FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patents [Amendment] Rules, 2006
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION
Mixotrophic Cultivation of Microalgae for the Production of Biofuel
2. APPLICANT
NAME : Indian Oil Corporation Limited
NATIONALITY : IN
ADDRESS : G-9, Ali Yavar Jung Marg, Bandra (East), Mumbai-400 051, India
3. PREAMBLE TO THE DESCRIPTION
Complete
The following specification describes the invention and the manner in which it is to be performed:

TRAINS AND USE THEREOF FIELD OF INVENTION
The present invention relates to a method of cultivation of micro-algae for the production of biofuel. Specifically, the present invention also relates to a method of mixotrphic cultivation of algae.
BACKGROUND OF INVENTION
Algae can be grown by both autotrophic and heterotrophic modes. The autotrophic mode has constraint of low biomass productivity due to light limitation at high cell densities and dark coloured (opaque) wastewaters. The heterotrophic mode of cultivation requires external organic carbon source and this is done under sterilized conditions, which leads to higher cost. Hence, there is need for development of algae cultivation system which overcomes deficiencies of autotrophic and heteretrophic mode of algae cultivation and has the benefits of open pond system with utilization of higher CO2 concentration and higher growth under light and dark conditions using inexpensive resources as external carbon sources.
EP1707641A2 provides methods employing iterative cycles of recombination and selection/screening for evolution of whole cells and organisms toward acquisition of desired properties. Examples of such properties include enhanced recombinogenicity, genome copy number, and capacity for expression and/or secretion of proteins and secondary metabolites.
The technique of genome shuffling combines the advantage of multiparental crossing allowed by DNA shuffling together with the recombination of entire genomes normally associated with conventional breeding, or through protoplast fusion that increases the recombination process.
US6531646 relate to method for the genetic modification and improvement of Porphyra species utilizing protoplast fusion is disclosed. The method of the invention features the use of conchoporangial branch conchocelis for at least one of the sources of protoplasts for

protoplast fusion. Protoplasts fusion method involves either a chemical fusing agent like polyethylene glycol (PEG) or electrofusion.
US20100162620 provides systems and processes for optimizing each type of algal-based production of bioproducts (such as oil) separately and independently, thereby improving overall production of oil, lipids and other useful products. This process is advantageous because it allows the optimization of the individual steps and growth phases in the production of oil from biomass. This also allows the use of different feedstocks and growth conditions for the different process steps.
US20120028338 to mixed algal compositions able to proliferate on industrial waste water, and to methods of obtaining an algal biomass from such cultures for use in generating a biofuel. The invention further encompass methods of cultivating mixed populations of freshwater and marine alga comprising a plurality of genera and species to provide a biomass from Which may be extracted lipids, or converted into biodiesel by such procedures as pyrolysis.
Deng et al. (2011), African J. Agri.Res. Vol.6(16), pp. 3768-3774 is a scientific publication which relates to effects of selective medium on lipid accumulation of chlorellas and screening of high lipid mutants through ultraviolet mutagenesis
Bhatnagar et al. (2011), Applied Energy, Vol.88, Issue 10, pp. 3425-3431 relates to mixotrophic growth potential of native microalgae namely Chlamydomonas globosa, Chlorella mimutissima and Scenedesmus bijuga isolated after long-term enrichments from industrial wastewater and cultured in media supplemented with different organic carbon substrates and wastewaters. The mixotrophic growth of these microalgae resulted in 3–10 times more biomass production relative to phototrophy.
Vigeolas et al. (2012), J. of Biotech. Vol.162, Issue 1, pp.3-12 is a scientific publication which relates to isolation and partial biomass characterization of high triacylglycerol (TAG) mutants of Chlorella sorokiniana and Scenedesmus obliquus, two algal species considered as potential source of biodiesel.

Pittman J.K et al. (2010), Bioresour. Tech. Vol.102, Issue 1, pp-17-25 is another scientific publication which relates to the potential of microalgae as a source of renewable energy for microalgal biofuel production. Wastewaters derived from municipal, agricultural and industrial activities potentially provide cost-effective and sustainable means of algal growth for biofuels.
Mohan S.V et al. (2011), Bioresour. Tech. Vol.102, Issue 2, pp-1109-1117 is also a scientific publication which provides an overview on the possibility of using mixed microalgae existing in ecological water-bodies for harnessing biodiesel. Microalgal cultures from five water-bodies are cultivated in domestic wastewater in open-ponds and the harvested algal-biomass was processed through acid-catalyzed transesterification.
Mixotrophic Algae and Their Consortia for the Production of Algae Biofuel Feedstock in Wastewater-fed Open Ponds (2010). University of Georgia relates to the use of mixotrophic algae to decontaminate heavily polluted wastewaters and, simultaneously, provide high-yields of starting materials for the production of biofuels and organic chemicals of commercial interest (http://www.ibridgenetwork.org/ugarf/mixotrophic-algae-and-their-consortia-for-the-production-of-a)
Pittman et al (2010), Bioresource Technology, Volume 102(1), January 2011; pages 17-25, relates to the potential of microalgae as a source of renewable energy has received considerable interest, but if microalgal biofuel production is to be economically viable and sustainable, further optimization of mass culture conditions are needed. Wastewaters derived from municipal, agricultural and industrial activities potentially provide cost-effective and sustainable means of algal growth for biofuels. In addition, there is also potential for combining wastewater treatment by algae, such as nutrient removal, with biofuel production. Here we will review the current research on this topic and discuss the potential benefits and limitations of using wastewaters as resources for cost-effective microalgal biofuel production.
Mohan SV et al. (2011) Bioresource Technol. Volume 102(2): pages 1109-17 relates to Biodiesel as an eco-friendly fuel is gaining much acceptance in recent years. This communication provides an overview on the possibility of using mixed microalgae existing in ecological water-bodies for harnessing biodiesel. Microalgal cultures from five water-bodies are cultivated in domestic wastewater in open-ponds and the harvested algal-biomass was

processed through acid-catalyzed transesterification. Experiments evidenced the potential of using mixed microalgae for harnessing biodiesel. Presence of palmitic acid (C16:0) in higher fraction and physical properties of algal oil correlated well with the biodiesel properties. Functional characteristics of water-bodies showed to influence both species diversity and lipid accumulation. Microalgae from stagnant water-bodies receiving domestic discharges documented higher lipid accumulation. Algal-oil showed to consist 33 types of saturated and unsaturated fatty acids having wide food and fuel characteristics. Simultaneous wastewater treatment was also noticed due to the syntrophic association in the water-body microenvironment. Diversity studies visualized the composition of algae species known to accumulate higher lipids
The prior art discloses random mutagenesis of algal strain for lipid and biomass productivity. Still, the major drawback in the microalgae for use in biofuel is the nonavailability of suitable strains and cost effective method for cultivation and harvesting. Hence, there is need to develop cheaper methods for cultivation and fast growing strains with tolerance to adverse environmental conditions and ability to utilize high concentrations of CO2 and lipid productivity with composition suitable for making fuels.
Hence, there is need for development of algae cultivation system which overcomes deficiencies of autotrophic and heteretrophic mode of algae cultivation and has the benefits of open pond system with utilization of higher CO2 concentration and higher growth under light and dark conditions using inexpensive resources as external carbon sources.
SUMMARY OF THE INVENTION
Accordingly the main embodiment of the present invention provides a method of mixotrophic cultivation said method comprising:
(a) preparing a mixotroph, said mixotroph being a mutated microalgae strain;
(b) adding the mixotroph of step (a) in a mixotrophic source/medium;
(c) adding a selected plant extract in the growth media;
(d) allowing the growth and multiplication of mixotroph in the mixotrophic source/medium under open pond conditions;
(e) determining the biomass and/or oil content from mixotroph.

