Team:GA State SW Jiaotong/Culturing
Algae Culturing
Through reading papers, we learned about the growth environment and morphological characteristics of Symbiodinium. The temperature range suitable for its growth was 16.72~28.40℃, and the temperature ecological range (tolerance limit) was 10.88~34.24℃. Culturing of Symbiodinium for this model was performed in Harrison's prepared artificial seawater with a salinity of 3.05% PSU and in F/2 medium with a concentration of 75 mol/L of nitrogen.
In addition, we determined the response model required for this experiment by referring to the data:
(Where is the maximum specific growth rate of algae, is the tolerance of environmental conditions, Topt is the optimal growth temperature of algae, T is the experimental temperature, (k, b) or a is the parameter.)
FIG.1 Expected temperature growth curves of Symbiodinium
According to the data in the literature and the culture requirements of Symbiodinium, we made the culture plan: the constant light intensity is (90±20) E, light period is 12L: 12D, F/2 medium was used, the initial concentration of algae was 5*103/ml.
Due to the impact of the pandemic, the experiment was divided into pre-experimental stage and experimental stage. Also, when we were at home during the summer, we luckily had access to a BSL-2 labratory to carry out the preliminary experiment. Therefore, the experimental stage is consisted of Phase I and Phase II.
In the pre-experimental stage, we procured original algae, F/2 medium, and equipment. We preliminarily simulated the culture environment, such as the configuration of artificial seawater, the measurement and time setting of LED lights, and temperature detection. In addition, we observed the morphology of Symbiodinium under the microscope and explored the preservation conditions of the original algae.
FIG.2 Equipment purchased for algae culturing
FIG.3-4 Set up of the algae culturing equipment
In the preliminary experiment, the shape of Symbiodinium was clearly observed through the microscope, and we learned that the algae could be fixed with Luge reagent to count them more easily.
FIG.5 Symbiodinium observed under the microscope
Experimental Stage---Phase I
After consulting with the manager of a Medical Science & Technology company, we luckily made access to its BSL-2 lab to continue our experiment. We are allowed to carry out the algae culture in the lab and use their microscope for concentration counting.
During this stage of experiment, we start with culturing Symbiodinium at 18℃, 22℃and 26℃. LED lamp is fixed in the aquarium, and we set up the illumination time as 12L: 12D. Heater and refrigerated container are used to maintain the temperature we want. Also, we made artificial seawater by ourselves and got seawater with salinity of 1.026. To prevent microbial contamination, we applied filtration to sterilize the sea water, in which way the salinity is not effected. Besides, air pump is employed to provide enough oxygen for algae.
After setting up all the equipment, original algae bought from Biology company was added into F/2 medium made from sterilized seawater.
Unfortunately, the Phrase I of experiment was not successful. Algae concentration continued dropping, and in the end, we observed many impurities and fungi in our medium, which means the medium is contaminated and the sterilization process is below standard.
Experimental Stage---Phase II
Back to campus, we continued our experiment. This time, the artificial climate chamber was used for culture. To avoid bacterial contamination like last time, we used gas-permeable membrane to seal the flask instead of using air pump. Most importantly, the F/2 medium was sterilized by autoclaves, and inoculation operation was carried out on the clean bench.
In the experiment,we used 100ml triangulated flask for the culture, and set the experimental group at 20℃,22℃, 24℃, 26℃, as well as the room temperature control group. F/2 medium, (90±20) E illumination intensity, 12L: 12D illumination time, and artificial seawater with the salinity of 1.026 was used. By setting groups at different temperatures, we’d like to get the growth data of Symbiodinium, and the parameters will be optimized by fitting them with the experimental model.
FIG.6 Medium after sterilization
FIG.7 Artificial climate chamber
FIG.8-13 Culture environment of each group
In the beginning of the experiment, the concentrations of Symbiodinium in each culture medium were calculated every 72 hours. Lugo's solution is used to fix the algae during counting. After two weeks, the concentration was counted every 24 hours.
FIG.14 Counting the concentration under the microscope
Over the course of 37 days of algal culturing, we recorded the algal concentration data 14 times. The data were preprocessed first, the abnormal data were eliminated, and then filled by the adjacent interpolation method. Then, taking the maximum specific growth rate of alginate species as the dependent variable, one-way anova was performed for different temperatures; the maximum specific growth rate was significantly affected by different temperatures at the confidence level of P<0.01.
In order to study the ecological amplitude properties of Symbiodinium, we established the ecological amplitude response relation formula according to the Shelf tolerance law. However, since there is no definite agreement on the response relation formula yet, we adopted three formats commonly used in literature to analyze the growth data of Symbiodinium. Matlab software was used to optimize the response formula with nonlinear least square method, and Newton iterative algorithm was used to estimate and solve the parameters. Finally, six sets of parameter estimates (three response relation models, two experiments repeated data) were obtained. There was no significant difference between different estimates, indicating equivalence between six groups’ estimates. The mean value for six groups’ estimate is taken for the actual value, thus greatly improving the data accuracy. According to the fitting result of the final actual data, the experiment succeeded.
Coral Culturing
In order to address and rectify the bleaching process depicted by corals and their algal symbionts in the marine environment, our project begins first with the successful culturing of Acropora spp, Seriatopora spp and Stylophora spp in tanks of various sizes. The success of coral culturing is entirely attributed to how well the coral are able to adapt to the makeshift oceanic environment presented within any amount of time. Temperature, nitrogen levels, salinity, pH, the presence of herbivores, and microbial balance are among a few of the aspects that allow for the optimisation of health for any coral organism. The phenomenon of coral bleaching is widely known to be attributed to fluctuations in temperature due to global warming. It was discovered through trial and error in our research that corals prefer a strict and unforgiving temperature range of about 76-79 degrees celsius, and that even the smallest of deviation could negatively affect the livelihood of all the organisms within the tank. The initial set up of our at-home coral culturing tanks began with salt water. It was then discovered that a UV light is just as important for mimicking a 24 hour day to night cycle for the organisms along with heaters, thermometers, protein skimmers, live rock and live sand for microbial balance. The addition of other organisms that can be found in a typical coral’s environment such as fish and crabs or snails to help correct nitrate and phosphate levels are also vital, and they aid in providing good nutrients, such as calcium and magnesium to the coral. Corals must also be equipped with a constant and steady air flow because still water will not optimize growth and healthy branching. The overall health and maintenance of the created marine environment of our tanks is protected by ensuring that weekly dosing and testing of the water’s elemental levels, and the replenishing of fresh water to the tank is kept as consistent as possible.
Future Plan
In the near future, we will start Phase III of our algae culture experiment. We have explored the optimum condition for temperature, and we did some comparison between algae growth under solar light and regulated light. Next, more groups with different light intensities will be set up. We hope to find out more information for Symbiodinium growth, thus contributing to better regulation on molecular level.