Research conducted in the laboratory of Joshua S. Yuan Mentored by Ryan D. Syrenne Bioenvironmental Undergraduate Research Scholars (BURS) Research conducted by Kriti Gaur

Biochemical Mechanisms of Chlamydomonas reinhardtii

Data Optical Density

Nile Red Fluorescence

Nile Red Lipids

Biomass

10mmoL 0.814293333

25mmoL 0.65332

50mmoL 0.48252

100mmoL 0.48364

00.10.20.30.40.50.60.70.80.9

Absorba

nce(nan

ometers)

AverageOp3calDensityat570NanometersforChlamydomonas

reinhard/i,CC-4425(D66)

10mmoL 0.614546667

25mmoL 0.52148

50mmoL 0.554213333

100mmoL 0.504726667

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Asborba

nce(nan

ometers)

AverageOp3calDensityat570NanometersforChlamydomonasreinhard/i,CC-503

10mmoL 3145.533333

25mmoL 5319.533333

50mmoL 5807.933333

100mmoL 6499.733333

0

1000

2000

3000

4000

5000

6000

7000

8000

Fluo

rescen

ce

AverageNileRedFluorescenceofChlamydomonasreinhard/i,CC-4425

(D66)

10mmoL 6154.4

25mmoL 6020.466667

50mmoL 5349.466667

100mmoL 6623.933333

0

1000

2000

3000

4000

5000

6000

7000

8000

Fluo

rescen

ce

AverageNileRedFluorescenceofChlamydomonasreinhard/i,CC-503

10mmoL 77929.2

25mmoL 132340.8

50mmoL 143513.7333

100mmoL 161818.1333

020000400006000080000

100000120000140000160000180000

Lipids

NileRedStainingofLipidsinChlamydomonasreinhard/i,CC-4425

(D66)

10mmoL 150566.8

25mmoL 149591.9333

50mmoL 133166.6

100mmoL 168147.1333

020000400006000080000

100000120000140000160000180000200000

Lipids

NileRedStainingofLipidsinChlamydomonasreinhard/i,CC-503

Biomass(mg)0mmoL 6.18

25mmoL 3.94

50mmoL 3.98

100mmoL 5.19

01234567

Milligrams

BiomassofChlamydomonasreinhard/i,CC-4425(D66)

Biomass(mg)0mmoL 6.5

25mmoL 5.05

50mmoL 4.54

100mmoL 4.21

012345678

Milligrams

BiomassofChlamydomonasreinhard/i,CC-503

Sample1

Sample2

Sample3

Sample4

Sample5

Sample6

Milligrams 15 36 33 34 25 42

0

5

10

15

20

25

30

35

40

45

50

Milligrams

BiomassofChlamydomonasreinhard/i,SQSmutant

Part 1: The Effect of Sodium Bicarbonate on the Carbon Concentrating Mechanism of Chlamydomonas reinhardtii for Enhanced Triacylglycerol Production

Part 2: The Extraction of Squalene from Squalene-Synthase Mutants of Chlamydomonas reinhardtii

•  Chlamydomonas reinhardtii is a model organism for genetic research and for algae biofuel research. Two different strains of Chlamydomonas reinhardtii were selected for this project, CC-503, a wild-type strain, and CC-4425 (D66), a strain of Chlamydomonas reinhardtii genetically altered without a cell wall, and thus notable in algae biofuel research.  

•  The primary objective of the experiment was to determine the effect of different concentrations of NaHCO3 on the carbon concentrating mechanisms in strains of Chlamydomonas reinhardtii, CC-503 and CC-4425 (D66), for enhanced triacylglyceride production.

•  The secondary objective of the experiment was to extract the squalene compound from a transgenic squalene-synthase mutant of Chlamydomonas reinhardtii, (SQS) in order to potentially analyze the use of squalene as a biofuel compound.

