Evaluation of common wastewaters on the growth of alga Spirulina

Abstract

To evaluate the suitability of wastewater on the growth of alga Spirulina, reclaimed wastewaters, fishpond wastewater, industrial wastewater and mariculture water were used to culture Spirulina subsalsa and Spirulina platensis. S. subsalsa showed better adaptability to fishpond wastewater and higher specific growth rate than that of S. platensis. Thus, effects of factors such as the amount of baking soda, inoculation, and sodium nitrate on the growth, biomass and protein content of S. subsalsa were evaluated through the single factors design. Results showed that S. subsalsa had good growth under conditions of 4–12 g/L baking soda, 1–2 g/L NaNO3, 25–40°C, and 6000–12000 lux illumination. The maximum dry biomass and protein content were 3.48 g/L and 33.08%, respectively, suggesting that it was feasible to culture S. subsalsa in freshwater aquaculture wastewater. Wongsnansilp and Phinrub: Journal of Applied Biology & Biotechnology 2022;10(2):33-38 34 feasibility of actually cultivating Spirulina using wastewater to guide the further outdoor large scale microalgae cultivation. 2. MATERIAL AND METHODS 2.1. Wastewater Collection Seven kinds of local common wastewaters in Sikao District, Trang Province, Thailand were collected for the test. Details were shown in Table 1. The reclaimed water was taken from the local sewage treatment plant. The domestic sewage was anaerobic fermented and then flowed into the three-stage series purification tank. The purified water of each stage was used as the water source for alga cultivation, recorded as reclaimed water 1, 2 and 3. Freshwater aquaculture wastewater was taken from the local fishpond in the fishing off season. Industrial wastewater was also taken from the outlet of the local sewage treatment plant. The sewage from the local industrial plant was treated by purification tank and then used for alga cultivation. The mariculture water was taken from the local mariculture plant. The seawater was mixed with fresh water with a ratio of 3:1, and then processed by primary and secondary sedimentation removing some heavy metal ions. 2.2. Microalgae culture S. subsalsa and S. platensis were provided by Faculty of Science and Fisheries Technology, Rajamangala University of Technology Srivijaya. Two algae were cultured in Zarrouk medium to logarithmic growth stage as inoculation mother solution [12]. Different baking soda (2, 4, 8 and 12 g/L) were added into seven collected local wastewaters respectively, then using 1 mol/L NaOH to adjust pH value of all solution to 9. All solution were stood overnight and then filtered by 300 mesh cloth sieves; 200 mL filtered water was taken into a 500 mL flask, and then inoculated by mother algal solution. The initial OD550 values of all treatments were around 0.06; the temperature was 30°C; the light illumination was 10000 lux; the light-dark cycle was 12 h: 12 h. All cultures were shaken regularly 3 times per day for 5 min each time, and recorded the color variance and OD550 value by ultraviolet spectrophotometer meanwhile. 2.3. Single Factor Test The environmental adaptability of alga was evaluated by single factor test under the conditions of edible baking soda, inoculation amount, sodium nitrate, illumination and temperature, respectively [Table 2]. S. subsalsa was cultured with freshwater aquaculture wastewater based on the result of each single factor test orderly, so as to find the best cultivation conditions for biomass and protein yield. The specific growth rate and the average growth rate were calculated by the equation of ln(AX/A0) and ln(AX/A0)/T, respectively, where Ax was the OD550 value on the day x, A0 was the initial OD550 value, T was the culture time to the harvest day. 2.4. Detection method 2.4.1. Wastewater quality analysis The detection methods for the contents of total nitrogen, ammonianitrogen, total phosphorus, and phosphate phosphorus in wastewater were applied according to standard methods as published by the American Public Health Association [13]. 2.4.2. Dry weight determination Algae solution colors were daily observed and algae were harvested when the color changed from dark green to yellow green or the OD550 value did not increase again. The algal solution was filtered by 300 mesh cloth sieve, and the alga was washed by fresh water to neutral. The alga was dried at 60°C until total weight stable. 2.4.3. Protein content determination About 0.15 g dry algae powder was taken and mixed with 30 ml distilled water, and put into a centrifuge tube. After repeated freezing at –20°C and thawing for 5 times, the solution was centrifuged at 4000 rpm for 15 min. The supernatant was taken to determine the protein content following the Coomassie brilliant blue method [10]. 2.5. Data Analysis All data statistical analysis was performed using the SPSS 19.0 software. All data displayed a normal distribution checked by Kolmogorov–Smirnov test. The results were given as a mean with standard deviation (± SD). Figures were created with Microsoft Excel 2010 software. 3. RESULTS AND DISCUSSION 3.1. Wastewater Quality Analysis