Another embodiment of the present invention provides for a method wherein the step (b) mixotroph microalgae is a single microalgae strain or combination of microalgae strains.
Another embodiment of the present invention provides for a method, wherein step (b), the mixotrophic microalgae is selected from the group comprising Chlorella vulgaris IOC-106, Chlorella vulgaris IOC-112, Chlorella sp. IOC-114, Scenedusmus sp IOC-110 and Synechocosccus sp. IOC 111.
Another embodiment of the present invention provides for a method, wherein step (b) the mixotrophic source or medium is selected from the group comprising lighted open pond, controlled stirred tank, photo-bioreactor, in vitro culture medium comprising growth medium with organic and/or inorganic carbon source, hydrocarbon processing plant, kitchen effluent, automobile industry effluent, fruit processing plant effluent, dairy effluents, municipal waste water, sewage treatment plant, biogas plant slurry, agricultural residues, industrial waste having acetate, sugars , low or high carbon source and/or the like.
Another embodiment of the present invention provides for a method, wherein step (c) the plant extract has anti-microbial property.
Another embodiment of the present invention provides for a method, wherein the mixotrophic cultivation is carried out in lighted open pond, controlled stirred tank, photo-bioreactor, in vitro culture medium comprising growth medium with organic and/or inorganic carbon source, hydrocarbon processing plant, kitchen effluent, automobile industry effluent, fruit processing plant effluent, dairy effluents, municipal waste water, sewage treatment plant, biogas plant slurry, agricultural residues, industrial waste having low or high carbon source and/or the like.
Another embodiment of the present invention provides for a process of producing biofuel from the mixotrophic cultivation of mixotrophic microalgae, said method comprising the steps of :
(a) preparing a mixotroph, said mixotroph being a mutated microalgae strain;
(b) adding the mixotroph of step (a) in a mixotrophic source/medium;
(c) adding a selected plant extract in the growth media;

(d) allowing the growth and multiplication of mixotroph in the mixotrophic source/medium; and
(e) isolating the mixotroph from the mixotrophic source/medium;
(f) obtaining the biofuel.
Another embodiment of the present invention provides for a process, wherein the step (a) mixotroph microalgae is a single microalgae strain or combination of microalgae strains.
Another embodiment of the present invention provides for a process, wherein step (b), the mixotrophic microalgae is selected from the group comprising the mixotrophic microalgae is selected from the group comprising Chlorella vulgaris IOC-106, Chlorella vulgaris IOC-112, Chlorella sp. IOC-114, Scenedusmus sp IOC-110 and Synechocosccus sp. IOC 111.
Another embodiment of the present invention provides for a process, wherein step (b) the mixotrophic source or medium is selected from the group comprising lighted open pond, controlled stirred tank, photo-bioreactor, in vitro culture medium comprising growth medium with organic and/or inorganic carbon source, hydrocarbon processing plant, kitchen effluent, automobile industry effluent, fruit processing plant effluent, dairy effluents, municipal waste water, sewage treatment plant, biogas plant slurry, agricultural residues, industrial waste having low or high carbon source and/or the like.
Another embodiment of the present invention provides for a process, wherein step (c) the plant extract has anti-microbial property.
Another embodiment of the present invention provides for a process, wherein the mixotrophic cultivation is carried out in lighted open pond, controlled stirred tank, photo-bioreactor, in vitro culture medium comprising growth medium with organic and/or inorganic carbon source, hydrocarbon processing plant, kitchen effluent, automobile industry effluent, fruit processing plant effluent, dairy effluents, municipal waste water, sewage treatment plant, biogas plant slurry, agricultural residues, industrial waste having low or high carbon source and/or the like.
Another embodiment of the present invention provides for a method of mixotrophic cultivation in an open pond, said method comprising the steps of:

(a) preparing a mixotroph, said mixotroph being a mutated strains of microalgae;
(b) adding the mixotroph of step (a) in an open pond;
(c) adding a selected plant extract in the growth media;
(d) allowing the growth and multiplication of mixotroph in the mixotrophic source/medium under open pond conditions;
(e) determining the biomass and/or oil content from mixotroph.
Another embodiment of the present invention provides for a method, wherein the step (b) mixotroph microalgae is a single microalgae strain or combination of microalgae strains.
Another embodiment of the present invention provides for a method, wherein step (b), the mixotrophic microalgae is selected from the group comprising Chlorella vulgaris IOC-106, Chlorella vulgaris IOC-112, Chlorella sp. IOC-114, Scenedusmus sp IOC-110 and Synechocosccus sp. IOC 111.
Another embodiment of the present invention provides for a method, wherein step (b) the open pond is selected from the group comprising lighted open pond, controlled stirred tank, photo-bioreactor tank, hydrocarbon processing plant pond, kitchen effluent storage pond, automobile industry effluent treatment plant pond, municipal waste water pond and pond of sewage treatment plant having acetate, sugars, low or high carbon source and/or the like.
Another embodiment of the present invention provides for a method, wherein step (c) the plant extract has anti-microbial property.
Another embodiment of the present invention provides for a method, wherein step (c) the plant extract stimulate growth of mixotrophic microalgae.
Another embodiment of the present invention provides for a method, wherein the mixotrophic cultivation is carried out in lighted open pond, controlled stirred tank, photo-bioreactor tank, hydrocarbon processing plant pond, kitchen effluent storage pond, automobile industry effluent treatment plant pond, municipal waste water pond and pond of sewage treatment plant having acetate, sugars, low or high carbon source and/or the like.

Another embodiment of the present invention provides for a method, wherein the mixotrophic cultivation is useful for production of biofuel.
Another embodiment of the present invention provides for a use of mutated mixotrophic microalgae strain for mixotrophic cultivation.
Another embodiment of the present invention provides to a use of mutated mixotrophic microalgae strain for mixotrophic cultivation, wherein the mutated mixotrophic microalgae is selected from the group comprising Chlorella vulgaris IOC-106, Chlorella vulgaris IOC-112, Chlorella sp. IOC-114, Scenedusmus sp IOC-110 and Synechocosccus sp. IOC 111 alone or in combination thereof.
Another embodiment of the present invention provides for a use of mutated mixotrophic microalgae strain for mixotrophic cultivation, wherein the mixotrophic cultivation is carried out in mixotrophic source or mixotrophic medium selected from the group comprising of lighted open pond, controlled stirred tank, photo-bioreactor, in vitro culture medium comprising growth medium with organic and/or inorganic carbon source, hydrocarbon processing plant, kitchen effluent, automobile industry effluent, fruit processing plant effluent, dairy effluents, municipal waste water, sewage treatment plant, biogas plant slurry, agricultural residues, industrial waste having acetate, sugars , low or high carbon source and/or the like.
Another embodiment of the present invention provides for a use of mutated mixotrophic microalgae strain for mixotrophic cultivation, wherein the mixotrophic cultivation is carried out in mixotrophic source or mixotrophic medium selected and wherein the mixotrophic medium also comprises of plant extract which stimulates growth of mutated mixotrophic microalgae and also has anti-microbial property.
Another embodiment of the present invention provides for a use of mutated mixotrophic microalgae strain for mixotrophic cultivation, wherein the mixotrophic cultivation is carried out in lighted open pond, controlled stirred tank, photo-bioreactor, in vitro culture medium comprising growth medium with organic and/or inorganic carbon source, hydrocarbon processing plant, kitchen effluent, automobile industry effluent, fruit processing plant

effluent, dairy effluents, municipal waste water, sewage treatment plant, biogas plant slurry, agricultural residues, industrial waste having low or high carbon source and/or the like.
Another embodiment of the present invention provides for a use of mutated mixotrophic microalgae strain for mixotrophic cultivation in an open pond using plant extract for production of biofuel.
Another embodiment of the present invention provides for a use of mutated mixotrophic microalgae strain for mixotrophic cultivation in an open pond using plant extract for production of biofuel, wherein the plant extract is useful in stimulating growth of mutated mixotrophic microalgae strain and also as anti-microbial agent.
Another embodiment of the present invention provides for a use of mutated mixotrophic microalgae strain for mixotrophic cultivation in an open pond using plant extract for production of biofuel, wherein the mutated mixotrophic microalgae is selected from the group comprising Chlorella vulgaris IOC-106, Chlorella vulgaris IOC-112, Chlorella sp. IOC-114, Scenedusmus sp IOC-110 and Synechocosccus sp. IOC 111 alone or in combination thereof.
Another embodiment of the present invention provides for a use of mutated mixotrophic microalgae strain for mixotrophic growth in an open pond using a mixotrophic medium and a plant extract, wherein the plant extract stimulates growth of the microalgae and reduces microbial contamination.
Another embodiment of the present invention provides for a mixotrophic cultivation using mutant microalgae strain alone or in combination, wherein the mutant microalgae carries both autotrophic and heterotrophic modes of cultivation.
Another embodiment of the present invention provides for algae cultivation system comprising atleast one mutant microalgae strain capable of both autotrophic and/or heterotrophic mode of growth in an open pond system wherein the mutant microalgae strain utilizes higher CO2 concentration and higher growth under light and dark conditions using inexpensive resources as external carbon sources.