•  Results of the NaHCO3 treatment on the Chlamydomonas reinhardtii CC-4425 (D66) and CC-503 were that, as molarity increased, optical density decreased, but Nile Red lipids and Nile Red fluorescence increased. This demonstrated that the Chlamydomonas reinhardtii were storing additional lipids as a storage reserve, as a reaction to environmental stress.

•  Overall, Chlamydomonas reinhardtii CC-4425 (D66) had higher lipid reserves than Chlamydomonas reinhardtii, CC-503, demonstrating that it could potentially be a better candidate for algae biofuels. A squalene extraction was attempted for the SQS strain of Chlamydomonas reinhardtii. Biomass samples were lyophilized, and a squalene extraction was attempted, however, the squalene extraction was unsuccessful, due to different factors. Future studies must be conducting on using Chlamydomonas reinhardtii, CC-4425 (D66) as an alternative source of biofuel, and for enhancing the efficiency of squalene production in Chlamydomonas reinhardtii, SQS.

Abstract

Introduction

•  3 different strains of Chlamydomonas reinhardtii were used in the experiment. For the component of the experiment studying the CCM of Chlamydomonas reinhardtii, strains CC-503 (wild-type) and strains CC-4425 (D66) were used. All Chlamydomonas reinhardtii was cultured in a sterile environment and grown into early stationary growth phase. 150 mL of algae was grown for each treatment of CC-503 and CC-4425 (D66).

•  A 1 molar saturated solution of NaHCO3 (Ci source) was made for the experiment, mixed to 84 grams in 1 liter.

•  The Wild-type strain and D66 strain of Chlamydomonas reinhardtii were exposed to 4 different treatments, being 0 mmoL, 25 mmoL, 50 mmoL, and 100 mmoL of NaHCO3, respectively.

•  Measurements were taken every day, in triplicates, for 5 days. The SQS strain of Chlamydomonas reinhardtii was not exposed to any NaHCO3 solution, and was simply grown throughout the experiment. Squalene produced by the algae was measured through a GC-MS machine.

•  Optical density was measured with a spectrophotometer, through a cuvette. Nile Red lipids and Nile Red fluorescence were measured through a plate reader, in which the algae stains were stained with Nile Red Dye.

Materials and Methods

•  Overall, the exposure of both strains of Chlamydomonas reinhardtii to increasing concentrations of NaHCO3 resulted in a decrease in optical density as the molarity increased.

•  The Chlamydomonas reinhardtii cells that died as a result of the increased molarity were unable to cope with the environmental conditions that were a result of the increasing molarity of NaHCO3. The Chlamydomonas reinhardtii cells that were able to survive, however, accumulated more triacylglycerides inside their cell body. This was due to the fact that the environmental stress caused the Chlamydomonas reinhardtii to store more lipids. Often, the onset of lipid storage in algae is caused due to environmental stress conditions.

•  It can be proposed that the increased molarity of the NaHCO3 caused a condition of environmental stress, which in turn, resulted in the Chlamydomonas reinhardtii storing additional triacylglycerides inside their cell body, as a food reserve.

•  Additionally, the Chlamydomonas reinhardtii, CC-4425 (D66) was able to produce a higher amount of lipids than Chlamydomonas reinhardtii, CC-503. This may have been due to the fact that Chlamydomonas reinhardtii, CC-4425 (D66), does not have a cell wall, and is thus able to grow more efficiently, and has higher photosynthetic efficiency than Chlamydomonas reinhardtii, CC-503. Since it does not have a cell wall, CC-4425 (D66) may also have the ability to store more carbohydrate in its cells, which can later be converted to lipids.

•  Thus, when exposed to environmental stress, Chlamydomonas reinhardtii will store increasing amounts of lipids in response to that stress, and that different strains of Chlamydomonas reinhardtii can have different reactions to environmental stress depending on the adaptations that a particular strain has, in order to survive in that environment. 

