[001] The present invention relates to development a new method for harvesting and boosting biomass productivity of microalgae by microalgae- yeast association resulting in nearly 96% harvesting within 3 hours.
BACKGROUND AND PRIOR ARTS OF INVENTION
[002] Harvesting of microalgae is a major cost limiting factor for biofuels production. It contributed about 20-30%> of the total cost of biofuel production. The main techniques used for harvesting algal cells include sedimentation, flocculation, flotation, Tris-Acetate- Phosphate-Pluronic (TAPP) gel, centrifugation and filtration. Microalgae can be efficiently harvested (about 90-98%>) by centrifugation, however this is too expensive. For low cost production of microalgae, centrifugation harvesting needs to be replaced. Certain alternative technologies capable of harvesting large volumes of culture medium are available but they are not used frequently due to their limitations.
[003] Flocculating fungi or bacteria can be co-cultivated with microalgae or separately. The use of bacteria or fungi as a flocculating agent avoids chemical contamination of the biomass, but results in microbial contamination of biomass which is not fit for food or feed use of microalgae.
[004] Prochazkova et al., used spent brewer's yeast as flocculant for harvesting Chlorella vulgaris. Diaz-Santos et al. also reported the flocculation of microalgae by yeasts and yeast extracted protein
[005] In the present work, for the first time Saccharomyces cerevisiae is co-cultivated with microalgae. Also, the effect of this co-cultivation on biomass and different biochemical parameters has been studied.

[006] US20120282651A1 discloses one of the most important technical barriers for algal biofuel is the substantial harvesting and extracting cost. The present invention includes a system and method for the pelletization of single cell microalgae through co-cultivation with filamentous fungi to enable the low cost separation of microalgae from liquid medium and to significantly increase the biomass and lipid yield. The approach represents a simple and straightforward method to significantly reduce the cost for algae-based biofuel production and enable the economically viable algae biofuel production.
[007] Encarnacion Diaz-Santos E. et al, in their work Study of bioflocculation induced by Saccharomyces bayanus var. uvarum and flocculating protein factors in microalgae, disclosed autoaggregation of flocculent microalgae in response to stressing conditions is poorly understood, but it is a promising approach to induce the aggregation of microalgae into floes and make microalgal harvesting a straightforward and cheap procedure. The effect of the self-flocculating yeast strain Saccharomyces bayanus var. uvarum on two chlorophytes: the model freshwater microalga Chlamydomonas reinhardtii and the novel marine microalga Picochlorum sp. HM1, has been investigated. The addition of Saccharomyces induces cell aggregation in both microalgal species studied; being the flocculating effect caused by anaerobically grown yeasts almost two-fold the effect of standard aerobically grown yeast. In order to gain more insights into the origin of yeast induced microalgal flocculation, proteins released into the culture medium by the flocculent yeast S. bayanus var. uvarum during the fermentative phase of growth were isolated and their ability to induce flocculation was tested. Addition of 0.1 mg mL-1 of concentrated flocculating excreted proteins resulted in recovery efficiency values of 95% and 75% for Chlamydomonas and Picochlorum respectively. The flocculating activity of some plant lectins on the chosen chlorophytes was also evaluated.
[008] Prochazkova G. et al, in their work regarding harvesting freshwater Chlorella vulgaris with flocculant derived from spent brewer's yeast discussed