Another embodiment of the present invention provides for development of algae cultivation system which overcomes deficiencies of autotrophic and heteretrophic mode of algae cultivation and has the benefits of open pond system with utilization of higher CO2 concentration and higher growth under light and dark conditions using inexpensive resources as external carbon sources.
Another embodiment of the present invention provides for method of algae cultivation using at least one mutant novel strain of microalgae alone or in combination which overcomes deficiencies of autotrophic and heteretrophic mode of algae cultivation and has the benefits of open pond system with utilization of higher CO2 concentration and higher growth under light and dark conditions using inexpensive resources as external carbon sources.
Another embodiment of the present invention provides for use of at least one mutant novel strain of microalgae alone or in combination for algae which overcomes deficiencies of autotrophic and heteretrophic mode of algae cultivation and has the benefits of open pond system with utilization of higher CO2 concentration and higher growth under light and dark conditions using inexpensive resources as external carbon sources.
DESCRIPTION OF INVENTION
While the invention is susceptible to various modifications and/or alternative processes and/or compositions, specific embodiment thereof has been shown by way of example in the drawings and tables and will be described in detail below. It should be understood, however that it is not intended to limit the invention to the particular processes and/or compositions disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternative falling within the spirit and the scope of the invention as defined by the appended claims.
The graphs, tables, formulas, protocols have been represented where appropriate by conventional representations in the drawings, showing only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.

The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.
The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that one or more processes or composition/s or systems or methods proceeded by "comprises... a" does not, without more constraints, preclude the existence of other processes, sub-processes, composition, sub-compositions, minor or major compositions or other elements or other structures or additional processes or compositions or additional elements or additional features or additional characteristics or additional attributes.
Definition:
For the purposes of this invention, the following terms will have the meaning as specified therein:
As used herein, the terms “Low quality water” or “Poor quality water” or “Water containing heavy metals”, when used in the context of the present invention refers water which cannot be used directly for drinking, agriculture, human or animal consumption or other purpose. Such water is a was waste from industrial effluents, water containing heavy metals, hydrocarbons, water with high salinity, sewage water, reject water of reverse osmosis (RO) plant, river water with higher COD and BOD, water with coloring agent and other industry effluent etc. Further in context of the present invention the “Low quality water” or “Poor quality water” also includes water, which is or is found to be undesirable and harmful to human, anima or aquatic life in resepct of drinking, living or for any other purpose related an organism’s survival or need.
As used herein, the term “High Value Products” when used in the context of the present invention refers to vitamins, pigments, anti-oxidants, omega-3 & omega-6 polyunsaturated fatty acids, DHA or EPA.
As used here, the term “Mutagenizing Agent/s or Mutagenic Agent/s or Mutagens”, when used in the context of the present invention refers to agent/s a chemical, ultraviolet light, or

a radioactive element, that can induce or increase the frequency of mutation in an organism.
As used here the term “Strain/s” or “Novel Strains”, when used in the context of the present invention refers to novel/new variants/strains of the microalgae produced or developed by the process of the present invention. These variants are genetically different in their control or parent or original forms. These variants are artificially developed and survive and perform better at extreme environmental conditions.
As used here the term “Chemical oxygen Demand or COD”, when used in the context of present invention refers to the test commonly used to indirectly to measure the amount of organic compounds in water. It determines the amount of oxygen required to oxidize an organic compound to carbon dioxide, ammonia, and water.
As used here the term “Biological Oxygen Demand or BOD”, when used in the context of the present invention refers to amount of dissolved needed by aerobic biological organism in a body of water to break down organic material present in a given water sample at certain temperature over a specific period.
As used here the term “Biofuells”, when used in the context of the present invention refers to a fuel that uses energy from a carbon fixation produced from microalgae. These fuels are made from a microalgae biomass conversion.
As used herein the term “Mutagenesis or mutagenized”, when used in the context of the present invention refers to a process by which the genetic information of an organism is changed in a stable manner, resulting in a mutation. In context of the present invention it achieved experimentally using laboratory procedure by exposing the microalgae to various mutagens.
As used herein the term “Protoplast fusion or Somatic Fusion”, when used in context of the present invention refers to genetic modification of microalgae from same species by fusing their protoplasts (for e.g. pooled samples of C. vulgaris fused with another pooled samples of C.vulgaris) to form a new hybrid plant with the characteristics of both, a somatic hybrid.

As used herein the term “Mutant strains” or “mutated strains” or “mutated microalgae strains”, when used in context of the present invention refers to modified microalgae by a mutagen and protoplast fusion, wherein the fusion has been carried out in the microalgae of same species or pool of microalgae of same species. The mutant strains of the present invention do not in any manner or meant to refer to transgenic mutants or transgenic microalgae or transgenics or transgenic material. In the present invention the strains do not comprise genes of any unrelated higher life-form/s or organism/s or unrelated microorganism/s. In the present invention even the microalgae from different genus have not be crossed or nor any genetical material from different microalgae genus have been fused. The microalgae as referred herein in context of the present invention are capable of mixotrophic cultivation and/or mixotrophic growth in mixotrophic medium or mixotrophic source.
As used herein the term “Mixotrophic cultivation”, when used in the context of the present invention refers to cultivation wherein both phototrophy/phototrophic and heterotrophy/heterotrophic processes or conditions are driven or carried out independent of each other and/or simultaneously by microalgae strains of the present invention. Further the mixotrophic cultivation of the present invention is also useful for production of biofuels. The mixotrophic cultivation in context of the present invention wherein the algae cultivation overcomes deficiencies of autotrophic and heteretrophic mode of algae cultivation and has the benefits of open pond system with utilization of higher CO2 concentration and higher growth under light and dark conditions using inexpensive resources as external carbon sources.
As used herein the term “Mixotrophs or “Mixotrophic microalgae” or “mutated mixotrophic strains” or “mutated mixotrophic microalgae strains”, when used in the context of the present invention refers to mutated microalga strain/s of present invention which are capable of carrying out mixotrophic cultivation under both phototrophic and/or heterotrophic conditions. Further the mixotrophs of the present invention are also capable of carrying out mixotrophic cultivation for production of biofuels. The mixotrophic microalgae of present invention during the mixotrophic cultivation can simultaneously be cultivated on inorganic and organic carbon source for the production of biofuel. The mixotrophs or mixotrophic microalgae in the present invention can be used alone or in

combination as mixture or composition for mixotrophic cultivation. These strains further have capability of stimulated growth in the presence of plant extract as described in the present invention.
As used herein the term “Phototrophy” or “Phototrophic” or “Phototrophic process”, when used in the context of the present invention refers to a process by which microalgae strains of the present invention which can use energy from sunlight or inorganic compounds to produce organic compounds such as carbohydrates, fats, and proteins from inorganic carbon dioxide.
As used herein the term “Heterotrophy” or “Heterotrophic” or “Heterotrophic process”, when used in the context of the present invention refers to a process by which microalgae strains of the present invention which use organic compounds or organic carbon source or organic carbon dioxide to produce organic compounds such as carbohydrates, fats, and proteins.
As used herein the term “Mixotrophic source” or “Mixotrophic medium” when used in the context of the present invention refers to lighted open pond, controlled stirred tank, photo-bioreactor, in vitro culture medium comprising growth medium with organic and/or inorganic carbon source, an open pond system, hydrocarbon processing plant, kitchen effluent, automobile industry effluent, fruit processing plant effluent, dairy effluents, municipal waste water, sewage treatment plant, biogas plant slurry, agricultural residues, industrial waste having low or high carbon source etc. More specifically the mixotrophic source or mixotrophic medium is source which provides the mixotrophs with inorganic or organic carbon source, thereby allowing higher carbon mitigation. Further the mixotrophic source or mixotrophic medium of the present invention in addition to carbon source as mentioned above also has plant extract as anti-microbial source.
As used herein the term “biobased” or “biobased products” when used in the context of the present invention refers to biobased products are product of biological origin like in this case may be lipids, neutraceuticals, colour, antioxidant etc.
As used herein the term “Plant extract”, when used in the context of the present invention refers to extract which is prepared by a method/process as described in example 3 of the

present invention. The plant extract described as herein stimulates the growth of mutated mixotrophic microalgae strains in the mixotrophic medium and/or in an open pond. The plant extract described as herein has anti-microbial property or functions as an anti-microbial agent to prevent contamination and/or growth of microbe (i.e. bacteria or fungi, etc.) which may grow in the mixotrophic medium or in the open pond when the mutated mixotrophic microalgae are used for mixotrophic cultivation or mixotrophic growth.
The present invention provides for method of algae cultivation system which overcomes deficiencies of autotrophic and heteretrophic mode of algae cultivation and has the benefits of open pond system with utilization of higher CO2 concentration and higher growth under light and dark conditions using inexpensive resources as external carbon sources.
The present invention also provides for overcoming the deficiencies of autotrophic and heteretrophic mode of algae cultivation and has the benefits of open pond system with utilization of higher CO2 concentration and higher growth under light and dark conditions using inexpensive resources as external carbon sources.
The present invention also provides the use of novel mutant strains of microalgae which are effective and surprisingly efficient in overcoming deficiencies of autotrophic and heteretrophic cultivation which is often seen with microalgae in general due to light limitation at high cell densities and dark coloured (opaque) wastewaters. The advantage of cultivation or mixotrophic cultivation of the novel mutant strains of microalgae is such that they can be surprisingly and efficiently be grown well in open pond systems, wherein they utilize higher CO2 concentration and higher growth under both light and dark conditions using inexpensive resources as external carbon sources for biofuel production.
The microalgae strains were collected from the selected from various locations in India as described Nannochloropsis oculata IOC-105 was collected from Aeration tank of effluent treatment plant of Indian Oil Panipat refinery, Panipat-Haryana, India, Chlorella vulgaris IOC-106 was collected from soil of Indian Oil Corporation, R&D Centre, Faridabad, India, Chlorella vulgaris IOC-112 was collected from Aeration tank, Effluent Treatment Plant, Indian Oil, Panipat Refinery, Panipat –Haryana-India; Chlorella sp. IOC-114 was collected from Yamuna River, near Kalindi Kunj-Delhi; Scenedusmus sp IOC-110 Aeration tank of effluent treatment plant of Indian Oil Panipat refinery, Panipat-Haryana, India and