•  A squalene extraction was attempted for the 6 samples of dried algae biomass, using a GC-MS machine, but was unsuccessful, meaning that the data was inconclusive. The squalene extraction may have been unsuccessful because some of the cells of Chlamydomonas reinhardtii was not successfully transformed with the genes for limonene and squalene production. Additionally, squalene is an extremely volatile compound. It may have been difficult to get a significant amount of squalene, due its nature, thus the extraction may have been unsuccessful for that reason.

•  Future applications of this research can be to expose other strains of Chlamydomonas reinhardtii to other forms of environmental stress, to enhance lipid production in the cell.

Discussion

Optical Density

Nile Red Fluorescence

Nile Red Lipids

f-value 29.85 9.98 9.36

p-value <.0001 <.0001 <.0001

Optical Density

Nile Red Fluorescence

Nile Red Lipids

f-value 5.64 2.19 2.45

p-value <.0001893 0.099324 0.072968

Statistical Analysis of Chlamydomonas reinhardtii, CC-4425 (D66)

Statistical Analysis of Chlamydomonas reinhardtii, CC-503

***Confidence interval set to 90%.

•  Chlamydomonas reinhardtii is a model organism for genetic research and for algae biofuel research. Algae, being a very important organism, is notable for its ability to produce lipids for algae biofuels, and to produce 70-80% of oxygen on Earth.

•  All types of algae have a mechanism in its cell known as the carbon concentrating mechanism, or CCM for short. The CCM allows algae to take in more amounts of inorganic carbon (Ci) through its pyrenoid and increase photosynthesis. Overall, the entire process is extremely efficient for algae , since the enzyme Rubisco also plays a role in this process, using both Ci and O2 as substrates. When the Ci goes through the pyrenoid, Rubisco essentially fixes the Ci so that the Ci is in the form that the algae can use for photosynthesis. With the CCM, algae can trap different forms of Ci in its cells, thereby speeding up the process of photosynthesis and potentially causing the algae to produce more lipids.

•  Additionally, algae are able to produce lipids known as triacylglycerides, when under adverse environmental conditions. Algae are known to produce these lipids as a storage reserve. These lipids are valuable for algae biofuel production.

•  Two different strains of Chlamydomonas reinhardtii were selected for this project, CC-503, a wild-type strain, and CC-4425 (D66), a strain of Chlamydomonas reinhardtii genetically altered without a cell wall, and thus notable in algae biofuel research.

•  Additionally, Chlamydomonas reinhardtii is known to produce isoprenoids by only using a MEP pathway, meaning that it is at an metabolic advantage to produce complex and volatile hydrocarbons, such as limonene, squalene, and terpinene to name a few. Such compounds can also be used for the production of algae biofuels, and even biosynthetic jet fuels. Thus, in order to produce these compounds, prior to the beginning of the experiment, a strain of Chlamydomonas reinhardtii was genetically engineered with genes for the biosynthesis of limonene and terpene, which would then subsequently produce squalene through the MEP pathway and squalene-synthase mechanism.

Figure 4: Diagram of Chlamydomonas reinhardtii Figure 3: Synthesis of triacylglycerols Figure 1: CCM in Chlamydomonas reinhardtii Figure 2: MVA and MEP pathways terpene biosynthesis in Chlamydomonas reinhardtii

Figure 1) Moroney, James V., and Ruby A. Ynalvez. "Proposed Carbon Dioxide Concentrating Mechanism in Chlamydomonas Reinhardtii." Eukaryotic Cell. American Society for Microbiology, Aug. 2007. Web. 15 July 2016. Figure 2) Ryan Syrenne, unpublished. Figure 3) N. D. Vaziri, C. H. Kim, B. Dang, Chang-De Zhan, K. Liang. "Downregulation of Hepatic Acyl-CoA:diglycerol Acyltransferase in Chronic Renal Failure." Home. American Physiological Society, 4 June 2004. Web. 16 Sept. 2016. Figure 4) "Pyrenoid." Wikipedia. Wikimedia Foundation, n.d. Web. 16 Sept. 2016.