that one of the key bottlenecks of the economically viable production of low added value microalgal products (food supplements, feed, biofuels) is the harvesting of cells from diluted culture medium. The main goals of this work were to prepare a novel flocculation agent based on spent brewer's yeast, a brewery by-product, and to test its harvesting efficiency on freshwater Chlorella vulgaris in different environments. The yeast was first autolyzed/ hydrolyzed and subsequently chemically modified with 2-chloro-N, N-diethylethylamine hydrochloride (DEAE). Second, optimal dosage of modified spent yeast (MSY) flocculant for harvesting C. vulgaris was determined in culture media of various compositions. It was found that the absence of phosphorus ions decreased (0.4 mg MSY/g biomass), while the presence of algogenic organic matter (AOM) increased (51 mg MSY/g biomass) the required dosage of flocculant as compared to complete mineral medium with phosphorus and without AOM (12 mg MSY/g biomass).
[009] Bhattacharya A. et al, in their work regarding a rapid method for fungal assisted algal flocculation: Critical parameters & mechanism insights disclosed a method for rapid flocculation of Chlorella pyrenoidosa cells with Aspergillus fumigatus pellets was developed. The process could flocculate 99% algal cells within 3 h. In order to identify the critical parameters, apart from the flocculation conditions (different fungal-algal ratios, flocculation temperature and agitation), the effect of cultivation time and various pretreatments (autoclaving, Cycloheximide exposure) for A. fumigatus was also investigated. Results revealed that 24 h old fungal pellets flocculated at 38 °C and 1:5 fungal-algal ratio showed the best flocculation efficiency. The cell viability assay showed that a viable and metabolically active fungal pellet is a prerequisite for flocculation. Scanning Electron Microscopy (SEM) studies confirmed that in addition to viability, intact and undamaged hyphae is also required for algal attachment. Fourier transform infrared spectroscopy (FTIR) data of the algal-fungal pellets compared to that of algae and fungi showed the involvement of specific groups in the interaction. Sharp decrease in peak intensity at 1024 cm-1 for the algal-fungal pellets

indicated the role of C-N groups in the flocculation process. The lipid content of the harvested algal fungal pellet was similar to the algal and fungal partners. Finally, this method was tested on wastewater grown algae, where 95% flocculation was achieved within 3.5 h. The algal-fungal pellets (1650 urn diameter) could be easily separated from the treated water. Hence, this process could serve as an alternative for concentrating microalgal cultures for biofuel production in a cost effective way. This report reveals critical parameters and new insights on algal-fungal flocculation apart from providing a rapid and feasible algal harvesting technique.
[0010] Jianguo Z et al. disclosed a novel method to harvest microalgae via co-culture of filamentous fungi to form cell pellets that while current approaches have limitations for efficient and cost-effective microalgal biofuel production, new processes, which are financially economic, environmentally sustainable, and ecologically stable, are needed. Typically, microalgae cells are small and grow individually. Harvest of these cells is technically difficult and it contributes to 20-30% of the total cost of biomass production. A new process of pelletized cell cultivation is described in this study to co-culture a filamentous fungal species with microalgae so that microalgae cells can be co-pelletized into fungal pellets for easier harvest. This new process can be applied to microalgae cultures in both autotrophic and heterotrophic conditions to allow microalgae cells attach to each other. The cell pellets, due to their large size, can be harvested through sieve, much easier than individual cells. This method has the potential to significantly decrease the processing cost for generating microalgal biofuel or other products.
[0011] Wrede D. et al. disclosed vide Co-Cultivation of Fungal and Microalgal Cells as an Efficient System for Harvesting, Microalgal Cells, Lipid Production and Wastewater Treatment the challenges which the large scale microalgal industry is facing are associated with the high cost of key operations such as harvesting, nutrient supply and oil extraction. The high-energy input for harvesting makes current commercial microalgal biodiesel production

