The Impact of pH on chlorella vulgaris

Figure 10 shows that the population of algae rose in pH solutions of 5.7, 8.1, and 11.4 while declining in pH solutions of 4.3 and 10.3. With a percentage rise of 861% – 245% and no algae present in pH 4.3 after the 14 days, it is possible to say with 95% certainty that the algae grew best in a solution of pH 8.1 from the solutions tested. This is because the confidence intervals in fig. 10 do not overlap. These findings would indicate with a 95% degree of certainty that Chlorella vulgaris development declines when the environmental pH moves away from the ideal range (pH 8.1), rejecting H0 and accepting H1. The qualitative findings, particularly Fig. 3, support this. Here, pH solution 8.1 may be seen to be green in color, indicating algal bloom. As opposed to pH 4.1, when the solution is colorless and no growth is indicated.

The experiment's main outlier was a batch of algae that had been produced in a pH 11.4 solution. The samples had an overall growth of +38% ± 36%, and the solutions had taken on a green color, as seen in fig. 3, which indicates the presence of algae. However, it would have been reasonable to have anticipated a decline in algal growth given the general trend, which saw a 50% decline in pH 10.3 population density. The various salts found in the various pH buffers could be the cause of this oddity.

My findings demonstrate that environmental pH does have an impact on Chlorella vulgaris population density. The ideal pH was determined to be 8.1, and any deviation from this ideal resulted in a reduction in population increase. 4.3 pH medium samples had no evidence of algae. The previously mentioned studies that show that the ideal pH for photosynthesis is 8.1 and the ideal pH for Acetyl CoA, which is responsible for the rate-limiting phase in the Krebs cycle, is 8.0, respectively, provide support for this (nzytech, 2012). When both of these processes proceed at their most efficient rates, a lot of ATP is generated, enabling rapid DNA metabolism and a dense population. Chlorella vulgaris grew more slowly under more acidic conditions; this may be because the higher H⁺ concentration caused changes in the tertiary structure of important enzymes, which changed the geometry of their active site and caused them to become denatured. As a result, the rate of photosynthesis and respiration decreases, leaving the algal cells with little energy to function and proliferate. Algal cells in samples cultivated at pH 4.3 perished because neither photosynthesis nor respiration took place. Cells found in pH samples of 10.3 and 11.4 may have been the same, but the change in structure was brought on by higher OH^- concentrations.

Even though a pH of 5.7 is regarded as acidic, fig. 10 does demonstrate an increase in population density. There was only a +267%31% growth between T1 and T6, as opposed to +861%245% in pH samples of 8.1, making it far less than the increases observed in those samples. This demonstrates that while growth did take place in acidic environments, productivity did suffer as a result. This has an impact on ocean acidification since ocean pH, which is currently 8.05, is 30% more acidic than it was at the beginning of the industrial revolution, and some estimates indicate that by 2100, ocean pH might be 7.8 to 7.9. (Rafferty, 2019). There may be a sharp reduction in algal populations worldwide as a result of these severe pH shifts. The potential wider consequences make this important. First, how it affects atmospheric CO₂ levels. Marine algae and phytoplankton are important components of the ocean's biological carbon pump (BCP).

This process involves storing in sediment the carbon that photosynthesis has removed from the atmosphere. In order to keep atmospheric carbon levels essentially constant, the BCP is crucial. Without this natural mechanism, which is made up of various fluxes, CO₂ levels would undoubtedly be much higher than they are now. An estimated one-third of all human CO₂ in the atmosphere has been absorbed by the ocean as a sink (Mackey, 2018). A positive feedback loop might arise, significantly escalating global warming and its adverse impacts on the entire planet, if the pH of the seas significantly changes and detrimental effects on growth are observed, comparable to those of Chlorella vulgaris in my experiment. Furthermore, algal populations play a crucial role in food webs and support a range of creatures. Other species that depend on them as a food supply may suffer if the population of these and other autotrophs declines.