Synechocosccus sp. IOC 111 was collected from soil of Indian Oil Corportation, R&D Centre, Faridabad.
The microalgae strains namely Nannochloropsis oculata (referred to herein as IOC-105) which was deposited with Culture Collection of Algae and Protoza (CCAP), UK under
Budapest Treaty on , 2013 and given accession number ;
Chlorella vulgaris (referred to herein as IOC-106) which was deposited with Culture
Collection of Algae and Protoza (CCAP), UK under Budapest Treaty on , 2013
and given accession number ; Chlorella vulgaris (referred to herein as
IOC-112) which was deposited with Culture Collection of Algae and Protoza (CCAP), UK
under Budapest Treaty on , 2013 and given accession number
;, Chlorella sp. (referred to herein as IOC-114) which was deposited with
Culture Collection of Algae and Protoza (CCAP), UK under Budapest Treaty on
, 2013 and given accession number ; Scenedusmus sp
(referred to herein as IOC-110) which was deposited with Culture Collection of Algae and
Protoza (CCAP), UK under Budapest Treaty on , 2013 and Synechocosccus sp.
(referred to herein as IOC 111) which was deposited with Culture Collection of Algae and
Protoza (CCAP), UK under Budapest Treaty on , 2013
The improved strains so developed are capable of growing in diverse harsh environments, specifically various low quality waters, low value organic sources, open pond/s, sewage treatment plant, biogas plant slurry, dairy effluents, municipal wastewater, etc to obtain biofuel and other value added products.
Thus the present invention provides for microalgae strains which are capable of carrying out both phototrophic and heterotrophic (i.e. mixotrophic cultivation) cultivation simultaneously. The unique characteristic feature of the microalgae of the present invention is such that they utilize both inorganic and organic carbon substrates for carrying out mixotrophic cultivation.
These microalgae strains of the present invention can carry out the mixotrophic cultivation by utilizing the both inorganic and organic carbon source to drive an additive or synergistic effect of both phototrophic and heterotrophic processes thereby enhancing the productivity of biofuels in waster waters and low quality water.

Another unique attribute of the present invention is the use of plant extract which functions as an anti-microbial agent, during the mixotrophic cultivation. During the mixotrophic cultivation of microalgae strains in any of the waste-water body or ponds or low quality water, there are high chances of microbial contamination, however, the plant extract prepared in the present invention having anti-microbial properties that prevent the growth and harboring of harmful microbial agents (for e.g. bacteria or fungi), thereby enhancing the mixotrophic cultivation of microalgae and their growth. Surprisingly, it has been found the plant extract used for the purpose does not inhibit the growth of mixotrophic microalgae but only acts against the microbial contamination. The plant extract, which is an alcoholic extract functions by minimizing the consumption of sugar by other undesired microbes. This extract not only inhibits the undesired microbes in the open pond or any un-sterilized reactors but also improves the growth of mixotrophs.
Thus, according to the present invention further provides a method for mixotrophic cultivation of algae including isolation and adaption of microalgae for cultivation under mixotrophic conditions, modification of strains for improved characteristic, cultivation under lighted open pond, controlled stirred tank, photobioreactor using inorganic like CO2 and organic source, addition of an alcoholic plant extract, harvesting of the algal biomass cultivated and cell disruption and oil extracting methods.
A unique characteristic feature of the present invention is that the during the mixotrophic cultivation the microalgae can grow extremely and surprisingly well in both inorganic and organic carbon source depending on the state of environment. The mixotrophic cultivation with the microalgae of present invention is such that it can be carried out both under controlled or open environment. Under the controlled environment external supply of growth medium (comprising both organic and/or inorganic carbon source) acts as the source of feedstock for production of algae mediated bio-based product. Under the natural environment condition both naturally present as well as externally provided inorganic or organic carbon source acts as the source of feedstock for the production of algae mediated bio-based product. or provide growth conditions or growth environment. The advantage of this is that the during the mixotrophic cultivation depending upon on the conditions of the source (i.e. whether the source of cultivation is natural body or controlled environment) the microalgae can utilization of CO2 act as a feedstock for production of algae mediated bio-based product.

According to the invention, further also provides for the oil/lipid extracted from the algae after mixotrophic cultivation is further converted to biofuel and/ or further down streamed to value added product. The algal residue after oil extraction can be used for production of gaseous and liquid biofuel using specific set of microbes. These microbes included but not limited to anaerobic bacteria of genus Clostridium and aerobic of genus Saccharomyces and Pichia.
The microalgae strains of the present invention are mutant strains of microalgae obtained by mutagenic and/or chemostat mediated adaptation. The microalgae strains were improved by using known process of mutagenesis using chemical and radiation mutagen followed by known process of recursive mutagenesis and protoplast fusion. The pooled mutant population is shuffled by homologous recombination using protoplast fusion followed by selecting improved progenies and subjecting the same to next round of selection. This process was carried out for six cycles
The microalgae were mutagenized by chemical (EMS, mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, benzo(a)pyrene and 4-nitroquinoline 1-oxide) and radiation mutagen (UV, gamma-rays) or their combination. The pooled mutant population is shuffled by homologous recombination using protoplast fusion followed by selecting improved progenies and subjecting the same to next round of selection. This process was carried out for six cycles. This accelerates directed evolution through recursive recombination of improved progeny, thereby improving multiple traits. Strains with higher growth, lipid productivity, salt, pH and heavy metal tolerance and with ability to grow under mixotrophic conditions were obtained. This accelerates directed evolution through recursive recombination of improved progeny, thereby improving multiple traits like higher growth and lipid productivity, carbon di-oxide utilization ability, biomas desired lipid composition salt, pH, temperature and heavy metal tolerance The composition of the lipid obtained from modified strains was suitable for biodiesel production.
For the process of mixotrophic cultivation pure culture alone and/or in different combination and ratio (cells numbers 0.01-5:1-100) were grown in wastewaters, low quality water, rainwater, bore well water, canal water and other water with and without supplementing carbon source in presence of CO2, nutrient mixture and light. These fast

growing algae utilize higher amount of organic and inorganic carbon source and dominates the algal population, if any present in water.
According to the present invention, the media which is used for cultivation of media contained nitrogen, phosphorus, CO2 and organic carbon source for growth along with trace element and vitamin source. According to the invention the microalgae strains of the present invention are used to sequester carbon di-oxide from various sources like flue gas, bio-gas plant exhaust and other source of concentrated CO2 having CO2 in the range of 0.05-100%, thereby helping in abating pollution.
According to the present invention, various sources of wastewater may be used for mixotrophic cultivation that includes effluent of hydrocarbon processing plant, kitchen effluent, automobile industry effluent, fruit processing plant effluent, dairy effluents, municipal waste wastewater, sewage treatment plant, biogas plant slurry etc. The organic material available in above sources of water also serves as a source of carbon for algal growth. Further, besides the above mentioned carbon sources, other sources of organic carbon used for cultivation include lignocelluloses biomass hydrolysate, plant starch, molasses, glycerol from biodiesel plant etc. in presence of carbon di-oxide from various sources like flue gas, bio-gas plant exhaust and other source and sunlight. The nitrogen, phosphorus and micronutrient sources which studied included corn steep liquor, fertilizers, yeast extract, poultry litter, soil extract etc.
Accordingly the main embodiment of the present invention provides a method of mixotrophic cultivation said method comprising:
(f) preparing a mixotroph, said mixotroph being a mutated microalgae strain;
(g) adding the mixotroph of step (a) in a mixotrophic source/medium; (h) adding a selected plant extract in the growth media;
(i) allowing the growth and multiplication of mixotroph in the mixotrophic
source/medium under open pond conditions; (j) determining the biomass and/or oil content from mixotroph.
Another embodiment of the present invention provides for a method wherein the step (b) mixotroph microalgae is a single microalgae strain or combination of microalgae strains.