economically unfeasible and can account for up to 50% of the total cost of biofuel production. Co-cultivation of fungal and microalgal cells is getting increasing attention because of high efficiency of bio-flocculation of microalgal cells with no requirement for added chemicals and low energy inputs.
[0012] Moreover, some fungal and microalgal strains are well known for their exceptional ability to purify wastewater, generating biomass that represents a renewable and sustainable feedstock for biofuel production. We have screened the flocculation efficiency of the filamentous fungus A. fumigatus against 11 microalgae representing freshwater, marine, small (5 mm), large (over 300 mm), heterotrophic, photoautotrophic, motile and non-motile strains. Some of the strains are commercially used for biofuel production. Lipid production and composition were analysed in fungal-algal pellets grown on media containing alternative carbon, nitrogen and phosphorus sources contained in wheat straw and swine wastewater, respectively. Co-cultivation of algae and A. fumigatus cells showed additive and synergistic effects on biomass production, lipid yield and wastewater bioremediation efficiency. Analysis of fungal-algal pellet's fatty acids composition suggested that it can be tailored and optimised through co-cultivating different algae and fungi without the need for genetic modification.
[0013] Muradov N. et al disclosed through fungal-assisted algal flocculation: application in wastewater treatment and biofuel production that the microalgal-based industries are facing a number of important challenges that in turn affect their economic viability. Arguably the most important of these are associated with the high costs of harvesting and dewatering of the microalgal cells, the costs and sustainability of nutrient supplies and costly methods for large scale oil extraction. Existing harvesting technologies, which can account for up to 50% of the total cost, are not economically feasible because of either requiring too much energy or the addition of chemicals. Fungal-assisted flocculation is currently receiving increased attention because of its high harvesting efficiency. Moreover, some of fungal and microalgal strains are well known for their ability to treat wastewater,

generating biomass which represents a renewable and sustainable feedstock for bioenergy production.
[0014] Prajapati SK et al disclosed vide Method for simultaneous bioflocculation and pretreatment of algal biomass targeting improved methane production, a novel method for simultaneous bioflocculation and pretreatment of algae is revealed. The method includes bioflocculation of precultured algae (Chroococcus sp.) using pellet forming filamentous fungus (Aspergillus lentulusFl 172995) resulting in nearly 100% harvesting within 6 h without addition of any nutrient and carbon source at the optimized fungal/algal (F/A) ratio of 1:3. The algal-fungal interactions require metabolically active fungus with opposite charge. The bioflocculation process is replicable at reactor scale under continuous aeration. Simple incubation of harvested algal-fungal pellets under controlled conditions was associated with significant cellulase production (> 0.4 FPU mL-1 ) by the fungus leading to soluble sugar release ( ~ 360 mg L-l) from algal cells. As a result, > 54%) enhancement in digestibility and marked increase in methane production (up to 50 %) from algal fungal pellets during anaerobic digestion was noticed. The invention is a unique process of its kind and has potential application in algae based biofuel production including biomethane, biohydrogen and biodiesel as well as in extraction of valuables from microalgal biomass.
[0015] CN103013833A discloses the invention discloses a novel high pH
induction and carbon dioxide emission reduction
coupling microalgae harvesting method. Microalgae cells are self-settled and efficiently concentrated by using high pH induction so that high-efficiency and low-cost harvesting is realized. A CO2 emission reduction carbon replenishing technology is coupled, acidic gas CO2 is efficiently absorbed by using harvested high-alkali supernate to ensure that the pH of the high-alkali supernate is reduced to be a level (pH>=8.2) suitable for microalgae growth, high-efficiency carbon replenishing of a culture medium is realized, the supernate after havesting can be

recycled, and the harvesting cost and fertilizer cost during large-scale cultivation of the microalgae are greatly lowered.
Reference:
Ummalyma SB. Mathew A. K., A. Pandey A. K., Sukumaran R.K. , 2016. Harvesting of microalgal biomass: Efficient method for flocculation through pH modulation. Bioresour. Technol. Bioresour. Technol. 2016, 213,216-221.
Toh P.Y., Azenan N.F., Wong L., Ng Y.S., Chng L.M , Lim J. , Chan D.J.C.,. The Role of Cationic Coagulant-to-Cell Interaction in Dictating the Flocculati on-Aided Sedimentation of Freshwater Microalgae. Arab J SciEng. 2017.
Seo, J.Y., Ramasamy P., K., Bohwa K., Jeong-Cheol ., , Ji-Yeon P., Jeong-Geol N., GooJ., S., Bin P., S., Kyubock L., You-Kwan O. Downstream integration of microalgae harvesting and cell disruption by means of cationic surfactant-decorated Fe304 nanoparticles. Green Chem., 2016,18, 3981-3989.
Vandamme, D., Pontes, S. C. V., Goiris, K., Foubert, I., Pinoy, L. J.J., Muylaert, K., 2011. Evaluation of electro coagulation-flocculation for harvesting marine and freshwater microalgae. Biotechnol. Bioeng. 2011, 108, 2320-2329.
Wan, C, Alam, M. A., Zhao, X. Q., Zhang, X. Y., Guo, S. L., Ho, S. H, Chang, J.S., Bai, F. W., 2015. Current progress and future prospect of microalgal biomass harvest using various flocculation technologies. Bioresour. Technol. 2015, 184, 251-257.

