Undergraduate Research Proposal Department of Chemical Engineering Faculty of Engineering Thammasat University

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1.

Topic Production of Biodiesel from Microalgae Grown in

Simulated Open Pond System

2.

Researchers

Ms.Rasita Wangsaekul ID. 5110753109 ID.

Ms. Keziah Avenido 5210751565

3.

Advisor

Dr. Phanida Saikhwan

4. Objectives

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4.1 To investigate the growth of Chlorella Sp. under uncontrolled environment conditions in order to study the possibility of growing the specie in open pond system 4.2 To make biodiesel from lipid obtained from

Chlorella Sp. 4.3 To test properties of biodiesel made

5. Research Scope

5.1 Microalgae used in the research is Chlorella sp. (Thailand Institute of Scientific and Technological Research, TISTR) 5.2 Chlorella Sp. is grown in simple batch reactors to simulate open pond system. 5.3 Properties of the biodiesel made are compared to ASTM standards

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6. Keywords
biodiesel, biofuel, biomass, Chlorella sp., microalgae, extraction, ASTM standards

open pond, alternative energy, lipid biodiesel testing, biodiesel quality,

7. Introduction

Due to the rapid increase of energy demand triggered by both commercial alternative and household consumptions, which are fuel to demand also rises dramatically. This eventually leads to emerging energy resources substitute fuel to balance the depleting ratio of fossil fuel versus energy production. Aside from the fact that fossil fuels are non-renewable resources of energy, it also raises the issue of environmental damage as it is burned before it can actually generate energy.

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One of alternative fuels under research is biofuel. Biofuels are derived from biomass which comes from organic matter. Most biomass are found in forests, prairies, marches, and fisheries. An example of biofuel is the biodiesel, which is classified as a non-toxic, biodegradable and renewable fuel. Biodiesel is produced from a wide variety of raw materials such as fresh or used vegetable oil, animal fats, oil seed plants and microalgae. These raw materials have varying oil contents and yield. The following table is a comparison of common vegetable and algae oil yield [1] : Table 1: Oil Yield Per Hectare of Land Of Different Crops Crop Corn Soybean Canola Jatropha Coconut Oil Palm Microalgae A (70% oil content) Microalgae B (30% oil 58,700 Oil Yield (L/h) 172 446 1190 1892 2689 5950 136,900

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content) It is quite obvious from the table that microalgae provide a promising amount of oil yield with less land area. Although biodiesel production is possible, a number of factors are to be taken into consideration when producing biodiesel. For example, equipment set-up costs, land area, and the amount of raw materials needed to produce an ideal volume of finalized product. There are still questions on the efficiency and lipid extraction especially under uncontrolled conditions. The problem lies with the inconsistencies of sample taken throughout the tank or container. Since an open pond is not ideal for laboratory space, algae can be cultivated on large-scale simple batch reactors instead. In addition to that, lipid yield is very limited although raw material comes in large quantities and production costs may not possibly be covered with the market cost. This then, beats the purpose of choosing algae as alternative. The research will investigate deeper on supporting feasibility of microalgae as raw material for biodiesel in simulated open pond system.

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8. Literature Review
8.1 Algae Research on algae was initiated during the 1970s and 1980s but was interrupted during the 1990s due to lack of funding. In general, algae can be described as simple organisms with either singular or multicellular structure. Algae can be classified into two main types namely; microalgae and macroalgae.. Most algae are considered photoautotrophs since they convert inorganic carbon such as carbon dioxide (CO2 ) with the aid of light, water and nutrients to organic molecules and thus, producing oxygen. Compared to terrestrial plants microalgal is more efficient in transforming light energy into biomass.The synthesis of carbohydrates (C6H12O6) from carbon dioxide (CO2) and water (H2O) to produce oxygen is shown in the following reaction [2]: 6CO2 + 6H2O --------------- (1) C6H12O6 + 6O2

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The photosynthetic reaction of algae composes of two main reactions. 1. Light absorbed by chlorophyll is trapped and used to separate water molecules into oxygen (O2 )and hydronium ions, and is converted as reduced NADPH and kept as chemical energy from ADP + Pi to ATP. photons NADPH + ATP 2. Dark reaction occurs where light energy is used up for production of carbohydrate through carbon fixation. CO2 + H2O carbohydrate