Another embodiment of the present invention provides for a method, wherein step (b), the mixotrophic microalgae is selected from the group comprising Chlorella vulgaris IOC-106, Chlorella vulgaris IOC-112, Chlorella sp. IOC-114, Scenedusmus sp IOC-110 and Synechocosccus sp. IOC 111.
Another embodiment of the present invention provides for a method, wherein mixotroph composition comprises the microalgae alone and/or in combination is in the ratio of 0.01-5:1-100 cells.
Another embodiment of the present invention provides for a method, wherein step (b) the mixotrophic source or medium is selected from the group comprising lighted open pond, controlled stirred tank, photo-bioreactor, in vitro culture medium comprising growth medium with organic and/or inorganic carbon source, hydrocarbon processing plant, kitchen effluent, automobile industry effluent, fruit processing plant effluent, dairy effluents, municipal waste water, sewage treatment plant, biogas plant slurry, agricultural residues, industrial waste having acetate, sugars , low or high carbon source and/or the like.
Another embodiment of the present invention provides for a method, wherein step (c) the plant extract has anti-microbial property.
Another embodiment of the present invention provides for a method, wherein the mixotrophic cultivation is carried out in lighted open pond, controlled stirred tank, photo-bioreactor, in vitro culture medium comprising growth medium with organic and/or inorganic carbon source, hydrocarbon processing plant, kitchen effluent, automobile industry effluent, fruit processing plant effluent, dairy effluents, municipal waste water, sewage treatment plant, biogas plant slurry, agricultural residues, industrial waste having low or high carbon source and/or the like.
Another embodiment of the present invention provides for a method, wherein the mixotrophic cultivation is useful for production of biofuel.
Another embodiment of the present invention provides for a process of producing biofuel from the mixotrophic cultivation of mixotrophic microalgae, said method comprising the steps of :

(g) preparing a mixotroph, said mixotroph being a mutated microalgae strain; (h) adding the mixotroph of step (a) in a mixotrophic source/medium; (i) adding a selected plant extract in the growth media; (j) allowing the growth and multiplication of mixotroph in the mixotrophic
source/medium; and (k) isolating the mixotroph from the mixotrophic source/medium; (l) obtaining the biofuel.
Another embodiment of the present invention provides for a process, wherein the step (a) mixotroph microalgae is a single microalgae strain or combination of microalgae strains.
Another embodiment of the present invention provides for a process, wherein step (b), the mixotrophic microalgae is selected from the group comprising the mixotrophic microalgae is selected from the group comprising Chlorella vulgaris IOC-106, Chlorella vulgaris IOC-112, Chlorella sp. IOC-114, Scenedusmus sp IOC-110 and Synechocosccus sp. IOC 111.
Another embodiment of the present invention provides for a process, wherein mixotroph composition alone and/or in combination is in the ratio of 0.01-5:1-100 cells.
Another embodiment of the present invention provides for a process, wherein step (b) the mixotrophic source or medium is selected from the group comprising lighted open pond, controlled stirred tank, photo-bioreactor, in vitro culture medium comprising growth medium with organic and/or inorganic carbon source, hydrocarbon processing plant, kitchen effluent, automobile industry effluent, fruit processing plant effluent, dairy effluents, municipal waste water, sewage treatment plant, biogas plant slurry, agricultural residues, industrial waste having low or high carbon source and/or the like.
Another embodiment of the present invention provides for a process, wherein step (c) the plant extract has anti-microbial property.
Another embodiment of the present invention provides for a process, wherein the mixotrophic cultivation is carried out in lighted open pond, controlled stirred tank, photo-bioreactor, in vitro culture medium comprising growth medium with organic and/or inorganic carbon source, hydrocarbon processing plant, kitchen effluent, automobile industry effluent,

fruit processing plant effluent, dairy effluents, municipal waste water, sewage treatment plant, biogas plant slurry, agricultural residues, industrial waste having low or high carbon source and/or the like.
Another embodiment of the present invention provides for a method of mixotrophic cultivation in an open pond, said method comprising the steps of:
(f) preparing a mixotroph, said mixotroph being a mutated strains of microalgae;
(g) adding the mixotroph of step (a) in an open pond; (h) adding a selected plant extract in the growth media;
(i) allowing the growth and multiplication of mixotroph in the mixotrophic
source/medium under open pond conditions; (j) determining the biomass and/or oil content from mixotroph.
Another embodiment of the present invention provides for a method, wherein the step (b) mixotroph microalgae is a single microalgae strain or combination of microalgae strains.
Another embodiment of the present invention provides for a method, wherein step (b), the mixotrophic microalgae is selected from the group comprising Chlorella vulgaris IOC-106, Chlorella vulgaris IOC-112, Chlorella sp. IOC-114, Scenedusmus sp IOC-110 and Synechocosccus sp. IOC 111.
Another embodiment of the present invention provides for a method, wherein mixotroph composition comprises the microalgae alone and/or in combination is in the ratio of 0.01-5:1-100 cells.
Another embodiment of the present invention provides for a method, wherein step (b) the open pond is selected from the group comprising lighted open pond, controlled stirred tank, photo-bioreactor tank, hydrocarbon processing plant pond, kitchen effluent storage pond, automobile industry effluent treatment plant pond, municipal waste water pond and pond of sewage treatment plant having acetate, sugars, low or high carbon source and/or the like.
Another embodiment of the present invention provides for a method, wherein step (c) the plant extract has anti-microbial property.

Another embodiment of the present invention provides for a method, wherein step (c) the plant extract stimulate growth of mixotrophic microalgae.
Another embodiment of the present invention provides for a method, wherein the mixotrophic cultivation is carried out in lighted open pond, controlled stirred tank, photo-bioreactor tank, hydrocarbon processing plant pond, kitchen effluent storage pond, automobile industry effluent treatment plant pond, municipal waste water pond and pond of sewage treatment plant having acetate, sugars, low or high carbon source and/or the like.
Another embodiment of the present invention provides for a method, wherein the mixotrophic cultivation is useful for production of biofuel.
Another embodiment of the present invention provides for a use of mutated mixotrophic microalgae strain for mixotrophic cultivation.
Another embodiment of the present invention provides to a use of mutated mixotrophic microalgae strain for mixotrophic cultivation, wherein the mutated mixotrophic microalgae is selected from the group comprising Chlorella vulgaris IOC-106, Chlorella vulgaris IOC-112, Chlorella sp. IOC-114, Scenedusmus sp IOC-110 and Synechocosccus sp. IOC 111 alone or in combination thereof.
Another embodiment of the present invention provides for a use of mutated mixotrophic microalgae strain for mixotrophic cultivation, wherein the mixotrophic cultivation is carried out in mixotrophic source or mixotrophic medium selected from the group comprising of lighted open pond, controlled stirred tank, photo-bioreactor, in vitro culture medium comprising growth medium with organic and/or inorganic carbon source, hydrocarbon processing plant, kitchen effluent, automobile industry effluent, fruit processing plant effluent, dairy effluents, municipal waste water, sewage treatment plant, biogas plant slurry, agricultural residues, industrial waste having acetate, sugars , low or high carbon source and/or the like.
Another embodiment of the present invention provides for a use of mutated mixotrophic microalgae strain for mixotrophic cultivation, wherein the mixotrophic cultivation is carried out in mixotrophic source or mixotrophic medium selected and wherein the mixotrophic

medium also comprises of plant extract which stimulates growth of mutated mixotrophic microalgae and also has anti-microbial property.
Another embodiment of the present invention provides for a use of mutated mixotrophic microalgae strain for mixotrophic cultivation, wherein the mixotrophic cultivation is carried out in lighted open pond, controlled stirred tank, photo-bioreactor, in vitro culture medium comprising growth medium with organic and/or inorganic carbon source, hydrocarbon processing plant, kitchen effluent, automobile industry effluent, fruit processing plant effluent, dairy effluents, municipal waste water, sewage treatment plant, biogas plant slurry, agricultural residues, industrial waste having low or high carbon source and/or the like.
Another embodiment of the present invention provides for a use of mutated mixotrophic microalgae strain for mixotrophic cultivation in an open pond using plant extract for production of biofuel.
Another embodiment of the present invention provides for a use of mutated mixotrophic microalgae strain for mixotrophic cultivation in an open pond using plant extract for production of biofuel, wherein the plant extract is useful in stimulating growth of mutated mixotrophic microalgae strain and also as anti-microbial agent.
Another embodiment of the present invention provides for a use of mutated mixotrophic microalgae strain for mixotrophic cultivation in an open pond using plant extract for production of biofuel, wherein the mutated mixotrophic microalgae is selected from the group comprising Chlorella vulgaris IOC-106, Chlorella vulgaris IOC-112, Chlorella sp. IOC-114, Scenedusmus sp IOC-110 and Synechocosccus sp. IOC 111 alone or in combination thereof.
Another embodiment of the present invention provides for a use of mutated mixotrophic microalgae strain for mixotrophic growth in an open pond using a mixotrophic medium and a plant extract, wherein the plant extract stimulates growth of the microalgae and reduces microbial contamination.