5
6. Banerjee, C., Ghosh, S., Sen, G., Mishra, S., Shukla, P., Bandopadhyay, R., 2013. Study of algal biomass harvesting using cationic guar gum from the natural plant source as flocculant. Carbohydr. Polym. 2013, 92, 675-681.
7. J. Zhang, Bo Hu. A novel method to harvest microalgae via co-culture of filamentous fungi to form cell pellets. Bioresour Technol 2012, 114, 529– 535.
10 8. Ndikubwimana, T., Zeng, X., Liu, Y., Chang, J. S., Lu, Y., 2014.
Harvesting of microalgae Desmodesmus sp. F51 by bioflocculation with bacterial bioflocculant. Algal Res. 2014, 6, 186-193.
9. Kumar V, Nanda M , Verma M. Application of agar liquid-gel transition
15 in cultivation and harvesting of microalgae for biodiesel production.
Bioresource Technology. (2017) 243; 163–168
10. T. M Mata, A. A Martins, N. S. Caetano. Microalgae for biodiesel
production and other applications: a review. Renew Sustain Energy Rev
20 2010,14,217–232.
11. E. Diaz-Santos, M. Vila, M. de la Vega, R. Leon, J. Vigara. 2015. Study of
bioflocculation induced by Saccharomyces bayanus var. uvarum and
flocculating protein factors in microalgae. Algal Res. 2015, 8, 23-29.
25
12. G. Prochazkova, P. Kastanek, T. Branyik, Harvesting freshwater
Chlorella vulgaris with flocculant derived from spent brewer’s yeast.
Bioresour. Technol. 2015, 177, 28-33.
30 13. Indian Patent, Indian Patent Ref. No. 1593/DEL/2015.
9

Table 1: Comparison of microalgal harvesting using various flocculation methods from the Prior Arts

method
Flocculation
Chemical
Nanoparticles/ Magnetophoretic separation Biopolymers Electrical method Plant product
Fungus
Bacteria
Agar gel

Disadvantages
Chemicals mixed with biomass
Costly, only for lab scale
Costly, only for lab scale
Biomass mixed with metals Large amount of plant product is needed
Reduced the growth of algae and time taken (12 and 72 h) Change in PH Heating required

References
1
2,3
4 5 6
7
8 9

5

OBJECTS OF THE INVENTION

[0016] An object of the invention is to develop a method for harvesting and boosting biomass productivity of microalgae.
10 [0017] Another object of the invention is to develop a method for harvesting and boosting biomass productivity of microalgae micro algae Chlorella sorokiniana UUIND6 using yeast Saccharomyces cerevisiae UUIND1.
[0018] Yet another object of the present invention is to show an alternative for 15 microalgae cultivation and harvesting of biofuel production in a cost effective way.
SUMMARY OF THE INVENTION
[0019] The present invention discloses and claims a process for enhancing 2 – 3
fold microalgae biomass productivity by yeast resulting in ~96% harvesting in 3
20 hours, said process comprising the steps of allowing the growth of microalgae
belonging to the class Chlorophyceae including Chlamydomonas and Chlorella,
10

