Selection Of High Oil-yielding Microalgae And Optimization Of Its Nitrogen And Phosphorus Removal And Lipid Accumulation

Recently, microalgae with inherent advantageous qualities such as rapid biomass concentration, high lipid content, and tolerance for extreme environment have attracted significantly increased interest as potential biodiesel feedstocks. However, low efficiency and high cost of oil production are the main bottleneck for large scale production of algae-biofuel. Utilizing nutrient and water resource from wastewater treatment for algae cultivation has became an important issue. Using wastewater will save nearly 94% of the nitrogen requirements and 90% of the freshwater which accounts for 25% of the total energy input.Therefore, in view of the broad development prospect of the combination of wastewater treatment and algae-biofuel production, microalgae with high oil-yielding ability and suitable biodiesel properties was screened and used for nitrogen and phosphorus removal in the artificial wastewater. The study investigated the effect of environmental factors on the nutrient removal and lipid accumulation ability of the microalgae and identified the suitable environmental conditions for the microalgae achieving the high nutrient removal efficiency and lipid yielding, beside studied the relationship among the growth rate, nutrient removal and lipid accumulation of the microalgae. At last, the mechanisms of environmental adaptation in the nutrient removal process were illustrated in detail. This application will realize the transformation of the sewage treatment system to the production system which could bring ecological and financial benefits.In this study, lipid-rich energy microalgae strains were screened. Ten microalgae strains were obtained from freshwater algae culture collection of the Institute of Hydrobiology in China, and they were evaluated for the potential of biodiesel production by determining the growth, oil-yielding ability and biodiesel capabilities. And results indicated that Phaeodactylum tricornutum was the most advantageous species coontained oil content of 61.43±0.95%, lipid productivity of 26.75 mg·L-1·d-1, the promising C16-C18 fatty acids of 74.50%, C16:1 of 22.34% besides favourable biodiesel capabilities such as greater cetane number of 55.10, minor iodine number of 99.2gI2/100g and rather small cloud point of 4.47℃.Furthermore, besides fatty acid profiles, significant biodiesel properties such as kinematic viscosity, specific gravity, cetane number, cold flow, oxidative stability, and higher heating value were analyzed to evaluate the potential of the microalgae candidates for biodiesel production. Using the predictive models based on fatty acid composition makes the estimation of fuel property easy and quick, which is of importance in biodiesel-based strain selection and the optimization of microalgae cultivation. Average degree of unsaturation, a computed fuel property, was proved to have high correlation with several other properties; an increase in average unsaturation leads to lower cetane number and poorer oxidation stability, but improved low temperature performance. To achieve the excellent biodiesel property, the average degree of unsaturation should keep from 0.2 to 1.4.Then, we explored the variations of growth, lipid and fatty acid yielding of P. tricornutum responded to gas liquid ratio (GLR). Results indicated that GLR had linearity with growth rate, oil content, and fatty acid methyl ester (FAME) content, respectively. The best GLR for P. tricornutum growth and accumulating oil is 1.5 vvm, under which conditions P. tricornutum achieved highest biomass production (227.09mg·L-1·d-1), highest lipid productivity (48.48 mg·L-1·d-1) and significant content of monounsaturated fatty acids (MUFA) of 48.79% and saturated fatty acids (SFA) of 50.16%. pH value was adjusted by the air flow and algal photosynthesis. CO32-/HCO3-ratio had an effect on the fatty acids production, particularly on the most promising constituent of C16:1, which account for 48.36%.Temperature of 20℃ was considered as the optimum temperature for P. tricornutum growth and yielding oil, with highest biomass productivity (98.64 mg·L-1), lipid productivity (24.66 mg·L-1·d-1) and fatty acid methyl ester production (5.19 mg·g-1). Temperature fluctuations every four hours had small effect on the growth rate and biomass of the algae, but significantly inhibited the lipid productivities, which were lower 50% than the lipid productivities at 20℃. Temperatures higher than the 20℃ were good for lipid accumulation.In the light:dark (12h:12h) cycles, low temperature in the night cycle could avoid the biomass loss and it was good for lipid accumulation. Under light temperature 20℃ and dark temperature 10℃ in light:dark (12h:12h) cycles,besides the highest lipid content (26.13%), P. tricornutum achieved the highest biomass (58.52 mg·L-1·d-1) and lipid productivity (15.29 mg·L-1·d-1), which were higher 1.6 times and 2.5 times, respectively, than the control and the treatment of night temperature of 20℃. So day temperature of 20℃ and night temperature of 10℃ were the optimum temperatures for P. tricornutum growth and lipid accumulation in day:night (12h:12h) cycles. The optimium pH for P. tricornutum ranges from 8.8 to 9.4. pH is 9.6 at the end of stationary phase.By the isolation and purification procedures, six pure unialgal strains were finally obtained from Quancheng Lake in Jinan City, Shandong Province. SDEC-6 and SDEC-8 were finally selected the most advantageous algae strains with high lipid production and suitable biodiesel properties. SDEC-6 attained the highest lipid content of 37.6%and SDEC-8 achieved the highest lipid productive of 13.52 mg·L-1·d-1. The fatty acid productivities of SDEC-6 and SDEC-8 were 11.7 mg·g-1 (DW) and 9.55 mg·g-1 (DW), respectively. The two strains were identified as Cyanobium parvum SDEC-6 and Scenedesmus quadricauda SDEC-8, respectively, using microscope, scanning