THE PATENTS ACT,1970
(39 OF 1970)
COMPLETE SPECIFICATION
(SEC-10, RULE 13)
"PROCESS FOR THE PRODUCTION OF BIOFUEL FROM ALGAE"
ABELLON CLEANENERGY LIMITED,
3rd Floor, Sangeeta Complex, Nr. Parimal Crossing, Ellisbridge, Ahmedabad-380006, Gujarat, India

Title: Process for the production of biofuel from Algae
Filed of invention
The present invention relates to method of cultivation of marine photosynthetic cells under heterotrophic and autotrophic growth condition and production of biofuel are described herein.
In addition to these heterotrophic cultivation vessels for marine photosynthetic cells can be any types, flasks, glass bottles, any types of fermenter. Autotrophic cultivation can be of any types such as but not limited to open & closed race way pond, Photobioreactor of any types, shape and length. More particularly any types of the fuel such as but not limited to bio-diesel, pellets for feed, pellets for food, pellets for combustion to generate energy and bio-fertilizer can be produce from cultivated marine photosynthetic cells, The present invention is not limited to Dunaliella tertiolecta strain but cultivation methods can be used for any of the marine or fresh water micro algae type.
Background of the invention
The continuous use of fossil fuels by mankind has caused immense damage to the environment. Fossil fuels are excess carbon that has been removed from the carbon cycle and sequestered within the earth's crust. This was nature's way of protecting itself, however the continuous removal of this sequestered carbon from the earth's crust causes a net increase in carbon in the atmosphere causing various global climatic events such as global warming (Sawayama et al. 1995). Fossil fuels have a limited supply and the known resources are reaching rapid exhaustion, sending crude oil prices to record night. These factors have initiated the global search for renewable, cleaner biofuels (Chisty 1980-81).

These environmental effects of burning fossil fuels and the increased crude oil prices have triggered increased interest in biofuels and alternative source of renewable energy. Among liquid biofuel, biodiesel is traditionally produced from oil seed crops, which have lower yields per land area and threaten food security when compared to algae which have high oil yields (~ 90 times more oil per area of land in comparison to the best oil seed crop) and do not require arable land for cultivation.
Solar energy is renewable compared to energy from fossil and nuclear origin. Solar energy can beneficially be explored for the application of photosynthetic processes to produce biomass that can be processed to produce fuels and other products through appropriate conversions. The utilization of solar energy for micro algae growth is attractive as compare to other traditionally used crop.
Micro algae require simlight, water ,and carbon dioxide to grow. Under optimal conditions, algal cultures can double in population size between two and three times per day. Lipids and fatty acids form a major part of an algal cell, as membrane components, metabolites and storage products (Princen 1982).
At present the process involving production of biofuel from plant derived oil is limited due to slow growth, limited availability of arable land and water in many parts of the world, low amount of oil produced per acre per year resulting in high cost of creating biofuel from oil. However, it is possible to reduce the cost of producing biofuel from plant-derived oils. Phototrophic organisms such as algae are of particular interest, as they are known to be one of the most efficient plants for converting solar energy into cell growth. Algae is particularly preferred due to other facts like comparative higher growth rates, possibility to use carbon dioxide from polluted gases such as emission from fossil fuel based power-plant, ability to use nutrients from waste streams, high oil content, no need for fertile soil and no need for fresh water in case of marine species. Also the oil produced by algae can

