Effect Of Environmental Factors On Growth Of Isochrysis Galbana

Isochrysis galbana, a species of marine microalgae, which is of substantial interest inaquaculture for its good nutritive characteristics, especially to mollusks larvae, fish andcrustaceans in the early stages of growth. Inorder to explore ways of high density culture, aseries of researches were carried out from the basic cultures in small bottles, high densitycultures in the photobioreactors, the isolation and identify of the growth-inhibitor to themechanism of dissolved inorganic carbon (DIC) utilization in Isochrysis galbana. The mainresults are summarized as following.1. The optimal medium and culture conditions of Isochrysis galbanaThe optimum medium: NaNO₃ 45.5 mg/L, KH₂PO₄ 2.195 mg/L, Na₂SiO₃ 0.8714 mg/L,NaHCO₃ 20 mg/L, MnCl₂·2H₂O 15.8336μg/L, CuSO₄·5H₂O 14.9808μg/L, ZnSO₄·7H₂O0.2013μg/L, Na₂MoO₄·2H₂O 0.7295μg/L, CoCl₂·6H₂O 23.795μg/L, FeCl₃·6H₂O 482.7μg/L, Na₂EDTA 0.665 mg/L, VB₁ 100μg/L and VB₁₂ 0.5μg/L were added to natural seawater.The optimum culture conditions: tempature 25℃, pH 8.0, light intensity 7500 1x andaeration rate 0.2 vvm. 1.3 g/L of dry cell biomass was obtained in eight days culture under theoptimal cultural conditions.2. The mode of high density culture in photobioreactorsThe optimal culture conditions and the fed-batch culture, semi-continuous culturesmode was established in the plate photobioreactors. Under the conditions of 5 cm light-path,7000 1x light intensity, 0.375 vvm aeration rate and semi-continuous culture mode, themaximal dry cell biomass of 2.2 g/L was determined in 8 days in plate photobioreactors. Theresults showed that the fed-batch culture, especially fed-batch culture with various feed rateand semi-continuous cultures, was more favorable for the growth of Isochrysis galbana.3. Isolation and identified a kind of microalgal growth-inhibitorA type of antialgal substanceswas found by the cultures of cell-free filtrates whichgotten from culture suspension at the three growth phases (exponential, stationary and deathphase) of Isochrysis galbana. Results showed that, as the age of Isochrysis galbana culturesadvances, the concentration of the antialgal substances in cultural liquid increases, and whichsuggested that Isochrysis galbana produced gradually antialgal substances from the exponential growth phase up to the death phase. The growth inhibitory rates of Isochrysisgalbana by cell-free filtrates at the three phases were 7.17%, 34.2% and 64.1% in day 10,respectively. The crude,antialgal substance (CEAE) was extracted out from the old culturalliquid by ethyl acetate, which also showed significant inhibitory effects on growth ofIsochrysis galbana. When CEAE concentrations was from 3.6 mg/L to 21.6 mg/L, the growthinhibitory rate of Isochrysis galbana by CEAE were 12.9%, 27.5%, 29.3%, 59.5% and 67.2%in day 6, respectively. The CEAE could significantly (P＜0.05) inhibit the cell growth of thesix species of marine feed microalgae too, at the concentration of 21.6 mg/L, the growthinhibitory rate of Chaetoceros muelleri, Chaetoceros gracilis, Phaeodactylum tricornutum,Nitzschia closterium, Platymonas elliptica, Dunaliella salina was 63.3%, 59.6%, 61.1%,66.2%, 68.3% and 48.5% in day 6, respectively. And the biochemical compositions in thecells of Isochrysis galbana and the six species of marine feed microalgae also could beaffected by CEAE.The growth-inhibitor was successful isolated and purified from CEAE. Molecularformula of the growth-inhibitor, C₂₂H₃₈O₇, was determined by FABMS, m/z 414 [M-H]-. InUV spectrum the maximum absorption was at 278 nm that suggested the presence ofO=C-C=C functionality. The ¹³C-NMR spectrum of the growth-inhibitor showed 22 carbonsignals, including three carbonyl carbon signals (6167.9, 167.9 and 167.89), threeoxymethylene carbon signals (δ72.0, 65.7 and 65.7), four methyl carbon signals (δ30.8, 30.8,19.8 and 19.8), one ethylene carbon signal (δ132.5 and 129.1), one quaternary carbon signal(δ77.4), one methine carbon signal (δ27.9) and eight methylene carbon resonances. Fourmethyl proton signals (δ1.72, 1.72, 0.99 and 0.99) were observed in the ¹H-NMR spectrum.The ¹H-NMR spectrum of the