Treatment Of Ammonium-rich Industrial Wastewater By The Mixotrophic Culture Of Galdieria Sulphuraria With Phycobiliprotein Co-production

Ammonium-rich industrial wastewater has the characteristics of large emissions,high salinity and strong toxicity,which leads to its poor biodegradability and difficulties in wastewater treatment.Traditional treatment methods exist inherent limitations of high energy consumption and cost,secondary pollution and low resource utilization.Galdieria sulphuraria is a kind of unicellular microalgae from the extreme environment of sulfuric acid hot springs,which can withstand the stress conditions such as strong acid,high temperature,and toxic metal.With flexible metabolic capacities,G.sulphuraria is able to grow autotrophically,and can also use a variety of organic carbon sources for heterotrophic and mixotrophic growth with the accumulation of phycobiliprotein.At present,its application studies mainly focus on different wastewater treatment and heat-resistant phycobiliprotein production.The treatment of ammonium-rich industrial wastewater by G.sulphuraria can not only achieve a goal of NH₃-N removal and water purifying,but also convert ammonia nitrogen into high-value biomass rich in protein,especially phycobiliprotein,so as to realize the resource utilization of wastewater and transform waste into treasure.In this study,when cultivated in the shaking flask system,standard medium was firstly used to optimize the mixotrophic culture conditions of G.sulphuraria.Then,the mixotrophic culture conditions for the ammonium-rich industrial wastewater treatment were further optimized by using wastewater medium,and the adaptive culture strategy of high ammonia nitrogen was established.In a 5 L photo fermentor,the synergism of the process parameters,ammonia nitrogen removal efficiency and phycobiliprotein production efficiency of the expanded culture system based on wastewater were systematically studied,and a stable and efficient photo fermentation technology with amplification was established.The main research results are listed as follows:1.In the shaking flask system,the effects of various nutritional and environmental factors on the mixotrophic growth of cells in standard medium were systematically studied.The results showed that the optimal mixotrophic conditions of G.sulphuraria were determined as:temperature of 30℃,light intensity of 150-230μmol/（m²·s）,initial glucose concentration of 20 g/L,ammonium sulfate of 5.24 g/L（carbon-nitrogen ratio of 6,mol/mol）,p H value of 2.0,initial cell density of 1×10⁸cells/m L.After 8 days of culture under optimized conditions,the maximum cell density and biomass of G.sulphuraria were 7.30×10⁸cells/m L and 13.07 g/L,respectively.2.In the shaking flask system,the effects of various process parameters on the wastewater treatment by the mixotrophic culture of G.sulphuraria with phycobiliprotein co-production were systematically studied.Results showed that in the culture system using sterilized wastewater,when the light intensity was 140-270μmol/（m²·s）,initial NH₄⁺concentration of wastewater was 3.38 g/L with an initial p H value of 3.0-4.0,and supplied with whole elements of standard medium（except for nitrogen）and glucose（glucose concentrations of 20 g/L in medium after supplementation）during cultivation,the average NH₄⁺removal rate was 0.39 g/L/d and the final NH₄⁺removal efficiency reached 99.05%after8 days of culture.And the phycobiliprotein content in recycled biomass and corresponding yield were 66.92 mg/g and 1786.72 mg/L,respectively.In the culture system using unsterilized wastewater,the optimal initial p H value was 1.0-3.0.In this case,when initial glucose concentration,p H value and cell density were 20 g/L,2.0 and 1×10⁸cells/m L respectively,the final cell density of G.sulphuraria reached 3.88×10⁸cells/m L after 4 days of culture while non-algal particle density was only 8.00×10⁵cells/m L.The average NH₄⁺removal rate was up to 0.38 g/L/d,and the maximum content and yield of phycobiliprotein were 163.50 mg/g and 1683.17 mg/L,respectively.In single batch culture in shaking flasks,the NH₄⁺removal rate in the unsterilized system was significantly lower than that in the sterilized system（p<0.05）.3.The adaptive culture strategy of high ammonia nitrogen for seed cells was studied in the shaking flask system,and the rates of NH₄⁺removal and phycobiliprotein production before and after the culture were compared.It was found that short-term adaptive culture could significantly improve the tolerance of seed cells to high ammonia nitrogen concentration,and the specific growth rate of cells and NH₄⁺absorption were significantly improved（p<0.05）.After adaptive culture,seed culture fluid was inoculated with an initial cell density of 1.47×10⁸cells/m L into the wastewater with NH₄⁺concentration of 6.78 g/L.Cultivated under the conditions of 30℃and the light intensity of 170-210μmol/（m²·s）for 4days,the average NH₄⁺removal rate reached the peak of 0.38 g/L/d,which was 1.8 times of that before adaptive culture.The maximum content and yield of phycobiliprotein were 141.25mg/g and 1963.32 mg/L,respectively.4.In a 5 L photo fermentor,the parameters of fed-batch culture were optimized,and it was verified that the alga could continuously and efficiently treat ammonium-rich industrial wastewater with initial NH₄⁺concentration of 3.52-5.46 g/L in the enlarged system.When the sterilized photofermentation system was adopted and the process parameters were set as:central light intensity of 150-230μmol/（m²·s）,aeration rate of 250 L/h,the stirring speed of200 r/min,glucose supplemental level of 17-35 g/L and the initial cell density of 5.74×10⁸cells/m L,the average NH₄⁺removal rate reached the peak（1.60 g/L/d）achieving NH₄⁺removal efficiency of 95.32%.At the end of culture,the recycled biomass was 61.55 g/L of which the phycobiliprotein content was 40.20 mg/g,and its yield and production rate were2474.59 mg/L and 691.17 mg/L/d,respectively.When the unsterilized photofermentation system was adopted and the process parameters were set as:central light intensity of 150-230μmol/（m²·s）,aeration rate of 250 L/h,the stirring speed of 200 r/min,glucose supplemental level of 17-35 g/L and the initial cell density of 5.03×10⁸cells/m L,the average NH₄⁺removal rate reached the peak（1.71 g/L/d）achieving NH₄⁺removal efficiency of 98.69%.After the end of culture,the recycled biomass was 60.30 g/L of which the phycobiliprotein content was32.54 mg/g,and the yield and production rate of phycobiliprotein were 1961.92 mg/L and555.07 mg/L/d,respectively.The results showed that NH₄⁺removal rate of photo fermentation in the unsterilized system using seed culture fluid after adaptive cultivation was significantly higher than that of the sterilized system（p<0.05）,which saved the energy cost of sterilization process and had more economic advantages in large-scale wastewater treatment.