be processed to produce biodiesel just as easily as oil from any land-based plant derived oil.
While algae efficiently transform solar energy into chemical energy via a high cell growth rate, a high rate of cell growth is not sufficient to make biofuel cost effectively, a higher rate of oil (lipid) production by algae is also required. Specifically the conditions necessary to facilitate a fast growth rate of algal cell have been found to hinder the production of lipids by algae cells. Similarly the conditions necessary to facilitate a fast rate of lipid production within algae cell have been found to hinder the growth of algae cells.
Algae cultivation methods include a closed system or Photobioreactor where in factors that affect growth like temperature, pH, oxygen concentration, carbon dioxide concentration, nutrient concentration, salt concentration, culture density, energy input (light intensity), pressure, liquid depth, shear rate, contamination that is entry of other unwanted organisms are controlled effectively. This results in high cell yields but the cost for Photobioreactor is very high. Other methods include an open system like circular pond or raceways ponds, which are perceived to be less expensive than Photobioreactor, because they cost less to build and operate. Although raceways are of low-cost, they have a low biomass productivity compared with Photobioreactor.
Apart from a conventional autotrophic growth in presence of light, number of algae species are also known to grow heterotrophically that is in absence of light by using organic substrates as their sole carbon and energy source (Wen, 2003). In micro algae culture heterotrophic growth can be a cost effective alternative to photoautotrophic growth (Chen, 1996). In heterotrophic culture, carbon sources such as, sugars or organic acids can be used as the sole carbon source. This mode of cultivation eliminates the requirement of light and, therefore, offers the possibility of greatly increasing cell density and productivity (Chen,. 1996) in

batch culture. A heterotrophic batduculture may be further modified as fed-batch, chemostat or perfusion culture to generate a high cell density.
The algae cell capture and store solar energy through photosynthesis holds great promise as a renewable energy solution for mankind. But for sustainable bio-fuels production one has to ensure a continuous supply, many efforts have been done to meet this requirement.
It is desirable to cultivate Dunaliella tertiolecta by present cultivation mode and produced continuous biomass which can be process into useful products such as a solid fuel, liquid fuel, feeds, food, gaseous fuel and compost.
Object of the Invention
The main object of the present invention is to produce bio-fuel from Algae, which tackles most of the issues, currently associated with conventional biofiiel.
Another object.of the present invention is to produce biomass production of Dunaliella tertiolecta by initially heterotrophic cultivation followed by autotrophic cultivation, and biodiesel production for the lipid (oil) extracted from dry biomass of Dunaliella tertiolecta.
Further object of the invention is to provide continuous production of Dunaliella tertiolecta biomass for liquid bio-fuel that can be obtained in sufficient quantities throughout the year.

Detail Description of invention
In the present invention production of bio-fuel is done from Algae. Algae are important because of their high protein content. In fact, they rival meat in.protein content. Algae (a Latin plural) are a large and diverse group of simple, typically autotrophic organisms, ranging from unicellular to multicellular forms. The largest and most complex marine forms are called seaweeds. They are photosynthetic, like plants, and "simple" because they lack the many distinct organs found in land plants. For that reason they are currently excluded from being considered (www.wikipedia.org visited on January 23, 2009) The embodiment of the invention are directed to use of oil from various fresh water or marine microalgae.
Structurally, the system includes a first stage where in algae is cultivated under heterotrophic conditions in closed reactor at a high cell density and a second stage where in algae is cultivated under photoautotrophjc conditions in an open system.
t . . . ■ * In the first stage algae is cultivated in a closed condition under heterotrophic
conditions using a suitable carbon source like, but not limited to glucose, glycerol,
and acetate or combination of any two or more such sources. The heterotrophic
cultivation can be carried out in a suitable bioreactor such as, but not limited to
stirred tank reactor, air lift reactor, or a tubular Photobioreactor installed indoors.
After achieving high cell concentration, a portion of the growth (inoculum) is
transferred to second stage for photoautotrophic cultivation in a Photobioreactor or
an open system such as circular, raceway or agitated raceway pond. The amount of
inoculum that is transferred from first stage to second stage is equivalent to about
5.0 to 50.0% of the total target cell mass achieved with such an open system. By
providing inoculum at higher cell mass level and with actively growing cells it is
expected that cell mass development would be faster and chances of
contamination commonly encountered in an open system would be eliminated.

