1 THE EFFECTS OF LIGHT INTENSITY ON THE GROWTH RATE OF C YANOTHECE By Kevaughn Aiken
2 Table of Contents Abstract .... 3 Introduction .. 3 4 Method and Material 4 6 6 9 Conclusion 9 ... 10
3 Abstract This research was directed towards investigating the effect of light intensity on the growth rate of algae in an aqueous environment. An exopolysaccharide producing cyanobacterium cyanothece was grown in a controlled photo bioreactor enviro nment while the specific growth rate and cell density were monitored at different light intensities. The light intensities varied between 40 1, actual values being: 44, 58, 93, 96, 289 and 306 The unique characteristics of the c yanothece provided great properties facilitating algae growth in varying conditions To ensure optimum harvesting, the salinity, pH and temperature of the reactor medium were controll ed, as the l ight intensity varied After two month s of cultivating, the highest cell and exopolysaccharide produced were recorded measuring 3.0 g/L and 2.6 g/L, respectively The experimental results showed that the specific growth rate and algae optical density increased as the light intensity increased In an effort to relate the microbial growth rates in the photo bioreactor environment to the concentration of a limiting nutrient, the Monod equation was used. Introduction As energy demands continues to increase, while traditional energy source s (such as fossil fuels, coal, oil and natural gas) decreases, more renewable resources are being explored. One of the most versatile energy sources is biofuel, based on its ability to be produced from so many renewable energy sources. Additionally, there are several advantages to producing biofuel over fossil fuel. Some of these advantages are that biofuels: produce less greenhouse gases overall than fossil fuels when they are burned, creates a greater fuel security for countries without oil reserves and plants are preserved in the production of biofuel. However, a complete transition from traditionally derived fuels to biofuel is impossible due to production capacity restraints and cost Though biofuels do not compete with traditional energy resources (S ingh et at. 2010), algae, as the third generation to produce biofuels, has several advantages to reduce the cost compared to t he first and second generations (Zhang, 2014). A few advantages are that it does not compete with food resources, does not use arable land, has higher growth rate, and can be cultivated in saline water or wastewater (Noraini et al., 2014; Chen et al., 2014). Due to the variety of algae species
4 and the applications, t he developmen t of methods for high throughput cultivation and efficient harvesting of m icroalgae has, over the past decades, constitu ted an active field of research ( Hassan, 2013 ) Cyanobacteria has high photosynthetic efficiencies and diverse metabolic capabilities th at provides the ability to convert solar energy into a variety of biofuel. The se unique characteristics of the unicellular cyanobacterium made it attractive for this research. Cyanothece a cyanobacterium isolated from a shallow lake in the Florida Keys C yanothece can fix nitrogen, tolerate high salinity and secrete an extracellular biomaterial which contains proteins and carbohydrates (Phlips et at. 1989; Moreira, 2014). Having the ability to fix nitrogen at near maximum rates, in medium s lacking nitroge n, helps to minimize cost of cultivation because no nitrogen is required. (Phlips et al., 1989 ; Zhang, 2014 ) Additionally, due to the tolerances for high salinity, fresh water resources can be conserved and the risk of contamination from other microorga n isms can be minimized (Zhang, 2014). There are many variable s that could contribute to algal growth, some of these includes the concentration of carbon dioxide /available air the concentration of phosphorous, temperature and light intensity However, the effect of light intensities on specific growth rate will be discussed in this paper. Method and Material There are two primary means of cultivating algae, open sys tems and closed systems. For this specific investigation, the closed system ( photo biorea ctors ) cultivation method were used I n order to mai ntain a controlled environment s ix 500 mL serum bottles were used as PBRs In the cultivation of the algae Allen medium without nitrogen (A Na medium) was developed by creating a series of stock solutio ns, this provided a controlled environment. The medium comprised of ethylenediaminetetraacetic acid disodium salt, dihydrate, potassium chloride, calcium chloride d ihydrate, dipotassium phosphate, magnesium sulfate heptahydrate, iron, vitamins, (molybdenum), sodium chloride and so dium bicarbonate as the buffer.