Strengths of Methodology

• The method was simple and easy to follow
• The method allowed multiple repeats to easily be done
• A quick and efficient method of calculating population density

Assumptions

Although there were many strengths to my methodology, some assumptions were made, these could have affected the reliability of my results.

• Chlorella vulgaris and other marine photosynthetic bacteria that may be impacted by ocean acidification can be compared.
  • However, a comparison can still be established because most unicellular algae share commonalities in the setting of pH homeostasis (Boron, 2004).
• As observed in Fig. 10, where the algae developed in a ball in the middle of the flask, the concentration of algae was the same throughout the sample.
  • Before taking a sample, the flasks were spun to reduce the risk.
• Each flask contained the same nutrients, so development was not constrained by these factors.
  • Since the experiment only lasted 14 days and the same batch of Bold's Basal media was used to minimize, technically, this should not have been the limiting issue.

Limitations of Methodology

Even though data were gathered at regular intervals over the course of the 14-day period, only T1 and T6 were included in the study since they are sufficient to demonstrate changes in algal growth in different pH solutions and enable me to answer my research question. Although conclusions can be drawn from the data obtained, as shown in figure. 11, with a 95% degree of certainty, extra statistical testing is required for definitive results. This cannot be done, though, as the experiment's sample size was insufficient.

Quantitative Data

The contains a table of the raw data. This displays the number of cells measured in a 0.04 mm² area's four corners, as seen in Figure. 2. The result of averaging the four numbers is depicted in A2 below.

The average population density per 1mm³ for T1 and T6 was computed using this data. To do this, divide the cell count (in 0.04 mm²) by 0.004. Figure 7 below displays this data. The beginning population for each sample was different, thus a percentage change of the original was calculated. This allowed for an accurate and straightforward comparison of the results, as shown in figure. 8.

I discovered a prior experiment where photosynthesis in plankton was evaluated using the Hills Reaction (Mystrica Ltd, 2012) through my study into the appropriate methodology. Electrons produced during the photosynthesis process' light-dependent stage are used to decrease DCPIP in the Hills reaction. Because the plankton is green, the solution's color should change from blue to colorless green in the DCPIP. To observe the impact of altering the buffer's pH, I modified the experiment's light intensity and wavelength. DCPIP's color change is typically marked with the naked eye, which can produce ambiguous results. To get over this restriction, I decided to run the reaction in a cuvette that was phin the Mystica colorimeter while using the light source as the source of light energy. This was linked to a computer and would have allowed for an objective evaluation of the color shift in real-time. A plankton suspension comprising phytoplankton was used to test this approach. In a cuvette, this was combined with DCPIP before being put in a Mystrica colorimeter.

Unfortunately, this strategy failed since the plankton's rate of photosynthesis was insufficient to lower the DCPIP. Low light intensity or bad plankton concentration could be to blame for this. Therefore, a volume was centrifuged to create a "plankton pellet" to boost the engagement of plankton in the sample. The turbidity of the solution, however, was too high when this solution was utilized. Therefore no light could get through when a colorimeter was used. It was decided not to employ this practice.

Further investigation into other techniques introduced me to the SAPS process, which involves growing samples of Chlorella Vulgaris and counting them using a hemocytometer (SAPS, 2019). Chlorella Vulgaris was chosen because it is a readily available, fast-growing algae (Sciento, 2019). Instead of employing the original methodology's temperature change, I used pH buffers to alter the growth medium's pH. Since this was the supplier's suggested growth media, a Basal Medium was employed. To demonstrate a range of outcomes, it was determined that the samples would be cultured at pH 4, 6, 8, and 12. The medium's pH was changed by adding 10cm³ 0. The pH of the solutions was then assessed using a pH probe and 1cm³ of pH buffer. To adjust the pH to the correct value, 15cm³ ± 0.1cm³ of the pad was required for pH 4, 10, and 12. The target pH is indicated in brackets in data tables together with the actual pH of the buffer solutions. The following hypotheses were put to the test in the experiment.

H0: Chlorella vulgaris population density will not change in response to pH changes in the environment.