Another aspect of the invention pertains to the supply of nutrients and carbon dioxide required for the growth of algae. During the first phase of heterotrophic cultivation, growth can be developed in batch or fed batch mode, apart from carbon source other nutrient like nitrogen, phosphorous or trace element which could become rate limiting at a point of time may also be provided. Particularly a fed batch process wherein nutrients are supplied at an incremental rate in response to cell mass and controlling other critical parameters like agitation, aeration, temperature would result in development of high cell density culture. Carbon source will be utilized for cell mass and carbon dioxide will be produced.
The carbon dioxide produced during heterotrophic growth will be supplied as a source of carbon for second phase of heterotrophic cultivation. Nutrients required for growth include nitrogen, phosphorous, salts like calcium, magnesium, manganese, zinc, copper, iron, potassium, cobalt, sodium chloride, was provided by recycling waste water such as municipal effluent or effluent from other industries. Additionally such nutrient may also be provided by using reject water from a water treatment plant or a cooling tower, supplemented with fertilizers or by using seawater supplemented with required nutrients for cultivation of marine algae.
After achieving target cell mass in the second phase cells were subjected to stress so as to achieve accumulation of lipid at high concentration in the cell. Such a physiological stress level will be achieved by allowing the algae cells to grow under condition of nutrient depletion. By this way it is proposed to achieve high overall productivity of lipids.
Once the algae culture has achieved a sufficient degree of growth, and lipid content the cells can be harvested, to isolate algae cells for further processing. Cell separation can be achieved by flotation, flocculation, filtration, and centrifugation or by combination of any of these methods.

Algae cell mass obtained can be further processed after drying or as a concentrated wet cell mass or slurry. Extraction of lipid from algae cells can be done by but not limited to a process of direct mechanical extraction using an expeller, solvent extraction or combination of both. After lipid extraction the algal cell mass can be recycled as a source of nutrient for the initial cultivation phase, as biofertilizer, as a feed supplement or as a feedstock for making biopellets for firing in a boiler. Another application- of extracted algal biomass could be .as a raw material for fermentation using suitable microorganism to produce bioethanol or biomethane.
Lipid or oil extracted from algae cells can be converted to fatty acid methyl ester by transesterification process and can be used as fuel as such or after blending with petroleum diesel.
Example 1: Collection & maintenance of of the algae strain
Liquid culture of Dunaliella tertiolecta CCMP 1320 was procured from the
Culture Collection of Marine Phytoplankton. Strains was further inoculated in F2-
Si containing salt as mention in; table -1, 2, 3, 4. Inoculate 5 ml algae, under
aseptically in to 100 ml flask containing 20 ml F2-Si medium, 8.0 pH was
maintained. Further expansion was carried out and used as inoculum for further
experiments.
Table - 1: Trace Element Stock:

Sr.No Ingredient Amount/L
1 Na2 EDTA 4.36 g
2 FeCI2.6H20 3.15 g
3 CuS04.5H20 0.01 g f
4 ZnS04.7H20 0.022 g
5 C0C12.6H20 0.01 g
6 MnCl2.4H20 0.18 g
7 Na2M04.2H20 0.006 g

Table - 2: Vitamin Stock:

Sr.No Ingredient i— Amount/L
1 Cyanocobalamin (Vitamin B12)g 0.0005 g
2 Thiamine HCL (Vitamin Bl) j 0.1 g
3 Biotin 0.0005 g
7
Table - 3: Artificial Sea water ( Add chemicals one after another):

Sr. No Ingredient Amount
/L
1 NaCl 23.926 g
2 Na2SO4 4.088 g
3 KC1 0.677 g
4 NaHCO3 0.196 g
5 KBr 0.098 g
6 H3BO3 0.026 g
7 NaF 0.003 g
8 SrCl2.6H2O 0.002 g
9 MgCl2.6H2O 0.053 g
10 CaCl2.2H2O 1.518 g"
Table - 4: Final Medium Composition:

Sr. No Ingredient Amount/L
1 Artificial Sea Water ILit
2 NaNOs 0.75 g
3 NaH2PO4.2H2O 0.00056 g
4 Vitamin Solution 1ml
5 Trace element Solution 1ml