8.1.1Chlorella Sp. as Raw Material of Biodiesel The focus of this research is based on a specific strain of microalgae known as the Chlorella sp. but to widen scope, comparison of various strains and data experiments would also be included as examples. In choosing the strain of algae the most important factor is its oil content and efficiency in converting light energy to biomass. Based on experiments conducted on

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biodiesel according to strain of microalgae, the following data was gathered; Species Chlorella Sp. Nitzschia Sp. Nannochloropsis Sp. Schizochytrium Sp. Oil Content (% based on dry weight ) 28-34 45-47 31-68 50-77

Table 2 shows a comparison of oil content found in green algae [3]. This research will focus mainly on Chlorella sp.

8.2 Microalgae Cultivation Methods Microalgae cultivation comes in many forms. The most well-known and tested gowth technology systems are :

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1.

Open Pond System

A system that uses large pools as medium but not necessarily mean that it cannot be covered, instead it can be partially closed to allow carbonation in high concentrations [4] Open ponds are the oldest and simplest systems for microalgae cultivation. The system uses a shallow pond at approximately one-foot deep. Microalgae is grown according to its natural environment. It consists of a paddleqay which stirs the algal cells and nutrients and a raceway made from concrete. These raceways are built by digging and laying a line of plastic liner in order to prevent the liquid to be absorbed in the. It also has baffles in which the flow is guided to minimize

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space. This system is continuously operated to ensure that the nutrients are well mixed with the microalgae.[5] . 2. Closed Pond Systems

A system very similar to open ponds but only differs where it is fully isolated to screen possible contaminations. The other option for cultivating algae is using a closed system, or photobioreactor (PBR). These systems have gained in popularity as more high value products have been produced from algae. PBRs tend to be more complicated and expensive compared to open pond systems but it allows better condition control. This type of system reduces the possibility of impurities contaminating the algae and is ideal for cultivation of single species.[6] (Amin, 2009, Chisti, 2007, Pulz, 2001a).

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3. Tubular Systems

Tubular reactors consists of a series of straight transparent tubes. The tubes are usually made out of glass or plastic and have a diameter of 0.1 meter or less. Mixing is done via circulation of air and pumping. [6] 4. Plastic Bag Systems

A system in an isolated setting using a series of transparent plastic bags connected in series

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5. Well Systems (especially for chlorophyll reduced microalgae)

A system designed like a well with controlled settings 6. Pyramid Photobioreactor System

A controlled system consisting a pyramid glass or plastic container connected to an automated mixing system Open pond systems are the most ideal settings for mass production of biodiesel but since it uses a huge portion of land area and is out in the open, risks of contamination and unpleasant weather conditions may
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affect the growth of the algae. In addition to that, it is harder to control compared to that of a closed system wherein conditions are kept at constant. However, closed systems are much more expensive than that of an open medium.

8.3 Lipid Extraction Oil Extraction from organic matters can be chemical, physical or a combination of both. Large scale extraction is mostly performed by mechanical cell disruption followed by solvent extraction. In instances where mechanical cell disruption is not possible, the method of Blye and Dryer (1959) can be used as a substitute. This process involves a polar and a nonsolvent polar molecule mixed together in order to obtain lipid fractions. However, extractions may not produce pure ideal lipid since contaminations may be hard to distinguish and thus may raise issues of its effectivity as an end product. In a research paper [7], six species of algae were tested to determine the differences in

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their oil yield under normal growth control conditions. The following table is a summary of the each species versus time: results of the mentioned evaluation and the fluorometric reading of