growth-inhibitor exhibited two doublets for two couplingprotons atδ7.53 andδ7.12, a OH group atδ3.11, a isopropyl group atδ4.08 (1H) andδ0.99(6H). According above information, the chemical structure of the growth-inhibitor wasidentified as 1-[hydroxyl-diethyl malonate]-isopropyl dodecenoic acid.The further investigation found that exogenously added GI significantly decreased thechlorophyll contents in Isochrysis galbana and the six species of microalgae (Chaetocerosmuelleri, Chaetoceros gracilis, Phaeodactylum tricornutum, Nitzschia closterium,Plytymonas elliptica and Dunaliella salina). And it had a very significant effect on the rationof extracellular protein to polysaccharide and activities of NR (nitrate reductase), SOD(superoxide dismutase) and POD (peroxidase) in the cells of all test microalgae. But it wasobvious to increase MDA (malonyldialdehyde) content in algal cells. The results gave hints toelucidate the species-specific antialgal mechanisms of GI. Inhibition mechanism of GI onmicroalgae is to decrease the chlorophyll contents, and decrease or increase activities of NR,SOD and POD of algal cells, and change the ratio of extracellular protein to polysaccharide, thus increase in ratio of extracellular protein to polysaccharide leads to the increase inhydrophobicity of algal cells, finally algal cells flocculate and subside. Affect uptake ofinorganic nitrogen by cells and photosynthesis might play a major in the inhibitorymechanism. And the excess active oxygens were produced under GI stress and active oxygenparticipated in the damage of GI to microalgae. It indicated that the physiological metabolismof microalgae was inhibited by GI.4. Relationship betweenthe the activity of carbonic anhydrase and utilization ofCO₂Carbonic anhydrase, a ubiquitous mental enzyme that can catalyze the transformationreaction between HCO₃^- and CO₂, is one of enzymes whose catalytic velocity is confirmed tobe the fastest. It has been taken as a component of CO₂ concentrating mechanism (CCM) andplays a role in photosynthetic CO₂ fixation. Extracellular carbonic anhydrase (CA) activity inIsochrysis galbana was influenced by several external factors. Our research demonstrated thatlight intensity, pH and ions such as NO₃^-, NH₂^-, NH₄⁺, H₂PO₄^- and HCO₃^- were importantexternal factors that could regulate the extracellular CA activity in the cells of Isochrysisgalbana. The activity of CA significantly depends on the light intensity, when the lightintensity increased from 0 to 8000 lx, the CA activity increased rapidly and arrived its highestpoint at 6000 lx. With the increasing of pH values, the CA activity significant increased. Atrange of NO₃^- concentration from 0.5 mmol/L to 4 mmol/L, the CA activity was higher, andarrived to the maximal value at the concentration of 2 mmol/L; the CA activity decrease whenNO₃^- concentration was above 4 mmol/L, but the CA activity still was higher than that of thecontrol. The CA activity was pronounced increased with the increasing of NH₂^-concentrations and arrived to the highest value at the concentration of 3.5 mmol/L, thenslowly decreased when NH₂^- concentration over 3.5 mmol/L. Low NH₄⁺(0.5-2 mmol/L)concentrations caused significant increase in the CA activity, but over 2 mmol/L, the CAactivity sharply decreased. The CA activity was lower under the conditions of phosphatestarvation, and a significant increase in the CA activity was observed with the increasing ofH₂PO₄^- concentration, and a maximum value was obtained at H₂PO₄^- of 0.05 mmol/L. But,against with the above factors, HCO₃^- inhibited the CA expression.According our data, the cells of Isochrysis galbana can also transport HCO₃^- directlydependent-ATPase as viewed by DIDS inhibition. Band three proteins and Na⁺/ HCO₃^-co-transport system have been found involving in the direct transportation since SITS hasaffection on carbon acquisition. Hence, external carbonic anhydrase dehydrating HCO₃^- toCO₂ is the main pathway of DIC utilization by Isochrysis galbana. In addition, dissolved inorganic carbon (DIC) utilization is significantly inhibited by Ez (ethoxyzolamide),suggesting internal CA is essential to photosynthesis in Isochrysis galbana.