and typical genera belonging to the class Cyanophyceae include Spirulina, Oscillatoria and Microcystis, preferably Chlorella sorokiniana UUIND6 (GenBank accession number: KY780616) for 4 days on Bold's Basal Medium (BBM) in YEPD (Yeast Extract Peptone Dextrose- glucose, 10; peptone, 5 5; yeast extract, 3 g/l) media, supplemented with supplemented with antibiotic 40 µl 25 mg/ml; adding 2 ml of said YEPD broth media containing yeast cells to 98 ml of said BBM containing 4 days old algae, thereby making it in 2:98 ratio of yeast and microalgae; and incubating for 10 days at 27°C, with a photoperiod of light- dark cycle of 16 hours light: 8 hours dark and irradiated with six (6) white 10 cool fluorescent tubes (200 μmol m s ).
BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
Fig. 1: Schematic illustration of association of yeast with microalgae cells,
15 resulting in pellets like structure, which gravimetrically settle at the
bottom, wherein 1: Yeast Cells; 2: Microalgae Cells; 3. Co-cultivation of
microalgae and yeast cells; 4: Growth of yeast and microalgae cells; 5:
Enhanced productivity of algal biomass by yeast; 6: Pellet like structures
from Yeast and Microalgae; 7: Gravitational settling down of yeast-algae
20 pellets within 3 hours; 8: Yeast-Algal Biomass; and 10: Biodiesel.
Fig. 2: Images of yeast-algal pellets
th
(A) Images of yeast-algal pellets at 10X and 40X on 10 day (B) Scanning electron micrographs (FE-SEM) (a) Control Cells (B) Yeast-algae cells.
25
Fig. 3: Microalgae kinetics
(A) Allowing microalgae of control and agar solution to gravimetrically settle down. (B) Microalgae biomass settles down in medium within 3h. (C) Kinetics of microalgae biomass. The data are mean ± S.D. for
30 triplicate (n=3) results (p < 0.05).
11

Fig. 4: Biomass/ lipid productivity
(A) lipid productivity of Chlorella sorokiniana UUIND6 cultivated for
14th days. (B) Comparison of biomass productivity. The data are mean ±
5 S.D. for triplicate (n=3) results (p < 0.05).
Fig. 5: Comparative analysis of (A) controls microalgae cell and yeast-algae
pellet size (B) lipid content, (C) carbohydrate content (D) protein content
Chlorella sorokiniana UUIND6 on the 14th day. The data are mean ± S.D.
10 for triplicate (n=3) results (p < 0.05). (E) FTIR analysis of Control, yeast
and yeast-algae cells.
Fig. 6: Fatty acid methyl ester (FAME) profile of control, yeast and yeast-algae.
15 DETAILED DESCRIPTION OF THE INVENTION
[0020] At the very outset of the detailed description, it may be understood that the ensuing description only illustrates a particular form of this invention. However, such a particular form is only exemplary embodiment, and without intending to imply any limitation on the scope of this invention. Accordingly, the description is 20 to be understood as an exemplary embodiment and teaching of invention and not intended to be taken restrictively.
[0021] Throughout the description and claims of this specification, the phrases “comprise” and “contain” and variations of them mean “including but not limited
25 to”, and are not intended to exclude other moieties, additives, components, integers or steps. Thus, the singular encompasses the plural unless the context otherwise requires. Wherever there is an indefinite article used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
30
12

[0022] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of 5 the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends 10 to any novel one, or any novel combination, of the features disclosed in this specification including any accompanying claims, abstract and drawings or any parts thereof, or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
15 [0023] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference. Post filing patents, original peer reviewed research paper shall be
20 published.
[0024] The present subject matter relates to a process for harvesting and boosting biomass productivity of microalgae by microalgae- yeast association is revealed. The method includes bio-flocculation of micro algae (Chlorella sorokiniana
25 UUIND6) using yeast Saccharomyces cerevisiae UUIND1 resulting in nearly 96% harvesting within 3 hours. Results revealed that on 14th day of yeast-algal co-cultivation period, the best flocculation efficiency was recorded. Scanning Electron Microscopy (SEM) studies confirmed the attachment between algae and yeast cells. This is seen as pellets under the microscope which settles down at the
30 bottom gravimetrically. This association increases the biomass productivity by 2.3 folds. Yeast-algal co-cultivation also increases protein productivity. FAMEs data
13