Table 3: Average Peak Fluorometric Reading

Table 4: Fluorometric Reading vs Time

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 C1-1, C1-2, and C1-3 refer to samples that were control, samples treated with 24 hours of hypo-osmotic conditions, and samples subjected to nitrogen deficient conditions. It can be seen clearly that the fluorometric readings have increased for a short period of time until it reaches its maximum value and fluctuated afterwards. [8] 8.4 Biodiesel Testing This research will include data of testing biodiesel quality and whether it will or will not meet standard requirements of ASTM and other related properties. Table 5 is the list of FAME properties and Table 6 is based on ASTM Standards. International Standards For Biodiesel
AUSTRI A Standard/ Specificati ons Test Value Unit ON C1191 FRANCE Journal Official GERMAN Y DIN E 51606 ITALY UNI 10635 SWE DEN SS 36 USA MBTI D02

15 54 6751~

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Date Applicatio n

1 July 1997

14 Sep 1997

1997

Sep

21 April 1997

Nov

27 10Jan Augus t 03 **

1996 2002 E** **

FAME** VOME** 15C g/cm

2 3

FAME** VOME** 0.875 0.90

VOM FAME FAME

Density Viscosity

0.85 0.89 3.5 5.0 >100 <0/-15 <0.02 <0.05 <0.02 -

0.87 0.90

0.86 - 0.87 0.90 3.5 5.0 >300 <360 >100 <0/-15 <0.01 <0.5 <0.01 <700 0.90 5.0 -

4.473 339 357 162 0.000 4

40C mm /s C C C C summe r C % Mass 100% 10% Mass % Mass Mass % Mass mg/kg % %

3.5 - 5.0 3.5 - 5.0 <360 >100 <10 <0.3 <200 >110 <0/-10/20 <0.01 <0.05 <0.03 <300

3.5 - 1.9 6.0 -

Distillation I.B.P. Distillation 95% Flashpoint CFPP Pour

>100 >130 <-5 <0.00 1 <0.01 -

point Total Sulphur CCR CCR Sulphate ash (Oxide) Ash Water

0.050 0.04 <0.02 0

<300 <300

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content Impurities total Cetane Number Neutral No. mg/kg mg g Mass % Mass Mass % Mass Mass % Mass Mass mg/kg NA/K mg/kg % % % % <0.20 <0.02 <0.24 <120 <20 <0.1 >96.5 <0.8 <0.2 <0.2 <0.02 <0.25 <115 <10 <5/5 <0.3 <0.8 <0.4 <0.4 <0.02 <0.25 <115 <10 <5 <0.2 >98 <0.8 <0.2 <0.1 <0.05 <10 <0.2 >98 <0.8 <0.1 <0.1 >49 >49 <20 >49 <20 >48 >47 0 52.9

KOH/

<0.8

<0.3

<0.5

<0.5

<0.6 <0.80 0.23

Methanol content Ester content Monoglyci des e Triglycerid e Free glycerol Total glycerol Iodine No. Phosphor Alkaline met. Diglycerid

<0.02 0.020 0.003 <125 <10 <10/1 0 0.240 0.146 <10 <120 <10 -

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**FAME: Fatty Acid Methyl Ester

**VOME: Vegetable Oil Methyl Ester

Table 6: ASTM International Standards

8.3.1 Properties Related and Method of Testing Flash Point D93

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Flash point temperature is one measure to test subject response to heat in controlled laboratory conditions. In accordance to OSHA, liquids with a flash point under 37.8 degree Celsius are considered flammable. This can be determined according to three procedures depending on subject matter categorized as 1. Procedure A is applicable to distillate fuels (diesel, biodiesel blends, kerosine, heating oil, turbine fuels), new and in-use lubricating oils, and other homogeneous petroleum liquids not included in the scope of Procedure B or Procedure C. 2. Procedure B is applicable to residual fuel oils, cutback residua, used lubricating oils, mixtures of petroleum liquids with solids, petroleum liquids that tend to form a surface film under test conditions, or are petroleum liquids of such kinematic viscosity that they are not uniformly heated under the stirring and heating conditions of Procedure A. 3. Procedure C is applicable to biodiesel (B100). Since a flash point of residual alcohol in biodiesel is difficult to observe by manual flash point techniques, automated