Another embodiment of the present invention provides for a mixotrophic cultivation using mutant microalgae strain alone or in combination, wherein the mutant microalgae carries both autotrophic and heterotrophic modes of cultivation.
Another embodiment of the present invention provides for algae cultivation system comprising atleast one mutant microalgae strain capable of both autotrophic and/or heterotrophic mode of growth in an open pond system wherein the mutant microalgae strain utilizes higher CO2 concentration and higher growth under light and dark conditions using inexpensive resources as external carbon sources.
Another embodiment of the present invention provides for development of algae cultivation system which overcomes deficiencies of autotrophic and heteretrophic mode of algae cultivation and has the benefits of open pond system with utilization of higher CO2 concentration and higher growth under light and dark conditions using inexpensive resources as external carbon sources.
Another embodiment of the present invention relates to a method of algae cultivation in an open pond system using at least one mutant novel strain of microalgae alone or in combination which overcomes deficiencies of autotrophic and heteretrophic mode of algae cultivation, wherein microalgae utilizes the benefits of open pond system by utilizing higher CO2 concentration and higher growth under light and dark conditions using inexpensive resources as external carbon sources.
Another embodiment of the present invention relates to use of at least one mutant novel strain of microalgae alone or in combination for algae for algae cultivation in open pond system which overcomes deficiencies of autotrophic and heteretrophic mode of algae cultivation wherein the microalgae utilizes the benefits of open pond system by utilizing higher CO2 concentration and higher growth under light and dark conditions using inexpensive resources as external carbon sources.
The following non-limiting examples illustrate specific embodiments of the present invention. They are not intended to be limiting the scope of the present invention in any way.

The invention will now be explained with the help of following examples. However, the scope of the invention should not be limited to these examples as the person skilled in the art can easily vary the proportion of the ingredients and combinations.
EXAMPLES
EXAMPLE 1 Isolation of algae:
The algae were isolated from diverse sources like hydrocarbon processing industry wastewater, sewage treatment wastewaters, rivers, ponds and soil. The collected water and soil samples were inoculated in media containing Na2CO3 g/l (1-5), NaHCO3 (0.5-2.5) KH2PO4 (0.5-4), K2HPO4 (0.5-4), MgSO4 (0.1-1.0), (NH4)2SO4 (0.25-0.50), KNO3 (0.15-4.75), ZnSO4 (0.2-2.1), NaCl (0.2-10) Trace element (2 ml to 15 ml of solution). The trace element solution (gram per liter) comprises Nitrilotriacetic acid (0.1-1.0), FeSO4.7H2O (0.01-0.15), MnCl2.4H2O (0.001-0.005), CoCl2.6H2O (0.005-0.02), CaCl2.2H20 (0.01-0.5), ZnCl2 (0.01-0.15), CuCl2.H2O (0.01-0.03), H3BO3 (0.002-0.02), Na2MoO4 (0.001-0.02), Na2SeO3 (0.005-0.02), NiSO4 (0.01-0.03), SnCl2 (0.01-0.03). Media also contained yeast extract (0.1-4), organic carbon source (0.2-50), and antibiotics including ampicillin (sodium form), streptomycin sulfate, and kanamycin sulfate (100 mgL-1 each). Each 1000 ml of flask contained 500 ml of above media was autoclaved. It was inoculated with 5-10% of soil or water sample. The flask was incubated at 45°C for 2–10 days in presence of light and continuously CO2 was sparged.
After completion of incubation the 1 ml culture was spanned at 3000 rpm for 5 minutes. The cells which are floating were inoculated on agar plate made by adding 2% agar to above media. Plated were incubated at 45°C in presence of light. Fast growing single greenish yellow colonies were picked and carefully transferred to a new plate. The purified colonies are selectively picked up and inoculated into flasks containing growth medium, including but not limited to components of basal medium, for further culture.
Isolated algae were then grown in presence of light and inorganic (CO2) and organic carbon source (2-10%) in above media for 120 days with transfer in fresh media at two weekly intervals. The algae were further adapted to tolerance to heavy metals and other contaminants

in wastewater by growing them in media containing heavy metals (Cr, Pb, V, Ni, Hg ( individually 1ppm-100 ppm and in different combinations, up to 1%) along with the inorganic and organic carbon source in presence of light by transferring in new media at week intervals. Subsequently, the algae were grown in media in which wastewater from hydrocarbon processing industry was used as solvent in place of water. The fast growing algal strains having ability to grow in mixotrophic conditions in presence of heavy metal and other contaminants present in wastewater were selected. These microalgae were further purified to get axenic culture and characterized.
The selected algal strains were characterized according to their 18S rRNA gene sequences, as well as some morphological characteristics. The resulting 18S rRNA gene sequences were aligned and compared to the nucleotide sequences of some known microalge in GenBank database of the National Center for Biotechnology Information by using Basic Local Alignment Search Tool (BLAST®).
Isolated algae were then grown in presence of light and inorganic (CO2) and organic carbon source (2-10%) in above media for 120 days with transfer in fresh media at two weekly intervals. The algae were further adapted to tolerance to heavy metals and other contaminants in wastewater by growing them in media containing heavy metals (Cr, Pb, V, Ni, Hg ( individually 1 ppm-100 ppm and in different combinations, up to 1%) along with the inorganic and organic carbon source in presence of light by transferring in new media at week intervals. Subsequently, the algae were grown in media in which wastewater from hydrocarbon processing industry was used as solvent in place of water. The fast growing algal strains having ability to grow in mixotrophic conditions in presence of heavy metal and other contaminants present in wastewater were selected. These microalgae were further purified to get axenic culture and characterized.
The microalgae strains were further improved by recursive mutagenesis and protoplast fusion.
Example 2
Preparation of Mutant Strains

The microalgae were mutagenized by chemical (EMS, mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, benzo(a)pyrene and 4-nitroquinoline 1-oxide) and radiation mutagen (UV, gamma-rays) or their combination . The pooled mutant population is shuffled by homologous recombination using protoplast fusion followed by selecting improved progenies and subjecting the same to next round of selection. This process was carried out for six cycles. This accelerates directed evolution through recursive recombination of improved progeny, thereby improving multiple traits. Strains with higher growth, lipid productivity, salt, pH and heavy metal tolerance and with ability to grow under mixotrophic growth conditions were obtained.
In one set of experiment: EMS was added into 5 ml of the log phase culture in a 15-ml centrifuge tube to a final concentration of 0.4 gL-1 and the culture suspension was further incubated in a water bath at 45°C for 15 min. Diluting the culture 20 times with pre-chilled, fresh mineral salts medium subsequently terminated the treatment. The mutated cells were centrifuged and transferred to the media having salt concentration 10%, heavy metal concentration (200 ppm), pH (10). After 24 h incubations at 45°C under mixotrophic conditions , the culture broth was serially diluted and plate on media agar plates having high salt concentration 10%, high heavy metal concentration (200 ppm), high pH ( 10), The plates were incubated at 45 0C for 48 hours in light and presence of CO2. Protoplast was prepared according to method known in prior art. For shuffling protoplasts were fused by suspension in buffer (0.5 M sucrose, 10mM Tris –HCl, 20mM MgCl2) containing 15% dimethyl sulphoxide and 60% PEG-6000. The resulting suspension was incubated at 250C for 50 min. The fused protoplast preparation was diluted with regeneration media (above media containing 0.5M sucrose) and protoplasts were harvested by centrifugation at 3500 rpm for 10 min at 250C. The protoplast cells floating was collected and were re-suspended in regeneration media and shaken at 200 rpm for 12 h before plating on agar plates higher salt concentration 12%, heavy metal concentration (250 ppm), pH ( 11) . The plated were scraped to generate a pooled fusion library. The formation of protoplasts, their fusion and their subsequent regeneration was repeated six times with pooled regenerated cells from one fusion being the inoculum for the subsequent protoplast culture. Non-shuffled controls were prepared by the recursive formation and regeneration of protoplasts without exposure to PEG. This process was carried out for six cycles. This accelerates directed evolution through recursive recombination of improved progeny, thereby improving multiple traits. Strains with higher growth, lipid productivity, salt tolerance ( up to 10%), pH (5-12) and heavy metal tolerance ( up to 1%) and with ability to grow under mixotrophic growth conditions were obtained.