5 To reduce potential contamination, the PBRs were autoclaved at 121 C for 30 minutes before used. 250 ml of the medium was then added to each PBR, in the presence of 1 mL of algae. The sys tem was them moni tor and controlled to maintain conditio ns. The pH of the solutions was maintained at 7.50.25. Instead of a stirring rod, air was pumped into each PBR, through a plastic tube and a filter stone, at 2 L min 1 To mitigate contamination, alc ohol was used to clean the air tubes and stones before being placed into solution environment. The PBRs were then placed in an algae cultivation chamber. A modified refrigeration, containing LED lights, was used as the cultivation chamber. The PBR systems were exposed to 13 hours of light and 11 hours of darkness per day during which the temperatures were controlled at 302 C during light cycle and 222 C during dark cycle The light intensities varied within the chamber, this allowed different cultures to operate under specific light intensities The light intensities ranged between 40 300 m 2 s 1 Sample testing and analysis was divided into two periods. The first period was one week long, while the other was for the duration of the cultivation period. For the first period, 1 mL samples for analysis were taken daily for one week. After the first wee k, samples were then taken every two days and analyzed throughout the cultivation period. The optical density, salinity and dry weights were measured and analyzed from the samples ; the pH of these samples was measured to maintain at 7.50.25. For the analy sis of the optical density, 1mL samples were taken and centrifuged at 10000 rpm for 15 minutes. Afterwards, the supernatant was separated from the biomass pellets. The biomass pellet was then resuspended by adding 1 mL of distilled water, after which the o ptical density was measured In the event that the optical density of the biomass was greater than 0.6, the sample was then diluted in effort to increase accuracy. The salinity was maintained at approximately 35 psu. A deviation in the salinity based on evaporation and other factors, resulted in the adding of autoclaved distilled water. The amount of water to be added for salinity deviation was calculated using equation (1) below:
6 (1) For the dry weight measurement of cells and exopolysaccharide (EPS), 15 mL algae culture was centrifuged at 10,000 rpm for 20 mintures. Then the sup ernatant was removed and 15 mL DI water was added to resupend the cell pellet. Then the resupsension was centrigued again until the salinity was lower than 2 psu. Then 10 mL of supernatant and cell was withdrawn, and placed on weighed aluminum dishes. The dishes were then placed into an oven, where the samples were dried at 105 C until constant weight. Then the samples were burnt at 550 C, the difference of the two weights was the volatile solid (VS) dry weight of the cells and the EPS. Because of an extra loss of the culture medium, the VS of the EPS should subtr act the loss of the medium to get the EPS dry weight. This mass was then measured, and observed throughout the algae cultivation. Light intensities were set for specific reactors throughout the course of the experiment. The light intensities ranging from l ow to medium, were used as the specific algae growth was observed. A table outline the light intensity associated with specific PBRs is illustrated below. Table 1. Illustration of light intensities associated with specific PBR Light Level High Medium Low Light Intensity 289 306 96 93 58 44 Reactor # R1 R2 R3 R4 R5 R6 Results and Discussion Algae was cultivated in PBR over a two month period. Periodically, the biomass was recorded for analysis. Over the course of the 60 day period, optimal biomass dry weight was recorded which correlated with the reactor with the highest light intensity. Figure 1. Illustrates a graph of the measured data.
7 Figure 1. Cell growth overtime Figure 2 Exopolysaccharide production overtime For the first five days of cultivation there were minimal difference s in the dry mass weigh t However, over the later day s the biomass dry weight increase d as th e light intensity increased. The cell and EPS dry weight recorded as high as approximately 3.0 g/L and 2.6 g/L, respectively (Figure 1 and 2) Based on the performance displayed by the data obtained, one can conclude that increasing light intensity increased the cell and EPS dry weight. Having a h igh EPS dry weight is critical for processing. The high carbon and hydrogen content makes lipids an energy
8 dense molecules. This feature makes it a favorable target for processing and usage as an alternate energy source to traditional fuels. Data from figure 1 was used to calculate the specific growth rate at the specified intensity. The equation used to calculate the specific growth rate is seen below: X OD me asured at time t (day) X 0 represents OD measured at t o t lag lag time to reach exponential phase Using the above equation, the specific growth rate s was calculated for the resistive light intensities. A table with the calculated values can be seen below in Table 2. Table 2 : Light intensity and specific growth rate per reactor In an effort to relate the microbial growth rates in the photo bioreactor environment to the concentration of a limiting nutrient, the Monod was used. The fundamental Equations governing this principle can be seen below. max Ma ximum specific growth rate (day 1 ) L Light intensity used for algal growth ( m 2 s 1 ) K L Va lue of L when max =0.5 The Monod equation was then used to compare modeled values to th e experimental results. Figure 3 below illustrates how well the model fits the experimental data. Therefore, the model holds true, an increase in light intensity towards algae result s in an increase in the specific Reactor # R1 R2 R3 R4 R5 R6 Light Intensity 288.9 305.7 95.95 93.2 57.6 44.3 run1 (day 1 ) 0.57 0.6 0.3 0.31 0.21 0.19
9 Figure 3 Relationship between specific growth rates for algae vs. light intensity Conclusion Though biofuel processes are usu ally more expensive than fossil the advantages of the third generation biofuel method, algae, makes it a favorable candidate. A few of these advantages includes algae : not competing with food resources, does not use arable land, has higher growth rate, and can be cultivated in saline water or wastewater. These advantages along with the unique characteristics of the cyanothece, p rovides the necessary properties to facilitate algae growth in varying conditions. To mitigate disturbances throughout the testing, bioreactors were used. Algae was harvested in a controlled environment to isolate the variab le of interest (light intensity) and en sure optimum harvesting. By operating within controlled parameters, the results showed that the specific growth rate and algae density increased as the light intensity increased. After two month s of cultivating, the highest cell and EPS biomaterial produced were approximately 3.0 g/L and 2.6 g/L, respectively. The experimental data was then fitted to observed. Therefore, the model holds true, an increase in light intensity towards algae results in
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