displayed that co-cultivation of yeast- microalgae increased the saturated fatty acids (SFA), mono saturated fatty acids (MUFA) and poly saturated fatty acids (PUFA) content compared to control. Hence, this microalgae- yeast association process could serve as an alternative for microalgae cultivation and harvesting for 5 biofuel production in a cost effective way.
[0025] Yeast Saccharomyces cerevisiae UUIND1 (GenBank accession number: KY385556) was maintained in YEPD (Yeast Extract Peptone Dextrose- glucose, 10; peptone, 5; yeast extract, 3 g/l) media. To avoid bacterial growth YEPD 10 media was supplemented with ampicillin 40 µl 25 mg/ml.
[0026] Chlorella sorokiniana UUIND6 (GenBank accession number: KY780616) was grown for 4 days on Bold's Basal Medium (BBM). All solvents and reagents used in this study were of HPLC grade.
15
[0027] To achieve pelletization and boosting the biomass growth, yeast cells are grown in YPD broth at 30°C for 36-48 h, when solutions of 1 g l-1 dry weight were equivalent to an optical density of 0•708 at 600 nm and a total cell count of 1•34 × 1010 cells l-.. 2 ml of YEPD broth media which containing yeast cells was
20 added to 100 ml of Bold's Basal Medium (BBM) containing 4 days old algae. After that algae was incubated for 10 days at 27 °C, with a photoperiod of 16 h: 8 h (light- dark cycle) and irradiated with 6 white cool fluorescent tubes (200 μmol m-2 s-1).
25 [0028] Lipids were extracted from yeast, mono cultural microalgae and yeast-algae pellets by a modified method of EG, Bligh and WJ Dyer [20].
[0029] Although some yeast strains have been recently used for flocculating
microalgal culture, but they suffer from the limitation of being time consuming
30 and cultivated separately. For cultivation separately, they need additional cost of
nutrient medium which can be overcome by co-cultivating it with microalgae
14

using our method. The previous studies and their salient features have been listed in Tables 1 and 2. Microalgae harvested by this method can be used as food or feed as it is free from bacterial and fungal contaminants.
5 [0030] Time required for yeast co-cultivation mediated rapid flocculation is higher than chemical methods, the process results in complete harvesting of the biomass while the chemical contamination is avoided (Table 2).
Table 2: Flocculation efficiencies of microalgae with yeast

Yeast Co-cultiv ated Micro alage Recov
ery
efficie
ncy
% Advantages Disadvantages Refer ences
S.
bayanus var. uvarum No Chlamy domona s sp 95 S. bayanus and by the proteins released into the culture medium during its fermentative growth Yeast is required to grow in separate medium and protein isolated 1
Sacchar omyces pastoria nus No Chlorel la
vulguri s 90 Spent brewer’s yeast Modified spent yeast (MSY) flocculant isolated 2
Sacromy
ces
cerevisia
e
UUIND
1 Yes Chlorel la
singula ris 96 Co-cultivation increase in biomass and protein
productivity of microalgae - Curre
nt
study
10 [0031] In the present study, yeast was added to microalgae and allowed to grow with algae for 10 days; flocculation time is only 3h under optimized conditions.
[0032] This method can also be employed in food industries where microalgae is used for single cell protein and production of other edible valuable products,
15