electron microscopy (SEM), and transmission electron morphology (TEM) combined with 16S rRNA and 18S rRNA identification. The partial 16S rRNA and 18S rRNA sequence from C. parvum SDEC-6 and S. quadricauda SDEC-8 were submitted to the GenBank, and the Accession Number of them were KU318705 and KF999643, respectively, these two strains were preserved in China Center for Type Culture Collection (CCTCC, Wuhan), the numbers are CCTCC NO:M2015769 and CCTCC NO:M2014448, respectively.We studied the potential of C. parvum SDEC-6 and S. quadricauda SDEC-8 for growth, lipid accumulation, FA compositional profiles and nutrient removal from wastewater. Results indicated that C. parvum SDEC-6 and S. quadricauda SDEC-8 showed good ability to yield oil and remove nutrients in the wastewaters samples. In b artificial wastewater (TN:40mg·L-1, TP:8 mg·L-1), the lipid content of C. parvum SDEC-6 achieved around 30%. And it achieved the highest saturated fatty acid content (SFA), biomass concentration and fatty acid methyl ester (FAME) content. Compared to other cyanobacteria and green algae, C. parvum SDEC-6 showed better quality of nutrient removal and lipid accumulation, in five days, it showed the highest nitrogen and phosphorus remove rate and higher lipid accumulation rate, which were 0.82 mg·L-1·d-1,1.43 mg·L-1·d-1 and 11.65 mg·L-1d-1, respectively.In the b (TN:40 mg·L-1, TP:8 mg·L-1) wastewater, the removal efficiency of total nitrogen and phosphorus were 84.76% and 65.09%，respectively. Total lipid content achieved 28.04%, and the lipid accumulation rate was ten times higher than the cyanobacteria reported before. Hence, C. parvum SDEC-6 was suitable for the treatment of high nitrogen and phosphorus wastewater with good characteristics that high efficiency of nitrogen and phosphorus removal, less incubation time and high lipid productivities.There is no significant difference of the lipid productions of S. quadricauda SDEC-8 in the three wastewaters with different nitrogen and phosphorus concentrations. At the 9th day, the removal efficiency of TN was over 90%, the removal efficiencies of TP ranged from 60% to 98%, and they were totally removed at 16th day. It showed better nitrogen and phosphorus removal ability than other algae in this study besides high lipid productivities of 10 mg-L-1·-d-1. In the b (TN:40 mg·L-1, TP:8 mg·L-1) wastewater, S. quadricauda SDEC-8 obtained the highest mono-saturated fatty acid content (MUFA) (35.35%), FAME content (59.57±0.02 mg·g-1 DW) and FAME productivity (17.53 mg·L-1·d-1). In addition, the TN and TP concentration of effluent treated by S. quadricauda SDEC-8 could reach the level of class A of water quality standard of surface water (GB3838-2002). The abilities of S. quadricauda SDEC-8 for TN and TP removal and lipid production were superior to those of other reports. Hence, S. quadricauda SDEC-8 was regarded as the promising species for the depth treatment of the secondary sewage.According to different characteristics of the two dominate species, we proposed cultivating the C. parvum SDEC-6 by campus sewage. We also proposed the use of organic carbon source for growth and lipid enrichment in Scenedesmus obliquus was grown in secondary wastewater with sodium acetate (NaAc). We investigated the effect of uptake of nutrient (Nitrogen and Phosphorus) on bio mass, lipid productivity and fatty acid profiles of the potential algae species in wastewaters. We evaluated the growth, nutrient assimilation and lipid production of the potential algae species in wastewaters. Finally, we proposed a better way to optimize the efficiency of nutrient uptake and lipid production of the algae species in wastewater system.Results showed that C. parvum SDEC-6 grew fast in the campus sewage. The lipid content achieved 26.8%, which was compared to that produced in the artificial campus sewage. The removal efficiency of TN, ammonium, and TP were 29.55%,41.17% and 95.62%, respectively. It showed the higher TP removal rate than that in artificial campus sewage. And at 6th day, over 90% of TP was removed. C. parvum SDEC-6 had C16:0 content of 69.10% and C18:1 content of 21.85%. The correlations among the remaining TN, NH4+-N, TP and biomass concentration showed good liner relationship. C. parvum SDEC-6 uptaked ammonium as the main nitrogen source in campus sewage and phosphorus deficiency resulted the low specific growth rate and low nitrogen and phosphorus removal efficiency.S. obliquus showed good abilities of growth, lipid accumulation and nutrient removal in domestic secondary effluent. Acetate addition improved the growth and fatty acid production besides the favorable biodiesel property of S. obliquus. The treatment with 20 mg L-1 of NaAC added showed highest biomass productivity of 129 mg L-1 d-1, saturated fatty acid (SFA) content of 177 mg g-1, monounsaturated fatty acids (MUFA) content of 61.5 mg g-1, and FAME productivity of 34.3 mg L-1 d-1. The MUFA content and FAME productivity were all 3 times higher than the control treatment without NaAC addition.The correlation among the remaining dissolved inorganic nitrogen (DIN), TP and biomass concentration in the secondary effluent showed obvious exponential relations. S. obliquus showed fast absorption of phosphorus, it may be caused by the large cell surface of S. obliquus. The enhancement of the biomass concentration of S. obliquus was mainly caused by the utilization of nitrite. When the N/P ratio was smaller than 16, specific growth rate and N/P ratio showed good linearship relation. Ammonium was better for improving the cell growth rate. When the N/P ratio was higher than 16, phosphorus limitation was that the main factors that inhibit the growth of S. obliquus cells. Considering nutrient removal, biomass production, incubation time and economic cost, in order to optimize lipid production and achieve 90% nutrient removal, these results indicate a steady state biomass of 350 mg L-1 and steady harvest rate of 20% d-1 should be maintained.