Example 2: Heterotrophic cultivation
Medium as per the mention composition (table -5) in sterile water, five liter flask containing 2 1 prepared medium was autoclaved at 121 °C for 15 min. After cooling, add 15 % of the seven days old algae culture was inoculated aseptically. Kept the flask on shaker at 25 °C under dark condition. Glucose was added in various quantity (0-3 g/1) during experiment. Optical density was measured at 670 nm and dry weight (Zhu & Lee, 1997) of washed algae cells were recorded after every 24 hr to 384 hr andj.recorded (Table 6). Microscopy at each step performed for any other algae contamination and gram staining perform for any bacteria] contamination. Table - 5: Heterotrophic Medium Composition:

Sr.No Composition , - Amount g/1
1 NaCl 29.2
2 KNO3 1.0
3 MgCl2.H2O 1.5
4 MgS04.7H2O 0.5
5 KC1 0.2
6 CaCl2 0.2
7 K2HP04 0.045
8 Tris (Hydroxy mehyl) aminomethane 2.45
9 EDTA2Na 1.89 mg
10 ZnSO4.7H2O 0.087 mg
11 H3BO3 0.61mg
12 CoCI2.6H2O 0.015 mg
13 CuSO4.5 H2O 0.06 mg
14 MnCl2 0.23 mg
15 (NH4)6Mo7O24.4 H2O 0.38 mg
16 Fe (ni).EDTA 3.64 mg
17 Glucose 2.0 g
pH -8 adjust with 1 N HCL

Table - 6: Cultivation record under heterotrophic condition.

Sr.No Incubation period hour Addition of Glucose g/i OD at 670 Dry weight nm g/l
1 0 0 0.060 0.05
2 24 0.5 0.068 0.05
3 48 0.5 0.081 0.06,
4 72 0.5 0.111 0.15
5 96 1.5 0.184 0.38
6 120 1.5 0.246 0.59
7 168 1.5 0.351 1.22
8 192 2.0 0.450 1.91
2.57
9 216 2.0 0.568

10 240 2.0 0.590 3.23
11 264 2.5' 0.640 4.64
12 288 2.5 0.680 5.45
13 312 2.5 0.754 6.78
14 336 3.0 0.825 8.06
15 360 3.0 0.948 9.12
16 384 3.0 0.952 10.86
Example 3: Autotrophic cultivation
Medium was prepared as per the ingredient mention in table 7. One week old heterotrophically grown algae culture was further diluted by artificial sea water to fifty percent and used as inoculum. Five liter flasks containing two liter autotrophic cultivation medium containing 15% inoculum, under open lab condition. Flasks were kept on shaker at 25 °C under white fluorescent light. After every 24 hr culture was monitored by optical density at 670 nm and dry weight (Zhu & Lee, 1997) and recored (Table 8). Microscopy at each step performed for any other algae contamination. Fully grown culture further expanded to five liter in transparent plastic tubs to 1000 L agitated open race way pond under partial sun Hght.

Table - 7: Medium Composition for Autotrophic cultivation:

Sr.No Ingredient Amount/L
1 Artificial Sea Water 1 Lit
2 NaNO3 0.75 g
3 NaH2PO4.2H2O 0.00056 g
4 Trace element Solution 1ml
pH -8 adjust with 1 N HCL
Table -8: Cultivation record under Heterotrophic condition:

Sr. No. Incubation period (hr) OD at 670
nm Dry weight g/1
1 0 0.040 0.02
2 24 0.042 0.02
3 48 0.071 0.08
4 72 0.110 0.17
5 96 0.173 0.29
6 120 0.271 0.45
7 168 0.315 0.72
8 192 0.384 0.94
9 216 0.412 1.15
10 240 0.489 1.28'
11 264 0.542 1.53
12 288 0.581 1.62
13 312 0.590 1.88
14 336 0.612 2.05
15 360 0.640 2.24
16 384 0.654 2.27

Example 4: Harvesting of algae biomass.
Harvesting of cultured algae biomass was performed in alum for reduction of the concentrate algae cells and followed by centrifugation at 4000 rpm for 10 min. For large volume alum treatment was given in fabricated settling tank, followed by centrifugation.
Alum concentration was used from 100 mg/1 to 800 mg/1, and after 15 min packed cell volume was recorded ( Table - 9). Harvested biomass washed with water and kept for drying at 45°C in hot air oven.
Table -9: Packed cell volume at different concentration of alum.