Example 3: Mixotrophic Microalgae growth in open pond
The selected strains were inoculated in the g/l of Na2CO3 (1-5), NaHCO3 (0.5-2.5), Di-ammonium phosphate (0.5-10), urea (0.1-5) MgSO4 (0.1-1.0), (NH4)2SO4 (0.25-0.50), ZnSO4 (0.2-2.1), NaCl (0.2-100) Trace element (2 ml to 15 ml of solution) and Multi vitamin solution (0.2- 2ml). The trace element solution (gram per liter) comprises Nitrilotriacetic acid (0.1), FeSO4.7H2O (0.01-0.15), MnCl2.4H2O (0.001- 0.005), CoCl2.6H2O (0.005-0.02), CaCl2.2H2O (0.01-0.5), ZnCl2 (0.01-0.15), CuCl2.H2O (0.01-0.03), H3BO3 (0.002-0.02), Na2MoO4 (0.001-0.02), Na2SeO3 (0.005-0.02), NiSO4 (0.01-0.03), SnCl2 (0.01-0.03). The multivitamin solution (g/l) includes Biotin 0.01-0.03, Folic acid (0.01-0.03), Pyridoxine HCl (0.5-0.2), Thiamine HCl (0.02-0.06), Riboflavin (0.01-0.04), Nicotinic acid (0.002-0.01), Ca-Pentotheonate (0.002-0.01), Lipoic acid (0.0025-0.0075), Vitamin B12 (0.0005-0.0015), PABA (0.0025-0.0075), peptone (2-10), yeast extract (2-7), lantana methanolic plant extract (2%), molasses (5-20%) . The pH and salinity of media was 10 and 10%, respectively. Sampling were done every day to estimate the dry cell weight, chlorophyll content and lipid content regularly. Algal cell yield can be determined using various methods, including but not limiting to light intensity measurement of the cell suspension, such as OD540 nm of cell suspension. Preferable conditions such as glucose concentration, different nitrogen sources in the basal medium, temperatures, and shaking rate during algal-seed-cells cultivation in shaking flasks are determined by real-time light intensity measurement of the cell suspension. A temperature of open pond between 10-50°C. CO2 was sparged continuously and with sun light.
Cell growth is measured by the absorbance of the suspension at 540 nm and dry cell weight. 1.5 ml of algal culture was taken in pre-weighed Eppendorf tubes, centrifuged at 8000 rpm for 5 minutes. The supernatant media was removal using micropipette and the algae pellet at the bottom was dried at 105°C until the constant weight was achieved. The dry weight of algae biomass was determined gravimetrically and growth was expressed in terms of dry weight. Lipid measurements were made by using a mixture of methanol, chloroform, and water.
A culture sample is collected at three points during the experiments for lipid analysis. The culture sample is centrifuged at 3,500rpm for 10 minutes in a large (200ml) plastic centrifuge tube; the pelleted cells along with 35ml of supernatant are then transferred to a plastic centrifuge tube (45ml) to be re-centrifuged again at 5000rpm for 10 minutes. The supernatant is removed

by pipette. The pellet is then resuspended with 4ml of DI H2O, then 10ml of methanol and 5ml of chloroform is added, resulting in a 10:5:4 ratio of methanol : chloroform : water. At this ratio, all solvents are miscible and form one layer. After overnight extraction on a shaker table, 5ml of water and 5ml of chloroform are added which results in a 10:10:9 ratio of methanol : chloroform : water. Tubes are centrifuged for 10 minutes at 5000rpm. At this solvent ratio, two layers are formed, a water methanol upper layer and chloroform lower layer. The chloroform lower layer which contains the extracted lipids is then removed by Pasteur pipette and placed into a pre-weighed vial. After the first extraction, 10ml of additional chloroform is added to conduct a second extraction. The additional 10ml of chloroform again results is a 10:10:9 methanol: chloroform : water ratio and two layers are formed. The tube is centrifuged at 3,500rpm for 10 minutes, and the lower chloroform layer is removed by Pasteur pipette and placed into another pre-weighed vial. The chloroform is evaporated by heating in a 55°C water bath under a constant stream of nitrogen gas. After 1 hour in a 105°C oven, vials are weighed again. The weight difference represents weight of lipids extracted from the culture sample. The extracted lipid was analysed by gas chromatography as per method described in prior art. The lipid showed fatty acid suitable for biodiesel production. The bacterial account was taken on nutrient agar plate by conventional spread plate plate subsequent to serial dilution. Tables-1 & 2 present the results from the novel mutated mixotroph microalgae strains and the wild strains microalgae.
Table-1: The Bio-mass and oil content of micro-algal species obtained mixotrophic

Strain
Biomass (g (DCW)/l) Oil Content (% w/w (DCW) Bacterial Count CFU/ml
Without With plant plant extract extract Without With plant Without plant extract extract plant extract With plant extract
Chlorella vulgaris
(IOC-106) 9.4 14.82 18.2 28.8 2.7 X 1011 <103
C. vulgaris (IOC-112) 7.6 10.01 19.5 29.2 9.3 X 1012 <102
Chlorella sp. (IOC-114) 8.4 11.84 20.3 29.0 7.3X 1012 <103
Scenedusmus sp.
(IOC-110) 9.2 19.5 21.5 30.5 4.2X 1013 <102
Nanochloropsis oculata
(IOC-105) 11.4 12.4 26.2 29.2 7.0X 1013 <102
Synechocosccus sp.
(IOC-111) 4.93 10.3 17.2 24.6 2.0X 1012 <102
Mixture of above ( in equal ratio) 12.3 28.3 27.4 34.9 2.1X 1012 <102
Table-2: The Bio-mass and oil content of non-mutated/wild micro-algal species under mixotrophic

Wild Strains Biomass (g (DCW)/l) Oil Content (% w/w (DCW) Bacterial Count CFU/ml
Without plant extract With plant extract Without plant extract With plant extract Without plant extract With plant extract
Chlorella vulgaris
(IOC-106)* 4.4 7.82 17.2 22..8 2.9 X 1010 <103
C. vulgaris (IOC-112)* 5.6 8.1 18.5 23.2 6.2 X 1012 <103
Chlorella sp. (IOC-114)* 3.4 7.8 19.3 23.0 4.2X 1012 <103
Scenedusmus sp.
(IOC-110)* 4.2 6.5 17.5 22.5 1.3X 1013 <103
Nanochloropsis oculata
(IOC-105)* 3.4 7.4 16.2 20.2 4.1X 1013 <102
Synechocosccus sp.
(IOC-111)* 2.3 4.3 14.2 19.2 1.7X 1012 <102
Mixture of above ( in equal ratio) 8.3 12.3 20.4 24.9 9.2X 1012 <102
* The accession IOC number given for wild strains is only for reference to show that the particular wild strain corresponds to related mutated strains.

Example 3: Preparation of Plant Extract
The composition of the invention includes extract of different part of the plants such as lantana, tobacco, neem, mahendi, vegetative and/or fruit plant material and/or mixtures thereof. Plant material includes the stem, leaves and fruit of the plant and any part of the plant. In a particularly important aspect, the plant material is dried, powdered and extracted with different organic solvents, water, acid water, alkali water separately, sequentially and/or simultaneously at different temperature, pressure to remove the compounds having ability inhibit the growth of undesired bacteria and stimulate growth of algae under mixotrophic conditions. The temperature ranges from 30-160°C, preferably 50-110°C and the pressure ranges from atmospheric to 15 lbs. The extracted material was further purified using known art like column chromatography and each fraction was evaluated for their ability to inhibit growth of undesired bacteria and stimulate growth of algae under mixotrophic conditions. The plant extract was effective in the concentration ranging from 1-7% (v/v) in media.

We Claim:
1. A method of mixotrophic cultivation said method comprising:
(a) preparing a mixotroph, said mixotroph being a mutated microalgae strain;
(b) adding the mixotroph of step (a) in a mixotrophic source/medium;
(c) adding a selected plant extract in the growth media;
(d) allowing the growth and multiplication of mixotroph in the mixotrophic source/medium under open pond conditions;
(e) determining the biomass and/or oil content from mixotroph.

