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Biofuel Engineering : Optimal Nitrogen Conditions for Algae Growth and Biofuel Production

Biofuel's Future : Optimal Nitrogen Conditions for Algae Growth and Biofuel Production 

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Executive Summary

For decades, fossil fuels and related nonrenewable energy sources have spurred international industrialization and manufactured the global network we live in. 

Historically, fossil fuels may have functioned as an auspicious boon, but environmental research and analysis over the past few decades have revealed the significant harms of such energy sources. 

By eroding the ozone layer and driving global warming, fossil fuels have accelerated an extreme  habitat transformation for a wide array of species and will continue to consume our atmosphere and planet until alternatives are implemented.  

To that end, biofuels have recently emerged as a viable alternative, with algae potentially serving as an effective source due to its ubiquity. 

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As algae is an organic material, nitrogen bearing nutrients efficiently stimulate algal growth. The purpose of this experiment will be to  evaluate the nitrogen conditions that will optimize algal growth. We will use C. reinhardtii and a  total of 18 test tubes with 6 different nitrogen concentration levels (0.000 g/mL, 0.012 g/mL,  0.021 g/mL, 0.045 g/mL, 0.075 g/mL, and 0.089 g/mL); as such, there will be three replicates per  level. 

We intend to measure the algal growth with a spectrophotometer and use a Spearman Rank  Correlation Test to analyze our data. The data will be represented on a graph, and we will use the  calculated p-value and correlation coefficient to deduce conclusions from the experiment. We  predict that the test tubes containing 0.075 g/mL will bear the greatest algal growth. 

The rationale for our predictions derives from nitrogen’s role as a limiting nutrient (and thus, an increased nitrogen concentration yields an increased the cell density) and its ideal range of 5%- 8% based on prior literature (Concepts for Bioenergy from Algae 2005). Therefore, we believe  that 0.075 g/mL will serve as the concentration that will best support C. reinhardtii growth and  thus produce the largest quantity of biofuel.


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In recent years, the development of climate change has accelerated to alarming levels. Largely due to fossil fuels, the concentration of greenhouse gases is growing while the protective ozone layer is diminishing, and dangerously warming our planet by several degrees. To mitigate  the effects of fossil fuels on our climate, researchers and government officials alike have  considered a transition to the budding sector of biofuels. 

With the potential to reshape our energy usage, biofuels present a source of renewable  energy. Its natural compatibility with the carbon cycle offers an alternative that can both sustain our energy demands while establishing a relatively harmless resource that may last for the  decades to come (Milledge and Heaven 2013). 

As algae is one of the most abundant forms of organic matter, it bears the unique ability to grow in extreme conditions; as such, algae may serve as a viable source for biofuels. 

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In order  to maximize the quantity of biofuel generated, we must thus determine the nutrients that  optimize the growth of algae. In this experiment, we will determine the effect of nitrogen on the  algae species C. reinhardtii

We will examine the growth of C. reinhardtii under several different conditions with varying nitrogen levels, while holding all other nutrient levels constant. At its core, the growth of  algae is a biochemical process closely linked with photosynthesis, which involves nutrients including nitrogen (Ross et al. 2018). 

In fact, nitrogen itself is a common macronutrient  necessary for protein formation and other integral biological processes (Sarvary 2020). Since nitrogen is generally less abundant in primary producers than other nutrients, it is typically  known as a limiting nutrient. We hypothesize that nitrogen is a limiting nutrient for algal growth, and thus an increase in nitrogen concentration will increase the algal density as measured by a  spectrophotometer. 

In particular, we suspect that the maximum growth for C. reinhardtii will be reached at a nitrogen concentration of 0.075 g/mL. With greater quantities of algae, we can  increase the amount of biofuel produced. 

Proposed Methods 

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In broad terms, we intend to test the growth of C. reinhardtii in separate test tubes, each  with a unique concentration of nitrogen. To do so, we require 18 test tubes, filtered lake water,  and a spectrophotometer to measure optical density. The experiment will be conducted with a  total of six levels, and three replicates per level. 

The six levels we will use will span across several different nitrogen concentration  degrees. According to an article published by the Food and Agriculture Organization of the  United Nations (FAO), algae roughly live in a 5-8% nitrogen environment, loosely approximate  to 0.050 g/mL to 0.080 g/mL (Concepts for Bioenergy from Algae 2005). As such, we will have  concentrations of 0.000 g/mL, 0.012 g/mL, 0.021 g/mL, 0.045 g/mL, 0.075 g/mL, and 0.089  g/mL. This information is displayed in the table below.


