243.Production of Clean Biofuel from Microalgae PROJECT ... of Clean Biofuel from Microalgae PROJECT REFERENCE NO ... Biodiesel is the fuel which is produced from different ... As

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  • 243. Production of Clean Biofuel from Microalgae







    1. Introduction

    To meet the growing fuel requirements, alternative fuels have been extensively researched.

    Biodiesel is the fuel which is produced from different feedstock like plant based oils or animal

    fat or grease. It is a clean burning and renewable fuel. It is currently used in blended forms

    with petroleum diesel in diesel engines. Even a 20% blend of biodiesel with 80% petroleum

    diesel reduces emissions significantly and helps the environment. One of the most recent

    break through has been biodiesel production from microalgae. To produce biodiesel from

    algal oil different microalgae species were reviewed. The conventional biodiesel production

    utilizes chemical catalyst in trans-esterification which poses many drawbacks. Hence, recent

    research has been focused on alternative eco-friendly catalyst like enzymes.

    Microalgae are microscopic photosynthetic organisms that are found in both marine and

    fresh water environments. Microalgae are organisms which efficiently convert solar energy

    into biomass. Micro algal fuel has high calorific value, low density and viscosity and hence

    makes it a better feedstock than plant based feedstock .The distinct characteristic of algae is

    that there are a number of species of algae which can be used and optimized to produce bio

    fuels of different characteristics.

    Biodiesel can be produced by any organic sources of oil like waste oil, animal fat and seed

    oils. Waste oils supply is limited hence it is not a feasible feedstock for biodiesel production.

    Using seed oil as feedstock reduces the seeds for food supply hence it is not viable. There is

    a need for a feedstock source which is abundant as well as a productive source of

    feedstock. The oil yield by different feedstock is shown in Table 1.1.The table shows that

    microalgae has the highest oil yield, hence it was considered as feedstock for this present


    Enzyme Trans-esterification:

    It is the process of conversion of triglycerides into methyl esters in the presence of enzymes

    as a catalyst. Enzyme trans-esterification has advantages like (a) Temperature conditions

    close to room temperature (b) Process of recovery of catalyst, product separation and waste

    water treatment is eliminated. (c) Environment friendly and biodegradable. Commonly used

    enzyme for trans-esterification is lipase. Pseudomonas sp. produces extracellular lipase

    which can be extracted conveniently as Pseudomonas is widely researched and validated

    microbe Lipases are group of enzymes that hydrolyse oils and fats in biological systems.

    Most species of animals, plants, fungi, bacteria produce lipase. Lipase from P.aeruginosa

  • has been most researched and widely used hence lipase was extracted from P. aeruginosa

    in this present work.

    Table 1.1: Comparison of oil yield between different feedstock

    CROP OIL YEILD (L ha-1


    Soybean 446

    Canola 1,190

    Jatropha 1,892

    Palm 5,950

    Microalgae 136,900

    Key words: Trans-esterification, Algal Biodiesel, Lipase, FFA-Free Fatty Acid.

    2. Objectives

    There is currently a lot of research to produce biodiesel from different feedstocks to get

    better oil content as well as yield of Biodiesel. Vast land area is required usually to produce

    feedstock. The comparison between different feed stocks with oil yield and land required is

    shown in Table 1.2.As observed in the table 1.2 algae has the highest oil yield and least land

    requirement hence in the present work algae was considered as oil feedstock.

    Table 1.2: Comparison of feed-stocks with oil yield and land required

    Crop Oil in Litres per hectare

    Jatropa 3400

    Castor 1413

    Sunflower 952

    Safflower 779

    Palm 5950

    Soy 446

    Coconut 2689

    Algae 100000

    Table 1.3: Oil content of various microalgal species


    Botryococcusbraunii 25-75

    Chlorella sp 28-32

    Crypthecodiniumcohnii 20

    Cylindrotheca sp. 16-37

    Dunaliellaprimolecta 23

    Isochrysis sp. 25-33

    Monallanthussalina 20

    Nannochloris sp. 20-35

    Neochlorisoleoabundans 35-54


  • Fig 1.1: Percentage of Land area for different feedstocks. It is seen that the land required for algae growth is least compared to other feedstock as

    shown in Fig 1.1.From the table1.3 it is observed that B. braunii has the highest oil yield

    compared to the other microalgae species. Hence in the present work B. braunii has been

    used to extract Algal oil. There are many drawbacks of chemical method of trans-

    esterification which are overcome by enzyme trans-esterification hence in this present work

    we have compared chemical and microbial trans-esterification of various oils to demonstrate

    the feasibility of enzyme trans-esterification over chemical method.

