Before I started this class, I knew that I wanted to study something along the lines of marine biology. Originally, I wanted to research something about coral bleaching, a catastrophic and very prevalent issue of modern times and climate change grows. However, the difficulties of obtaining the necessary materials and resources, especially due to living in a land-locked stated, meant I had to drop the topic. Afterwards, I discovered the topic of ocean darkening, in which the ocean has seen a shrinking of the photic zone as its light absorption has increased, leading my to my next organism: phytoplankton, one of the main reasons for such developments. I wanted to see how a change in their environment affects the level of light absorbance they cause. At first, I focused on their general population, where I considered altering their nutrient input or nutrient input schedule would impact the growth rates of phytoplankton. I soon, however, found that similar research had already been conducted and was not novel. I then moved on to learn about dissolved organic matter (DOM), carbon-based compounds produced from phytoplankton, in particular, chromophoric dissolved organic matter (DOM), the part of DOM that absorbs light, therefore relating it back to ocean darkening. I decided to combine it with ocean acidification, establishing my gap: while studies have been performed to further understand the production of DOM by phytoplankton and refine methods of measuring CDOM, little research has been done on how a reduction in pH affects the production of CDOM specifically. Would it cause an increase in the quantity of CDOM, a decrease, or no significant change at all? Such curiousities became the foundation of my research project.  

Previous research has been key in shaping my methodology and defining my understanding of phytoplankton and CDOM. Foundational studies like Thornton (2013) synthesize current knowledge of the composition of DOM released by phytoplankton and its mechanisms. There are two separated and classified types: passive leakage across cell membranes and active exudation, where phytoplankton release compounds into the environment. DOM release is not simply a passive process, but rather something that relates to the physiological state of the phytoplankton, their growth phase, and even environmental stress, which could change considering climate change. Research like Rochelle-Newall and Fisher (2002) compares several different species of phytoplankton, culturing them under controlled conditions, such as light and nutrient input, then measures the changes in CDOM quantity of each culture over time. Their results showed that CDOM fluorescence increased primarily during the stationary phase of phytoplankton growth.

Other papers helped in the development of my methodology. Creswell (2010) and Perumal et al. (2012)  explain the process of inoculation and the basics of stock cultures, including how to prevent contamination during transfers. Hurd et al. (2009) mentions different experimental methods of lowering pH, such as CO2 bubbling and the addition of HCl. Mannino et al. (n.d.) and Nima et al. (2019) describe the process of using spectrophotometry to analyze CDOM and establish 440 nm as a key wavelength to track CDOM absorption.

My experimental design and study focus is largely based off this research, aiming to effectively study and analyze CDOM release by phytoplankton after a change in pH levels. 

Culturing of Chaetoceros muelleri

• Organism: Chaetoceros muelleri, a species of marine diatom, will be used due to their documented higher productions of CDOM .

• Inoculation of Cultures: Stock cultures will be kept on 25 mL test tubes. Initial starter cultures of 125 mL will be inoculated from one stock culture each,  then 10 final starter cultures of 10 mL (5 control and 5 experimental) will be inoculated into 25 mL test tubes from one initial starter culture each. In order to obtain at least 60 final cultures, this process will repeated 6 times. 

• Maintenance of Conditions: Guillard's F/2 will be used as the culture medium in order to supply necessary nutrients.  All starter cultures will be maintained at a temperature of 25 - 30°C and a light level of 8,000-10,000 lux. Lighting will be set for 16:8 hours light/dark per day to 24-hour illumination. pH should also be monitored, with a pH level of around 8.1.

Manipuation of pH

• Process of Acidification: Every final experimental starter culture will have its pH manipulated to 7.8 through the gradual addition of HCl. pH will be checked after each addition using a pH monitor until 7.8 is reached and will further be maintained daily. Final starter cultures will then be allowed to reach the stationary phase before data collection. 

Collection of Absorbance Coefficient through Spectrophotometry

• Isolating CDOM: Each 10 mL culture will be filtered through 0.2 μm polycarbonate (or PES) filters to remove particles and phytoplankton cells and isolate the CDOM. CDOM samples should be refrigerated and kept out of light until ready to be analyzed. Once ready, they should be allowed to reach room temperature. 

• Measuring CDOM Quantity through Spectrophotometry: Instrument settings should be data points every 1 nm, a scan speed of approximately 100 nm per minute, and a slit width of approximately 4 nm.  For each data point, the absorbance of a blank at 440 nm and the absorbance of a sample at 440 nm will be found. The null point by averaging the absorbance in the 650 nm to 680 nm region. Then, these values will be used to calculate the absorbance coefficient.

Data Analysis

• Statistical Comparison Between Control and Treatment: The average value of the absorbance coefficient of the control group and the average value of the absorbance coefficient of the experimental group to determine if a significant difference exists.