Sr.No Alum concentration mg/1 Packed cell vol
1 100 mg 1.2%
2 200 mg 3.94 %
3 400 mg 5.26 %
4 600 mg 1.84%
5 800 mg 0.52 %

Example 5: Oil extraction from dried biomass and fatty acid profile.
Dried algae biomass further grinded into fine powder and lipids were extracted by a modified methods described by Xu and Beardall. Dry 10 g of fine algae powder were extracted twice with a mixture of WFI (water for injection) water Chloroform and methanol in 8:10:10 v/v respectively. Sonicate these mixture for 15 min. Filter the mixture with wharman glass microfiber filters. Add chloroform 10 ml and WFI 10 ml to filtrate and sonicate again for 10 in. The resultant solution was filtered and washed by 30 ml of 5 % NaCl solution, then the lower layer of CHC13 was separated and dried over anhydrous sodium sulphate. The solvent was removed through evaporation at 40°C under reduced pressure. Then, the total lipid were weighed and stored at - 20°C until analysis. Fatty acid profiling of extracted oil was performed as per the method described by Farag R.S. et al 1986.

Table -10: Fatty acid composition

SnNo Fatty acid Relative %
1 C10.0 0.13
2 C12.0 0.97
3 C14.0 0.77
4 C16:lw7 0.82
5 C16.0 4.91
6 C17:lw8 0.16
7 C17.0 0.08
8 C18.0 4.40
9 C18:lw9 0.80
10 C18:lw5 72.31
11 C18:l w7 0.25
12 C20:4w6 0.10
13 C20.0 0.19

Example 6: Transesterification and bio-diesel production
Extracted oil were used for transesterification for biodiesel production by methods described by National Biodiesel Board, 2002. USA (www.biodiesel.org). Catalytic mixture was prepared by mixing of 25 g KOH in 25 ml of methanol. The mixture of catalyst and methanol was poured into the algal oil in a conical flask and kept on shaker at 300 rpm for 3 h. After shaking solution kept for 16 h to settle the biodiesel and sediment layers clearly. After 16 h settling lower layer (glycerol, pigment, etc) were separate carefully, and upper layer (biodiesel) was washed with water until it was become clean. After washing biodiesel was dry using dryer and finally kept under the running fan for 12 h, and percentage of biodiesel calculate as shown in Table,- 11.
1 Table -1:1 Summary of trans csterification

Sr.No Parameter Value Percentage -of production
1 Oil 30 g -
2 Bio-diesel 27.6 g 92%
3 Glycerol & other pigment 2.4 g 8%
Example 6: Analysis of algae biomass after oil extraction
After oil extraction algae biomass dried in oven at 45°C Proximate analysis (Ash content, fixed carbon, moisture content, volatile mater ), Energy content, total sulphur and bulk density analysis were analyzed as described by James GS (James, G. S. 2005) and record values shown in table 12.

Table-12: Analysis of algae biomass

Sr. No Parameter Values
Proximate analysis
IMoisture 7.6%
2Ash 7.0%
3Volatile matter 71.5%
4Fixed Carbon 13.9 %
Energy Content
SHigher Heating Value (HHV) 4012 Kcal/kg
Other analysis
6Total Sulfur 0.67%
7Bulk density 643g/l
Example 7: Cultivation of Algae by C02 produced from heterotrophic cultivation.
As described in example 2, algae were grown under heterotrophic condition. Along with inoculated flask one flask without inoculation containing medium for autotrophic condition were prepared. Under aseptically "U" joint was established so produced C02 from heterotrophically grown flask was bubbled in flask containing autotrophic medium. Then, these flask is inoculate with 15% inoculum and recored the growth parameter. Due to C02 pH of medium become acidic which was set to 8.5 before inoculating algae. Along with C02 enriched flak controlled flask without C02 enrichment was used as control. And flask were observed for 120 hr. Encouraging growth of algae was found in the C02 enriched medium (Table 13).