2. The method as claimed in claim 1, wherein the step (b) mixotroph microalgae is a single microalgae strain or combination of microalgae strains.
3. The method as claimed in claims 1-2, wherein step (b), the mixotrophic microalgae is selected from the group comprising Chlorella vulgaris IOC-106, Chlorella vulgaris IOC-112, Chlorella sp. IOC-114, Scenedusmus sp IOC-110 and Synechocosccus sp. IOC 111.
4. The method as claimed in claims 1-3, wherein mixotroph composition comprises the microalgae alone and/or in combination is in the ratio of 0.01-5:1-100 cells.
5. The method as claimed in claim 1, wherein step (b) the mixotrophic source or medium is selected from the group comprising lighted open pond, controlled stirred tank, photo-bioreactor, in vitro culture medium comprising growth medium with organic and/or inorganic carbon source, hydrocarbon processing plant, kitchen effluent, automobile industry effluent, fruit processing plant effluent, dairy effluents, municipal waste water, sewage treatment plant, biogas plant slurry, agricultural residues, industrial waste having acetate, sugars , low or high carbon source and/or the like.
6. The method as claimed in claim 1, wherein step (c) the plant extract has anti-microbial property.

7. The method as claimed in any one of claims 1-6, wherein the mixotrophic cultivation is carried out in lighted open pond, controlled stirred tank, photo-bioreactor, in vitro culture medium comprising growth medium with organic and/or inorganic carbon source, hydrocarbon processing plant, kitchen effluent, automobile industry effluent, fruit processing plant effluent, dairy effluents, municipal waste water, sewage treatment plant, biogas plant slurry, agricultural residues, industrial waste having low or high carbon source and/or the like.
8. A method as claimed in claims 1-7, wherein the mixotrophic cultivation is useful for production of biofuel.
9. A process of producing biofuel from the mixotrophic cultivation of mixotrophic microalgae as claimed in claim 1, said method comprising the steps of :

(a) preparing a mixotroph, said mixotroph being a mutated microalgae strain;
(b) adding the mixotroph of step (a) in a mixotrophic source/medium;
(c) adding a selected plant extract in the growth media;
(d) allowing the growth and multiplication of mixotroph in the mixotrophic source/medium; and
(e) isolating the mixotroph from the mixotrophic source/medium;
(f) obtaining the biofuel.

10. The process as claimed in claim 9, wherein the step (a) mixotroph microalgae is a single microalgae strain or combination of microalgae strains.
11. The process as claimed in claims 9-10, wherein step (b), the mixotrophic microalgae is selected from the group comprising the mixotrophic microalgae is selected from the group comprising Chlorella vulgaris IOC-106, Chlorella vulgaris IOC-112, Chlorella sp. IOC-114, Scenedusmus sp IOC-110 and Synechocosccus sp. IOC 111.
12. The method as claimed in claims 1-11, wherein mixotroph composition alone and/or in combination is in the ratio of 0.01-5:1-100 cells.

13. The method as claimed in claim 9, wherein step (b) the mixotrophic source or medium is selected from the group comprising lighted open pond, controlled stirred tank, photo-bioreactor, in vitro culture medium comprising growth medium with organic and/or inorganic carbon source, hydrocarbon processing plant, kitchen effluent, automobile industry effluent, fruit processing plant effluent, dairy effluents, municipal waste water, sewage treatment plant, biogas plant slurry, agricultural residues, industrial waste having low or high carbon source and/or the like.
14. The process as claimed in claim 9, wherein step (c) the plant extract has anti-microbial property.
15. The process as claimed in claims 9-14, wherein the mixotrophic cultivation is carried out in lighted open pond, controlled stirred tank, photo-bioreactor, in vitro culture medium comprising growth medium with organic and/or inorganic carbon source, hydrocarbon processing plant, kitchen effluent, automobile industry effluent, fruit processing plant effluent, dairy effluents, municipal waste water, sewage treatment plant, biogas plant slurry, agricultural residues, industrial waste having low or high carbon source and/or the like.
16. A method of mixotrophic cultivation in an open pond, said method comprising the steps of:

(a) preparing a mixotroph, said mixotroph being a mutated strains of microalgae;
(b) adding the mixotroph of step (a) in an open pond;
(c) adding a selected plant extract in the growth media;
(d) allowing the growth and multiplication of mixotroph in the mixotrophic source/medium under open pond conditions;
(e) determining the biomass and/or oil content from mixotroph.
17. The method as claimed in claim 16, wherein the step (b) mixotroph microalgae is a single
microalgae strain or combination of microalgae strains.

18. The method as claimed in claims 16-17, wherein step (b), the mixotrophic microalgae is selected from the group comprising Chlorella vulgaris IOC-106, Chlorella vulgaris IOC-112, Chlorella sp. IOC-114, Scenedusmus sp IOC-110 and Synechocosccus sp. IOC 111.
19. The method as claimed in claims 16-18, wherein mixotroph composition comprises the microalgae alone and/or in combination is in the ratio of 0.01-5:1-100 cells.
20. The method as claimed in claims 16-19, wherein step (b) the open pond is selected from the group comprising lighted open pond, controlled stirred tank, photo-bioreactor tank, hydrocarbon processing plant pond, kitchen effluent storage pond, automobile industry effluent treatment plant pond, municipal waste water pond and pond of sewage treatment plant having acetate, sugars, low or high carbon source and/or the like.
21. The method as claimed in claim 16-20, wherein step (c) the plant extract has antimicrobial property.
22. The method as claimed in claims 16-20, wherein step (c) the plant extract stimulate growth of mixotrophic microalgae.
23. The method as claimed in claims 16-20, wherein the mixotrophic cultivation is carried out in lighted open pond, controlled stirred tank, photo-bioreactor tank, hydrocarbon processing plant pond, kitchen effluent storage pond, automobile industry effluent treatment plant pond, municipal waste water pond and pond of sewage treatment plant having acetate, sugars, low or high carbon source and/or the like.
24. A method as claimed in claims 16-23, wherein the mixotrophic cultivation is useful for production of biofuel.
25. Use of mutated mixotrophic microalgae strain for mixotrophic cultivation.

26. The use as claimed in claim 25, wherein the mutated mixotrophic microalgae is selected from the group comprising Chlorella vulgaris IOC-106, Chlorella vulgaris IOC-112, Chlorella sp. IOC-114, Scenedusmus sp IOC-110 and Synechocosccus sp. IOC 111 alone or in combination thereof.
27. The use as claimed in claims 25-26, wherein the mixotrophic cultivation is carried out in mixotrophic source or mixotrophic medium selected from the group comprising of lighted open pond, controlled stirred tank, photo-bioreactor, in vitro culture medium comprising growth medium with organic and/or inorganic carbon source, hydrocarbon processing plant, kitchen effluent, automobile industry effluent, fruit processing plant effluent, dairy effluents, municipal waste water, sewage treatment plant, biogas plant slurry, agricultural residues, industrial waste having acetate, sugars , low or high carbon source and/or the like.
28. The use as claimed in claim 27, wherein the mixotrophic medium also comprises of plant extract which stimulates growth of mutated mixotrophic microalgae and also has antimicrobial property.
29. The use as claimed in claims 25-28, wherein the mixotrophic cultivation is carried out in lighted open pond, controlled stirred tank, photo-bioreactor, in vitro culture medium comprising growth medium with organic and/or inorganic carbon source, hydrocarbon processing plant, kitchen effluent, automobile industry effluent, fruit processing plant effluent, dairy effluents, municipal waste water, sewage treatment plant, biogas plant slurry, agricultural residues, industrial waste having low or high carbon source and/or the like.
30. A use of mutated mixotrophic microalgae strain for mixotrophic cultivation in an open pond using plant extract for production of biofuel.
31. The use as claimed in claim 30, wherein the plant extract is useful in stimulating growth of mutated mixotrophic microalgae strain and also as anti-microbial agent.

32. The use as claimed in claims 30-31. wherein the mutated mixotrophic microalgae is selected from the group comprising Chlorella vulgaris IOC-106, Chlorella vulgaris IOC-112. Chlorella sp. IOC-114, Scenedusmus sp IOC-110 and Synechocosccus sp. IOC 111 alone or in combination thereof.
33. A use of mutated mixotrophic microalgae strain for mixotrophic growth in an open pond using a mixotrophic medium and a plant extract, wherein the plant extract stimulates growth of the microalgae and reduces microbial contamination.
34. A method of algae cultivation in an open pond system using at least one mutant novel strain of microalgae alone or in combination which overcomes deficiencies of autotrophic and heteretrophic mode of algae cultivation, wherein microalgae utilizes the benefits of open pond system by utilizing higher CO2 concentration and higher growth under light and dark conditions using inexpensive resources as external carbon sources.
35. A use of at least one mutant novel strain of microalgae alone or in combination for algae for algae cultivation in open pond system which overcomes deficiencies of autotrophic and heteretrophic mode of algae cultivation wherein the microalgae utilizes the benefits of open pond system by utilizing higher CO2 concentration and higher growth under light and dark conditions using inexpensive resources as external carbon sources.