H1: Variations in the pH of the environment will have an impact on Chlorella vulgarises population density.

For each pH, five flakes with a buffer medium volume of 40 cm³ and a Chlorella vulgaris suspension volume of 2 cm³ and 0.05 cm³ were set up. The flasks were maintained close to one another on a sunny ledge to encourage photosynthesis, which in turn encouraged respiration and cell division. Additionally, cotton wool was placed within the flask to inhibit the introduction of microbes while allowing air to enter. Each day, flasks were gently spun to stir up the algae and encourage growth. Before sampling, the flasks were also carefully swirled to spread the algae uniformly. Since I didn't sure how long population growth would take, I chose to take samples every two days for the first seven days, then every three days for the first two collections. This proved that the population density on day 14 (T6) had changed enough to allow for a valid comparison to the initial population. Using a hemocytometer, the population size of each flask was determined. Data were consistently taken from the four corners of a 0.04mm² area. The number of cells would be tallied in the squares with a red outline, as shown in Figure. 2. Since they are outside of the corner square in Figure. 6, the circled group of cells would not be tallied. 

Possible Extensions of Methodology

More samples were taken for each pH solution. Further statistical analysis would be possible as a result, improving the consistency and dependability of findings.

Use the same process with a variety of different freshwater and saltwater species of photosynthetic bacteria. This would make it possible to compare all marine microorganisms more accurately, which would have broader effects on ocean acidification.

In order to identify and draw conclusions on the long-term impacts of changing environmental pH, samples should be monitored over a longer period of time.

Limitations & Strengths of data

As seen in the figure. 12, the estimated confidence intervals did not result in any overlap in the error bars. Therefore, a judgment might be drawn with 95% accuracy. In the data gathered, a clear correlation between environmental pH and Chlorella vulgaris population density could also be noticed; this is depicted in the figure. 12. Chlorella vulgaris grown in media with a pH of 11.4 has been recognized as an anomaly in the data that was gathered. There have also been offered explanations. The initial population number was different for each sample, hence the percentage change in population was used as the data for Figure. 12's analysis.

Although judgments could be drawn with a 95% degree of certainty, the sample size was too small for accurate statistical analysis. Although there is a definite trend in the data, which was also gathered over a 14-day period, it is still believed that the trend will last moving forward. Additionally, samples grown in pH 11.4 media did exhibit an increase in population density, which is inconsistent with the trend. It would be necessary to conduct additional analysis, such as repeating the test several times at this pH and holding the samples for a longer period of time. In order to draw valid conclusions on changes in Chlorella vulgaris population density in this ambient pH.

Calculating Errors
Unfortunately, a useful statistical analysis could not be performed because of the experiment's tiny sample size. Confidence intervals were therefore computed to see whether accurate inferences could be drawn from the data gathered.

Using the formula...

𝐶𝑜𝑛𝑓𝑖𝑑𝑒𝑛𝑐𝑒 𝐼𝑛𝑡𝑒𝑟𝑣𝑎l = \(\bar x \ ±\) \(t\frac{S}{\sqrt{n}}\)

Equ.3 (Maths is Fun, 2017)

The percentage change values given in Fig. 8 were used to create confidence intervals at a 95% level. The results are displayed in Fig. 9. The data was then displayed as a bar graph below after these were applied to the mean percentage change.

𝐶𝑂₂ + 𝐻₂O  ⇌  𝐻₂𝐶𝑂₃  ⇌  𝐻^⁺ + 𝐻𝐶𝑂₃⁻ ⇌  𝐻⁺ + 𝐶𝑂𝐶𝑂₃^2-

Equation 2 (Golda, 2017)

As a result, carbonate ions become less common (P. Rafferty, 2016), which results in softer shells for crabs and weaker skeletons for corals. Phytoplankton and algae, two marine microorganisms, may be impacted by pH variations. There is concern that this will lead to a positive feedback system, with reduced photosynthesis leading to an even greater increase in atmospheric CO₂ levels. (Cruse, 2019) The larger animals, like squid, could also be impacted by the lower pH levels, which could increase the quantities of carbonic acid in their bodies and cause respiratory and reproductive issues (P. Rafferty, 2016).