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apparatus with electronic flash point detection have been found suitable. Water and Sediment D2709 Test method used as an indication of water and sediment in middle distillate fuels such as Grade Nos. 1 and 2 fuel oil (Specification D396), Nos. 1-D and 2-D diesel fuel (Specification D975), and Nos. 0-GT, 1-GT, and 2-GT gas turbine fuels (Specification D2880). Kinematic Viscosity D445 Test method determining the kinematic viscosity, , of liquid petroleum products, both transparent and opaque, by measuring the time for a volume of liquid to flow under gravity through a calibrated glass capillary viscometer. The dynamic viscosity, , can be obtained by multiplying the kinematic viscosity, , by the density, , of the liquid. Other properties are commercially available on standard ASTM manuals and will be discussed further in the completion of research.
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8.4

Advantages and Disadvantages Like every other newly discovered alternative

source of energy, using microalgae for large-scale biodiesel production would require detailed evidence and support to be considered valid for the industry. Facts are to be laid down in accordance to what is the expected outcome and whether it would give more benefit than problems. The main advantage of the microalgae comes from its simple structure in which scientists are able to genetically engineer its properties, fast growth rate and high oil contents in certain strains. In addition to that, algae also consume large quantities of carbon dioxide in the atmosphere mostly released by the industrial corporations, thus, reducing emissions. Not only that but they also adapt easily into the environment growing even in low quality water. It is non-toxic and renewable. Although experiments prove that producing biofuels produce the same amount of carbon dioxide emissions as burning fossil fuels, environmentalist still accept the idea due to the fact that as it produces

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carbon dioxide, it also takes up carbon dioxide at the same time. In summary, almost no further carbon dioxide is added to the atmosphere. This means that with proper genetic modification and development the algae can possibly one of our future stabilized energy resource [9]. On the other hand, production costs of algae depend more on biological rather than engineered relates issues meaning the analysis must focus more on the natural capability of the chosen strain to convert light into biomass rather than funding its modification. At present, large-scale biodiesel production still faces problems because cost of production is two times higher than that of the current petroleum diesel costs. Land issues also play a minor role in this concept given that in cultivating microalgae for mass production, it would require open pond operations to meet the expected quantities of product. Open pond application at present emphasizes more on inland and controlled conditions rather than natural water settings and would obviously take up a considerably wide portion of land for successful manufacturing.

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9. Experimental Methods 9.1 Cultivation of Chlorella sp. The culture of Chlorella sp as raw material was duplicated from a sample provided by TISTR. Sample was introduced into 1 liter Duran bottles premixed with food and nutrients (formula is listed below in Table 7a and 7b). The containers were initially kept at room temperature with aid of magnetic stirrers to allow thorough mixing and even growth of specimen.

Table 7a: A recipe of food medium 21 (TISTR) Chemicals Na2HPO4.2H2O KH2PO4 CaCl2 Amount (mg) 260.00 740.00 10.00 10.00 50.00

Fe.EDTA

MgSO4.7H2O

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KNO3

1000.00 1.00 ml Amount (g) 3.58 12.98 1.83 3.20

Trace element mixture* Chemicals (1L) Al2(SO4)3.18H2O MnCl2.4H2O CuSO4.5H2O ZnSO4.7H2O

Table 7b: A trace element mixture* (TISTR)

9.1 Growth of Chlorella sp.

To obtain considerable amount of Chlorella sp.

required in producing a sufficient amount of samples for testing, the small Duran bottle samples are later on transferred to larger containers with an estimated volume of 15 liters each and is introduced to a electric stirrer. The concept of this research will focus on a model of uncontrolled conditions almost similar to that of an open pond. Growth of algae is expected to have constant growth within a period of 22-25 days. The optical density (OD, 682 nm approximately fixed at starting point of 0.01) will be