    As discussed above, to overcome the problems following objectives were chosen for the

    present work

    1. To isolate P.aeruginosa from Soil & water

    2. To isolate and culture B. braunii

    3. To extract and purify the Lipase from P.aeruginosa

    4. To extract the algal oil from dried algal mat

    5. Microbial trans-esterification of algal oil to produce Biodiesel

    3. Materials & Methodology

    Materials used: The materials used in the present work are summarized and provided in the following section under different headings. 1. Media: LB Agar (Hi-media laboratories), EMB Agar (Hi-media laboratories), Spirit Blue

    agar(Hi-media laboratories), Kings B Agar (Hi-media laboratories) for culturing, isolating

    and screening of P.aeruginosa and Lipase. Kuhls Basal Media for culturing B.braunii.

    2. Sample collection for algae: water sample containing algae traces was collected from

    Ulsoorlake, Bengaluru

    3. Culture and growth of B.braunii: Aquarium of 8 liters capacity with aerator was

    procured from local stores, Vijayanagar, Bengaluru.

    4. Sample collection for P.aeruginosa: Soil sample was procured from Football ground,

    RVCE and tap water was procured from biotechnology laboratory, RVCE. Standard

    culture was procured from Radiant Research Lab Pvt. Ltd, Peenya, Bengaluru.

    5. Experimental setup for Extraction, Purification of Lipase and Trans-esterification:

    Remi Refrigeration Centrifuge, Simtronics- Hot Water bath, Remi Bacterial Incubator and

    0.5 m pore size dialysis membrane bag with MWCO-20kDa

    6. Algal Oil Extraction: Closed type Soxhlet extraction apparatus. 4:1 Hexane: Petroleum

    Ether (v/v) solvent.

    7. Trans-esterification: RankemH2SO4, Methanol, NaOH pellets(fisher), Lipase enzyme

    extracted, water bath, phenolpthalein indicator.

    8. Oils for comparative studies: Pongamia oil, Groundnut oil and Rice bran oil were

    procured from Biodiesel Lab, RVCE.

  • 9. Testing of Biodiesel properties: ASTM D 445 Cannon-Fenske Capillary Viscometer

    Tube, ASTM D 93 Pensky-Martens Flashpoint Apparatus, ASTM D 664 Potientiometric

    Titration setup

    Preparation of Lipase Preparation of Algal Oil

    Fig 1.2: Workflow of methodology

    1) To isolate P.aeruginosa from Soil &water : P.aeruginosa was isolated by serial diluting

    1gm of soil in distilled water and sample from all the dilutions were plated onto equal

    number of petriplates containing LB Agar and incubated overnight. The colonies obtained

    in LB Plates were sub-cultured by inoculating colonies onto EMB agar and kept for

    overnight incubation. Resultant colonies in EMB plates were then plated onto petriplates

    containing Mac-Conkey medium and incubated overnight. For selective isolation of

    lipolytic bacteria P.aeruginosa, colonies from Mac-Conkey plate were then transferred

    into Kings B media, which is specific to lipolytic bacteria and incubated for 24 hours. After

    the incubation, Biochemical and spirit blue test were conducted for screening of


    2) To isolate and culture B. braunii: After following standard operating procedure, sample

    was collected from Ulsoor lake and inoculated into modified kuhls medium for the growth

    of B.braunii and kept in tissue culture rack for about 2-3weeks. Microalgae obtained after

    Culture& isolation of


    Screening of P.aeruginosa

    Extraction, Isolation and

    screening of Lipase

    (2 weeks)

    Purification of Lipase

    Extraction of Algal oil from dried

    algal mat

    Growth of B.braunii mat

    Media optimization & culture of


    (3 weeks)

    Purification of Algal oil

    Microbial Trans-esterification

    Screening of Trans-esterification

    Qualitative tests of Biodiesel properties

  • 2 weeks of incubation was screened by microscopic observation. At the end of the 3rd

    week, fresh medium was added and continued incubation with aerator and proper light

    and dark photoperiod. The culture was then used to extract oil for the production of


    3) To extract and purify the Lipase from P.aeruginosa: Colonies from Mac-Conkey plate

    were transferred onto Kings B broth and incubated