243. Production of Clean Biofuel from Microalgae
PROJECT REFERENCE NO.: 39S_B_BE_006
COLLEGE : R.V. COLLEGE OF ENGINEERING, BANGALORE
BRANCH : BIOTECHNOLOGY
GUIDES : PROF. SHIVANDAPPA
STUDENTS : MS. VIDYASHREE S.
MS. NAVYASHRI S.
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
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
Key words: Trans-esterification, Algal Biodiesel, Lipase, FFA-Free Fatty Acid.
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
Table 1.3: Oil content of various microalgal species
MICROALGA OIL CONTENT
Chlorella sp 28-32
Cylindrotheca sp. 16-37
Isochrysis sp. 25-33
Nannochloris sp. 20-35
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
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
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
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
Purification of Lipase
Extraction of Algal oil from dried
Growth of B.braunii mat
Media optimization & culture of
Purification of Algal oil
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