The UN stated last year that we only have 12 years until an increase in the global temperature of more than 1.5°C is inevitable (Doyle, 2019). Professor of environmental science at the Stockholm Resilience Center, Johan Rockström, has described the possibility of a 2°C rise as "hazardous" (Rockstrom, 2018). These increases are brought on by the rising quantities of CO₂ and other greenhouse gases in the atmosphere. Ocean acidification may harm marine photosynthetic organisms, which could lead to uncontrolled CO₂ levels in the atmosphere. As I've already explained, the amount of carbon in our atmosphere and the rate of global warming are controlled by photosynthetic marine creatures like phytoplankton. I wished to investigate the danger posed by ocean acidification.

A colony's population density and the development rate of algae like Chlorella vulgaris are both influenced by the rates of photosynthesis, respiration, and DNA metabolism, as I discovered via my research. Glucose is produced during photosynthesis and it to create ATP through a succession of enzyme-catalyzed events during respiration. For DNA metabolism to take place, which will allow the cell to reproduce and hence raise population density, ATP is then required.

Research question:

What is the effect of environmental pH on the population density of Chlorella vulgaris?

I found 95% assurance that ambient pH affects Chlorella vulgaris growth. Fig.10 shows that Chlorella vulgaris grows best at pH 8.1. From T1 to T6, 14 days, pH 8.1 population density multiplied by 861% with a confidence interval of ±245%.

Algal microorganisms like Chlorella vulgaris reproduce asexually via mitosis. Nuclear division occurs during mitosis, the cell cycle's final phase before cytokinesis. DNA metabolism demands ATP energy in algae cells. Respiration in mitochondria produces ATP. During photosynthesis, algal cells create carbohydrates for respiration. Algal population growth is maximized when these three processes perform optimally. Since my experiment showed that pH variations affected algal growth, at least one of these processes must have been affected.

Two enzymes control photosynthesis. The light-independent Calvin Cycle in photosynthesis is enzyme-controlled. This mechanism happens in the chloroplast stroma, where the best circumstances are 8.1 pH and 25°C (Werdan K, 1975; Siddhartha Dutta, 2009). My own experimental investigation indicated that 8.1 pH increases Chlorella vulgaris population density.

The Calvin Cycle involves carbon fixation, reduction, and ribulose regeneration. Each is controlled by substrate-specific enzymes.

Rubisco catalyses carbon fixation. Carbon dioxide is fixed to ribulose biphosphate, generating an unstable six-carbon molecule that breaks into two glycerate-3-phosphates. (2013) Rubisco is a quaternary enzyme with eight hydrogen-bonded polypeptide chains. Ionic bonding, hydrogen bonds, and hydrophilic interactions connect alpha helixes and beta-pleated sheets in polypeptide chains (Department of Molecular Biology, Swedish University of Agricultural Sciences, 2008). Fig.11 shows Rubisco's structure.

Enzymes accelerate processes by offering an alternative activation energy source. The "lock and key" paradigm says enzymes match substrates precisely. Enzymes work best in a solution with a specific pH and temperature, which varies by the enzyme.

H⁺ or OH^- ions can disrupt ionic connection when pH changes. As mentioned, an enzyme's active site complements the substrate's. However, interfering with the tertiary structure's ionic interactions can modify the beta-pleated sheets and alpha helices depicted in fig.11 and the active site's form. The active site no longer complements the substrate, so enzyme-substrate complexes cannot form and the process is no longer catalyzed. Due to Calvin Cycle enzyme denaturing, Chlorella vulgaris and other algae species' photosynthesis decreases when pH changes outside the optimal range. Sugar production decreases with photosynthesis. This reduces respiration, ATP production, DNA metabolism, and growth.

Thus, photosynthetic enzyme denaturing may reduce Chlorella vulgaris population density when environmental pH drops below 8.1.