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constantly measured in a UV spectrophotometer (UV1201, Shimadzu, Japan). 9.2 Extraction of lipid To get lipid from algae, extraction would be in batch due to large quantities of cultivated algae and the limitation of laboratory apparatuses. Chlorella sp extraction is possible through liquid-liquid extraction. Procedure is as follows: 1. An electric centrifuge is set at 3000 rpm for 120 minutes to enable separation of microalgae and food 2. Algae is dried for 2-3 hours at temperature settings of 80-100C 3. The dried algae and a 96% ethanol solution (60 ml) are mixed thoroughly and continuously for 24 hours at room temperature. By performing filtration, dried algae is separated and mixed with hexane (32ml, 60ml). 4. There will be clear layer separation in which the lipid will be in the hexane layer
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5. Distill lipid out of hexane layer. 9.3 Measurement of lipid quality by using Acid Value Measurement [10] 1. Prepare 250 ml solution of 50% Isopropyl alcohol (IPA) and 50% toluene. 2. Use solution from 9.4.1 for 25 ml and add 1 g of lipid. A. Add Phenolphthalein indicator 1 ml. B. Titrate the solution in 9.4.3 with 0.0016 mol/l KOH, notice color change from clear to pink C. Calculate FFA AV% = FFA% = where sample (ml) A = Volume of KOH used in titration

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B = Volume of KOH used in titration blank (ml) MW = Molecular weight of KOH (56.1) W = mass of lipid (g)

9.4 Biodiesel Extraction from Obtained Lipid by Transesterefication 1. Prepare 1 L of lipid solution and heat it to 50 degree Celsius in a hot plate stirrer. 2. While heating, prepare 200 ml of methanol mixed with 4 g of lye (NaOH) to form sodium methoxide. Both chemicals are toxic so be sure to wear glasses, gloves and an apron. Make sure NaOH crystals are dissolved 3. If the oil reached set temperature then immediately mix it with sodium methoxide.

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4. Stir for 20-30 minutes in capped bottle and leave for a few hours. 5. Separate the upper layer of biodiesel from glycerin layer at the bottom. A portion of the product will be sent to a certified research lab for further verification of biodiesel claim and as for the remaining samples, tests are to be conducted in comparison with ASTM standards and FAME.

10.

Expected Outcome 1. Understand more about algae biodiesel and its origin 2. Produce a sample grade algae biodiesel and learn the procedures in testing its quality 3. Clearly compare controlled and uncontrolled conditions of environment and its effect to Chlorella sp. growth cycle

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4. Test the possibility of large-scale biodiesel production by simple batch reactors

Reference
[1] Lauren DElia; Andrew Keyser; Craig Algae Young,

Biodiesel Faculty of WORCESTER POLYTECHNIC 2010 [2] Blankenship, R. E. (2002) Molecular Mechanisms of Photosynthesis. Blackwell Science, Oxford.

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[3] Campbell. Biodiesel: Algae as a Renewable Source for Liquid Fuel. Guelph Engineering Journal, Guelph University (1), 2 7, 2008 [4] Microalgae Growth Technology Systems,Soley Institue, Dr. Mustafa Kizililsoley and Dr. Soner Helvacioglu, 2008 [6] Patil, V.; Reitan, K.I.; Knudsen, G.; Mortensen, L.; Kallqvist, T.; Olsen, E.; Vogt, G.; Gislerd, [5] Microalgae as a Feedstockfor Biofuel Production, Zhiyou

Wen, Virginia Cooperative Extension engineer, Biological Systems Engineering, Virginia Tech Engineering, Virginia Tech

Michael B. Johnson, graduate student, Biological Systems

[6] Algae and aquatic biomass for a sustainable production of 2nd generation biofuels, Mr. Raffaello Garofalo [7] Algal growth in horizontal tubular Reactors, P.J.M. van Beveren, Wageningen university , agrotechnology and food sciences [8] Microalgal Oil Extraction and In-situ Transesterification, Justin M. Ferrentino; Ihab H. Farag; Leland S. Jahnke, Chemical Engineering, University of New Hampshire (UNH) Durham, NH Plant Biology, UNH 2007

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[9] Effects of the availability of CO2 on growth, nutrient uptake, and chemical composition of the marine microalgae Chlorella sp. and Nannochloropsis oculata, two potentially useful strains for biofuel production, Gabrielle R. Faria, Caroline R.P.S. Paes, Dominique J.F.A. Castro, Natlia A.B. Tinoco, Elisabete Barbarino and Sergio O. Loureno, Fluminense Federal University, Department of Marine Biology 2012 [10] . . 2550.

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