited _Chlorella_ growth (data not shown). However, cellobiose should be hydrolyzed to glucose in order to improve utilization of sugar for microalgae growth. In our study, the presence of cellobiose in wood enzymatic hydrolysate was not detected (data not shown). The effect of wood enzymatic hydrolysate on fatty acid production in heterotrophic _Chlorella_ culture, was evaluated (Table 1). Fatty acid compositions in _Chlorella_ biomass were different during different cultivation profiles. A remarkable change in fatty acid composition was observed between _Chlorella_ cultivated in TGP medium and TGP-Enz10 or TGP-Cit10. In TGP medium, the primary  
Figure 2: Effect of neutralized (pH = 7) citrate buffer on _Chlorella_ growth in TGP (1 g/L glucose) medium. Different neutralized citrate buffer loadings: 0%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20% are marked: Cit0, Cit0.5, Cit1, Cit2, Cit5, Cit10, Cit15, Cit20, where 100% neutralized citrate buffer is \(\sim\)0.05 M. Heterotrophic (D-Dark) growth in photobioreactor at 0 μE m\({}^{-2}\) s\({}^{-1}\). Mixotrophic (L-Light) growth in photobioreactor at 75 μE m\({}^{-2}\) s\({}^{-1}\). Temperature of cultivation: 25 \({}^{\circ}\)C. Number of independent experiments: 3.  
fatty acid was linolenic acid (C18:3), followed by palmitic acid (C16:0) and linoleic acid (C18:2). In TGP-Enz10 and TGP-Cit10, C18:2 was a primary fatty acid (a remarkable increase in % composition), followed by C16:0 and C18:3 (a remarkable drop in % composition). Results suggest that the neutralized citrate buffer present in enzymatic hydrolysate and synthetic TGP medium causes a shift in fatty acid composition.