All organisms breathe. Algae are autotrophic chemotrophs that respire (including oxidative phosphorylation) using complex organic chemicals they synthesised from simple inorganic molecules during photosynthesis. Respiration involves glycolysis, link reaction, Krebs cycle, and oxidative phosphorylation. The last phase releases energy for ATP synthase to make ATP from ADP and Pi. Reduced NAD from the Krebs Cycle is needed for oxidative phosphorylation.

The Krebs Cycle is an enzyme-controlled closed loop that condenses acetyl CoA and oxaloacetate to create citrate and oxidizes it to release carbon dioxide and reduced NAD (Lumen Candela, n.d.). Enzyme-catalyzed reactions require temperature and pH. These settings beyond an enzyme's optimal range will slow reaction rates and denature the enzymes. Citrate synthase is the most important Krebs cycle enzyme. It condenses acetyl CoA and oxaloacetate to produce citrate, the rate-limiting step (Albert L. Lehninger, 2017). Its optimal pH is 8. (nzytech, 2012). This matches my experiment and fig.10.

The pH 8.1 samples had the biggest population density rise. My experiment's pH adjustments may have put algal mitochondria outside citrate synthase's optimal range. This would slow the Krebs cycle's rate-limiting condensation step, reducing reduced NAD production.

This would reduce ATP production, reducing DNA metabolism, Chlorella vulgaris development, and population density. Another reason why ambient pH fluctuations from 8.1 severely influenced Chlorella vulgaris development in my experiment.

G1, synthesis, G2, and mitosis comprise the cell cycle. Algal cell reproduction depends on DNA metabolism during synthesis. A sophisticated enzyme system replicates DNA semiconservative. Enzymes play different roles in replication. DNA polymerase 1 replicates DNA.

This enzyme replaces lagging strand RNA primers with DNA nucleotides. DNA polymerase 1 preferred pH 7.4. DNA replication requires lots of acidic deoxyribonucleotide triphosphates, making the optimum mildly acidic. DNA metabolism would slow if the nucleus' internal pH changed. RNA primers on the lagging strand would not be replaced, preventing DNA synthesis and algal cell reproduction. However, Schizosaccharomyces pombe, a fungus employed as a model organism in cell and molecular biology, had a nuclear pH reduction of 0.13 pH units in external pH solutions from 6.5 to 3.5. (Young, 2001). This shows that pH homeostatic mechanisms regulate nucleus pH. Protein channels carry H+ ions to adjust internal pH in many unicellular algae (Golda, 2017). Some unicellular algae lack these channels. Thus, algae and most marine microorganisms prefer pH homeostasis via CO2 diffusion into the open system (Boron, 2004). This energy-intensive strategy is short-term (Golda, 2017). My research showed that Chlorella vulgaris modulates nuclear pH via both methods (Golda, 2017). Thus, ATP deficiency, not enzyme denaturing, likely slowed DNA metabolism. ATP deficiency slowed DNA metabolism, preventing algal growth outside pH solution 8.1.

Ocean Acidification
Ocean acidification, according to John P. Rafferty (Rafferty, 2019), is the evolution of alkaline seawater to a more neutral pH rather than the seawater actually becoming acidic (pH below 7).

Rafferty claims that the growing levels of carbon dioxide in the earth's atmosphere are the main contributors to ocean acidification. The dissolved concentration of CO₂ in the oceans and the atmospheric concentration are in equilibrium. As a result of the equilibrium shifting to the left in equation 1, a rise in atmospheric CO₂ causes an increase in dissolved oceanic CO₂.

CO₂(g) ⇌ CO₂(aq)

Equation 1

After entering the oceans, CO₂ interacts with water to produce carbonic acid. After that, carbonic acid separates into hydrogen and bicarbonate ions. The pH of the seas is decreased by an increase in hydrogen ion concentration. depicted in Fig. 1 and streamlined in Equ. 2.

Ethical Issues – There are no major ethical issues with this experiment, despite living organisms being involved.

Environmental Issues – There are no major environmental issues with this experiment