Table - 13 Algae cultivation in medium enriched with CO2 produced during heterotrophic condition.

Sr, No Incubation period (hr) With CO2 Enrichment Without CO2 enrichment

OD at 670 nm Dry weight g/1 OD at 670 nm Dry weight g/1
1 0 0.033 0.02 0.031 0.02
2 24 0.047 0.02 0.044 0.02
3 48 0.070 0.08 0.064 0.07
4 72 0.124 0.22 0.89 0.15
5 96 0.145 037 0.127 0.23
6 120 0.216 0.41 0.184 0.29

We Claim:
1. A process for cultivation of suitable marine or fresh water micro photosynthetic cell/strain (called as Algae, a Latin plural) under heterotrophic to autotrophic growth condition using any type of fermenter for extraction of Lipid (oil) from dried mass of Algae for producing bio-diesel and Algae biomass used as bio-fertilizer, as high quality and high energy content biofuel such as pellets of biofuel for combustion to generate energy, pellets for feed and pellets for food and the said process comprising the following steps,
(a) A suitable marine Algae cell is cultivated in a closed system heterotrophic conditions using a suitable carbon source in a suitable Photobioreactor installed indoors;
(b) After achieving high cell concentration/density, a portion of the growth inoculum is transferred for photoautotrophic cultivation in a Photobioreactor or an open system;
(c) Carbon dioxide produced during heterotrophic growth is supplied as a source of carbon for heterotrophic cultivation;
(d) Nutrients required for growth was provided by recycling waste water such as municipal or other industrial effluent;
(e) After achieving target Algae cell mass, Algae cell were subjected to stress so as to achieve accumulation of Lipid at high concentration in the Algae cell;

(f) Algae culture has achieved a sufficient degree of growth and Lipid content, Algae cell can be harvested, to isolate Algae cell for further processing. Algae cell separation can be achieved by flotation, flocculation, filtration, and centrifugation or by combination of any of these methods;
(g) Algae cell mass obtained can be further processed after washing, drying or as a concentrated wet mass or as slurry as per requirement;
(h) Algae cell mass being high quality and high energy content biomass obtained at step (g) used for manufacturing of biofuel such as bio-diesel, as pellets of biofuel, as bio-fertilizer, as pellets for feed and pellets for food.
2. A process as claimed in claim 1, wherein Lipid from A\gae cell is extracted by a process of direct mechanical extraction using an expeller, solvent extraction or combination of both.
3. A process as claimed in claim 1, wherein Lipid or oil extracted from Algae cell is used to produce fatty acid methyl ester by transesterification process and can be used as biofuel as such or after blending with petroleum diesel.
4. A process as claimed in claim 1, wherein after Lipid extraction, Algae cell mass can be recycled as a source of nutrient for the initial cultivation phase, as bio-fertilizer, as a feed supplement or as a feed for making bio-pellets as biofuel of different shapes, odor and color.
: i
5. A process as claimed in claim 1, wherein extracted Algal cell mass is used
as raw material for fermentation using suitable micro-organism to produce
bio-ethanol and/or bio-methane.

6. A process as claimed in claim 1, wherein Algae cultivation is not limited to Dunaliella tertiolecta strain but cultivation methods can be used for any type of marine or fresh water micro Algae and uses thereof.
7. A process as claimed in claim 1, wherein heterotrophic cultivation can be through any dypes of cultivating vessels but not limited to fermenter, brown glass bottle, plastic bottle and flaks of any shape and size.
8. A process as claimed in claim 1, wherein autotrophic cultivation can be
. through any types but not limited to open system like circular pond,
raceway pond or closed system or through Photobioreactor of any shape and length.
9. A process for cultivation of suitable marine or fresh water micro
photosynthetic cell, called Algae under heterotrophic to autotrophic growth
condition for producing as bio-diesel, bio-fertilizer and high quality and
high energy content biofuel such as pellets of biofuel, pellets for feed and
pellets for food such as herein described with reference to foregoing
examples.