Nitrogen Concentration and Replicates for Each Treatment 

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Figure 1: The table above shows the nitrogen concentrations and replicates for each of the six treatments 

We will begin by filling the test tubes with filtered lake water and ensure that each tube  contains a constant quantity of other essential nutrients (eg. phosphorus) of C. reinhardtii  growth. Then, we will input a constant quantity of C. reinhardtii in each test tube to maintain  consistency across all 18 test tubes. 

Finally, we will add ammonium sulfate to each test tube at  varying concentrations in order to add the respective concentrations of nitrogen to each test tube.  

The independent variable of our experiment will be the concentration of nitrogen (g/mL) in the  test tubes, whereas the dependent variable will be the algal cell density (cells/microliter). Our  control group will be three test tubes with 0.000 g/mL nitrogen concentration because it  represents the effect of no nitrogen on algae growth.

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To record the data for our dependent variable, we will use a spectrophotometer, which  will allow us to approximate the concentration of algae cells in each test tube. We can then analyze our results using a Spearman Rank Correlation test, since we are investigating the correlation between the nitrogen concentration (our independent variable) and the algal cell density (our dependent variable). 

We will use the statistical software R to obtain statistical  measures including the p-value and correlation coefficient. From here, we can analyze the data to determine the correlation between nitrogen concentration and algal cell density, and ultimately determine the optimal nitrogen concentration. 

Despite the fact that the experimental design covers all bases, we will have to ensure that all confounding variables are taken into consideration, such as light or time.

Despite the fact that the experimental design covers all bases, we will have to ensure that  all confounding variables are taken into consideration, such as light or time, As both of these could potentially prevent us from reaching a firm conclusion, we will ensure that all test tubes are measured under the same lighting conditions and over same time period. 

Additionally, we should be sure to use the same type of cuvette for each trial and ensure that we only handle the cuvette with clean gloves and paper towels. Failing to do so will result in erroneous data points  due to the tainted glass which will interfere with the spectrophotometer. 

Anticipated Results  

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We expect that the test tubes with the 0.075 g/mL of nitrogen will yield the greatest  growth of algae. In the aforementioned FAO article, it was estimated that algae lives in 5-8%  nitrogen concentration (Concepts for Bioenergy from Algae 2005). Prior literature also holds that nitrogen is essential to algae growth as a chemical process, and thus higher nitrogen levels would  result in greater algae growth (Savage et al. 2020). Given that algae primarily live in 5-8% nitrogen concentration environments, the highest concentration of nitrogen we tested that is within the range determined by literature is 0.075 g/mL. We believe that any greater nitrogen  concentrations would hinder the growth of algae, and may even serve as catalysts for the  negative marine life health effects of excessive algal bloom (Savary 2020). 

Ultimately, we hope to find optimal conditions to support the growth of algae. The results  of this experiment will shed light into this matter because they would inform us of the exact  concentration of nitrogen that would best stimulate algae growth. By deducing the optimal nitrogen concentrations to produce algae, we will have further insight into the feasibility of algae  to generate biofuels.

Written by Avhan Misra

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Concepts for Bioenergy from Algae. 2005. Food and Agriculture Organization of the United  Nations. [accessed 2021 March 31st]. Milledge JJ, Heaven S. 2013. A review of the harvesting of micro-algae for biofuel production.  Rev Environ Sci Biotechnol. 12(2):165–178. 

Ross ME, Davis K, McColl R, Stanley MS, Day JG, Semião AJC. 2018. Nitrogen uptake by the  macro-algae Cladophora coelothrix and Cladophora parriaudii: Influence on growth,  nitrogen preference and biochemical composition. Algal Research. 30:1–10.  

Sarvary MA. 2020. Growing microalgae for biofuel production, carbon sequestration, and food. In:Sarvary MA, editor. Investigative Biology: A Laboratory Text. Plymouth:  Hayden-McNeil Publishing. 1-5. 

Savage E, Nagle N, Laurens LML, Knoshaug EP. 2020. Nitrogen derived from Combined Algal  Processing supports algae cultivation for biofuels. Algal Research. 50:101987.