| Microalgae are natural resources which hold various economic values. The cultivation and the exploitation of their products may aid to solving the problems met currently in food, energy, environments among others. However, the traditional autotrophic cultivation mode of microalgae has some limitations, for example, low cell density, high harvesting cost and so on, which have hindered the industrialization of microalgae. More and more researches have shown that the heterotrophic and mixotrophic cultivation modes can effectively improve the microalgal growth and greatly increase microalgal biomass production, thus becoming effective modes of cultivation. A small portion of microalgae can servive heterotrophic or mixotrophic cultivation while a large number of microalgae can not do so. The reasons underling such pnenomenon remain unclear until now. Studied in a wide range of species are highly appreciated in order to understand the physiological and biochemical mechanism of heterotrophic and mixotrophic growth of microalgae, and to explor the high value-added active substances.This study was carried out in an autotrophic and oil-producing microalga Nannochloropsis oculata and a bait microalga Tetraselmis chuii. These two microalgal species purified with streak plate method in combination with antibiotics. The two microalgae were able to grow heterotrophically and mixotrophically when the carbon source were suitable. The best variety and concentration of carbon source were identified. Cell microstructure, cell growth and biochemical components of microalgae species were also investigated. The findings provided the basic information of microalgal nutritional styles. In addition, these findings paved the ways of microalgal resource evaluation and protection.TV. oculata may easily be contaminated by bacteria. Such scenario may be worsened when the carbon concentration increased. At a high carbon concentration (50 mM), bacteria may bloom in a short time, which will certainly affect and even stop the growth of microalgae. At a low carbon concentration (5 mM), microalgae may establish a symbiotic relationship with bacteria, reaching a cell density much higher than that in autotrophic mode. However, the existence of symbiotic bacteria may fluctuate microalgal growth, making their growth deviate from the normal.By cloning and sequencing the 16S rRNA gene (rDNA hereafter), the symbiotic bacterium was identified as Alteromonas macleodii. Bacterium free microalgae were obtained by plate streaking and antibiotics treating. N.oculata cultured in different modes showed different antibiotics sensitivities:Autotrophic N.oculata was not sensitive to kanamycin below 200 μg/mL, but sensitive to chloramphenicol at concentrations ranging from 20 to 200 μg/mL. Kanamycin from 120 to 200 μg/mL may remove the symbiotic Alteromonas macleodii, of which 120 μg/mL was the best for microalgal growth. Chloramphenicol below 100 μg/mL functioned less in removing bacteria while kanamycin at 120 μg/mL and chloramphenicol at 20 μg/mL in combination can completely remove bacteria without influencing the mixotrophic growth of N.oculata.Pure culture of N. oculata can grew mixotrophically by selectively utilizing organic carbons. High concentrations of sucrose, glycerol, ethanol and sodium acetate inhibited the mixotrophic growth of this alga while sucrose at 10-20 g/L, glocuse at 5-20 g/L, glycerol at 20-30 mM and ethanol at 5-10 mM benefited its growth and enhanced its lipid accumulation. The microalgal cells cultured in sodium acetate expanded to a round shape, with diameters of 3-4, larger than those cultured in autotrophic mode. In addition, the whole cells appeared to be bright green after staining with the dye BODIPY 505/515, suggesting the lipid content of cell was high. The sodium acetate at concentrations higher than 0.1 g/L suppressed cellular growth and lipid accumulation. The higher the concentration was, the higher the lipid content was. Under restricted heterotrophic conditions, the optical density of N. oculata remained fairly constant; however, N. oculata returned to the normal growth when it was moved back to light after 2-week in 5 g/L glucose, suggesting that N. oculata was not yet dead at the heterotrophic mode, but grew at a extremely low speed.With the aid of 18S rDNA and rbcL genes and through molecular phylogenetic analysis, a green microalga collected from south Yellow Sea was identified as T. chuii. Glycerol is the best carbon source for the mixotrophic growth of T. chuii. Glycerol at concentrations ranging from 1 to 10 g/L can significantly improve its growth, and 15 g/L sucrose and 10-25 g/L glocuse,10-20 mM ethanol and 0.1 g/L glycine promoted also on the mixotrophic growth of T. chuii. In contrast, sodium acetate higher than 0.1 g/L and acetic acid inhibited the growth of T. chuii. T. chuii did not use sucrose, acetate sodium, glycine, glycerol, ethanol and acetic acid under strict heterotrophic growth; however, it was able to metabolize glucose for growing heterotrophically. Further, cell density increased with the increase of the concentration of glucose.The growth parameters and biochemical components of the microalga T. chuii were compared in different autotrophic modes and at different ratios of carbon to nitrogen (C/N). The results showed that autotrophic T. chuii grew very slow as its cell density was low. The microalga reached its stationary stage faster than it did in heterotrophic or mixotrophic cultures. Ranging from 4 to 24, with the increase of the ratio of C/N, lipid percentage continued to rise until protein content continued to drop in either heterotrophic or mixotrophic mode. The ratio of C/N 12 was likely to be the turning point of biomass accumulation and polysaccharide synthesis in either heterotrophic or mixotrophic cultures where T. chuii had the maximum biomass and the minimum polysaccharide content. T. chuii had low lipid content. The proportion of lipid under heterotrophic culture was extremely low, thus such culture mode may not suitable for the large scale oil production. Polysaccharide was relatively rich biochemical components, and was believed to have the highest scaling-up feasibility in heterotrophic mode.In conclusions, we obtained rich information of autotrophic microalgal culture modes, and provided scientific basis and technical supports for the utilization of microalgal cultivation at high density, development of high value-added products, and establishment of the best cultivation mode. In addition, our findings may also provide a theoretical reference for elucidating the mechanisms of heterotrophy and mixotrophy of microalgae. |
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