Optimization of cultivation conditions and engineering by random mutagenesis for high lipid production in Schizochytrium sp. S31

Schizochytrium sp. is widely studied microalgae to obtain high content of polyunsaturated fatty acids especially docosahexaenoic acid (DHA). They contain various bioactive compounds that can be used as pharmaceutical raw material, food additive, aquaculture and animal feed. They also play a crucial role in biofuel production which can be used as an alternative energy source. Schizochytrium sp. which is grown rapidly and produced high amount of DHA can be used as an alternative to fish oils. In this study, cultivation conditions were optimized to increase biomass and total lipid productivity of the Schizochytrium sp. S31 by using response surface methodology. After optimizing growth conditions, large scale production of Schizochytrium sp. S31 under favorable conditions were performed and downstream process including cell lysis, drying and lipid extraction were compared and optimized to maximize total lipid specially DHA extracted from Schizochytrium sp. S31. Finally, random mutation was applied by ultraviolet (UV) radiation or chemical mutagen and high yield of lipid accumulating mutants of a Schizochytrium sp. were selected by flow cytometric-based selection. Many factors may affect the cultivation conditions of Schizochytrium sp. such as medium composition, pH, salinity and temperature. Since polyunsaturated fatty acids have gained significance due to their role in human health, industrial and commercial usage, research on enhancement of biomass and lipid production has increased. Therefore, optimization of medium composition and environmental conditions to improve biomass and total lipid content of the organism is important. Applying a statistical strategy was an effective tool for optimization of the production and extraction process, which would also reduce the production costs through maximizing the yield. Statistical methodologies such as Plackett burman and central composite design have been extensively used to optimize several factors and their interactions. Plackett burman design was used recently as the first step in optimizing different bioprocesses to identify the factors with a significant effect on desired responses. Following the selection of the most significant factors, response surface methodology with central composite design is used to determine the optimum values of these factors. Based on contour plots and canonical analysis, a maximum biomass production of 26.86g·L-1 was obtained with 2.29 g.L-1 monosodium glutamate (MSG), pH 5.8 cultivation conditions. Maximum lipid production of 35% was obtained with 0.49 g.L- 1 MSG at 17.6 °C temperature conditions. Schizochytrium sp. S31 can be grown heterotrophically in bioreactor by supplying with alternative raw materials such as sugars, organic acids and alcohols. Thus, heterotrophic systems assure a cost effective way to obtain valuable microalgae produced DHA by using cheap substrates on large scale. Large scale heterotrophic growth technologies has lower contamination risk, lower harvesting costs, eliminated light limitation, high degree of process control and reproducible. In most instances, heterotrophic culture commonly results in much higher cell densities and lipid productivity than phototrophic culture because there is no light limitation in heterotrophic culture and controllable. According to large scale production results, 65 g/l of cell dry weight with the initial growth rate of 0.312 h-1 was obtained. C/N ratio of the Schizochytrium sp. culture was calculated as 19.32. The total lipid content was 27.5 % of the cell dry weight after applying optimized conditions. DHA percentage of the Schizochytrium sp. culture in 5 liter was calculated as 30.18%. Biomass, lipid and DHA productivity was calculated as 0.65 g/l.h, 0.17g/l.h and 0.053g/l.h respectively. Downstream processing steps required to obtain lipid from microalgal biomass once large scale production process is completed. Downstream process include harvesting, dewatering, cell disruption, lipid extraction. Harvesting microalgal culture includes centrifugation, filtration, and flocculation methods to concentrate microalgal culture. Drying step is known as post-dewatering step in which the cell pellet is completely dewatered. The cell pellet is exposed drying process for enhancing the efficiency of subsequent lipid extraction and lowering the cost of downstream process. Cell disruption is a key step in influencing lipid extraction yields. Sonication, high-pressure homogenizers, grinding, enzymatic reactions, chemical hydrolysis are the most known cell disruption methods applied for microalgae. Different types of solvents and extraction methods have been used in the literature to recover microalgal lipids. Hexane, methanol, ethanol, isopropanol are the typical solvents used for lipid extraction. According to the cell lysis and lipid extraction results, ultrasonication with hexane method increased the total lipid yield significantly with clear appearance. Sonication resulted in a 1.4-fold increase in lipid yield when compared with solvent alone. However, these traditional lipid extraction methods use large amounts of solvents that are mostly toxic. Supercritical liquid extraction (SFE) generally uses carbondioxide as a solvent at high pressure to extract lipid and/or nutraceutical products with higher selectivity in shorter extraction times. The effect of extraction temperature, pressure and time of SFE on the lipid yield and %DHA amount were investigated by using RSM. According to SFE results, pressure and temperature have significant effect (p <0.05) on total lipid yield and DHA concentration. Based on contour plot analysis, optimum extraction conditions were found to be 425 bar pressure at 40.5°C for 97.5min. After optimization of pressure, temperature and time variables of SFE, 30.2% lipid yield was obtained. Isolation of high yield lipid accumulating mutants of microalgae by flow cytometricbased selection can be performed by introducing mutations in the genome. There are mainly two mutation agents for random mutagenesis: UV radiation and chemical mutagenizing agent. Both methods do not require any genetic information about microalgae. Fluorescent activated cell sorting is a high throughput technique for selection of mutants with desired phenotype. This study highlighted that UV mutagenesis and high throughput selection improved lipid productivity in Schizochytrium sp. S31. Nutrient limitation conditions, such as nitrogen (N), phosphorus (P) starvation, and temperature limitation induced lipid accumulation in Schizochytrium sp. According to characterization results, mutants which were treated to UV for 30 second accumulate lipid faster than wild type. Time course experiment were conducted to understand the lipid accumulation profile difference between mutants and wild type. Total lipid was increased by 28.4 % for Mutant1 and 10.8% for Mutant2 comparing to wild type. The results obtained from gas chromatography mass spectrometry analyses confirmed the results obtained by flow cytometry, showing an increase in DHA in UV treated cultures (30sec) compared to untreated controls. There was a 17.9% and 12.1% increase compare to wild type in terms of DHA percentage in two different mutants respectively.