because with current harvesting method it increase the protein contents and contamination of algal biomass by Saccharomyces cerevisiae is not harmful.
[0033] Any microalgae belonging to the class Chlorophyceae including
5 Chlamydomonas and Chlorella, and typical genera belonging to the class
Cyanophyceae include Spirulina, Oscillatoria and Microcystis, preferably
Chlorella sorokiniana UUIND6 (GenBank accession number: KY780616) kept
for 4 days on Bold's Basal Medium (BBM), is selected for the study.
10 [0034] Co-cultivation of Saccharomyces cerevisiae UUIND1 with algae is an excellent tool for harvesting microalgae cells from the cultured medium. Moreover, the use of yeast also increased the algal biomass productivity and protein and carbohydrate contents. Algae grow with yeast in large size pellets, which could be easily separated from medium by simple gravity settling. The bio-15 flocculation of yeast-algae pellets was fast and showed 96 % harvesting within 3h. The presently study can provide energy efficient method for harvesting microalgae for biofuel. This association increases the biomass productivity by 2.3 folds. This method can also be employed in food industries where microalgae is used for single cell protein and production of other edible valuable products, 20 because with current harvesting method it increase the protein contents and contamination of algal biomass by Saccharomyces cerevisiae does not harmful. The following examples are given to illustrate the process of the present invention and should not be construed to limit the scope of the present invention:
25 Example 1
Pelletization and yeast-assisted flocculation
[0035] To achieve pelletization and boosting the biomass growth, yeast cells
grown in YPD broth at 30°C for 36-48 h, when solutions of 1 g l-1 dry weight
were equivalent to an optical density of 0•708 at 600 nm and a total cell count of
30 1•34 × 1010 cells l-. . 2 ml of YEPD broth media which containing yeast cells was
16

added to 100 ml of Bold's Basal Medium (BBM) containing 4 days old algae. After that algae was incubated for 10 days at 27 °C, with a photoperiod of 16 h: 8 h (light- dark cycle) and irradiated with 6 white cool fluorescent tubes (200 μmol m-2 s-1). Microalgae mono-culture was also grown on BBM as control. The yeast-5 algal mixture and control were shaken after every 6 h at 250 rpm for 10 min. All of the experiments were repeated at least three times. For flocculation efficiency analysis algal samples were analyzed after rotation (Scheme 1 in Figure 1). Table 3 shows the effect of different algal-yeast co-cultivation on on pigments of microalgae on 10th day.
10 Table 3: Effect of different algal-yeast co-cultivation on pigments of microalgae on 10th day

Chl a* (μg/ml) Chl b** (μg/ml) Car*** (μg/ml) Chl a + Chl b
Control 1.37±0.01 1.78±0.03 0.272±0.01 3.29±0.01
Algal-Yeast 1.48±0.02 1.30±0.03 0.520±0.01 2.53±0.04
* Chl a – Chlorophyll a. ** Chl b – Chlorophyll b. *** Car – Carotenoids. The data are mean ± S.D. for triplicate (n=3) results (p < 0.05).
15
Example 2
Total lipids Extraction and analysis
[0036] Lipids were extracted from yeast, mono cultural microalgae and yeast-20 algae pellets by a modified method of EG, Bligh and WJ Dyer [20]. Briefly, 50 ml of culture broth was transferred into centrifuge tubes and centrifuged at 3500 rpm for 5 min and the supernatant was discarded. The pellet was washed two times with distilled water, after that biomass was sonicated at 20 kHz for 5 min followed by addition of 10 ml of chloroform: methanol (2:1; v/v) and stirred for
25 30 min. Extract obtained was treated with 0.034% MgCl2, centrifuged at 3500
17

rpm for 5 min. Supernatant was washed two-three times with 1 ml of 2 N KCl/ methanol (4:l,v/v). 5 ml of chloroform/ methanol/ water (3:48:47, v/v/v) was added to it. The bottom chloroform layer was transferred to a new test tube and lipids yield was measured gravimetrically. Lipid production and percentage of lipid was calculated by the following equations:
[0037] Lipid yield %=Lipid content (g)/ Dry algae biomass (g)
[0038] Lipid productivity = Biomass productivity x Lipid yield (%)/ 100
[0039] For triacylglycerols (TAGs) detection lipid sample (5ul) was spotted on silica gel plate and TAGs were visualized according to A. Patel et al. method [21].

We Claim:

A process for enhancing 2-3 fold microalgae biomass productivity by yeast resulting in -96% harvesting in 3 hours, said process comprising the steps of:
a) allowing the growth of microalgae belonging to the class Chlorophyceae including Chlamydomonas and Chlorella, and typical genera belonging to the class Cyanophyceae include Spirulina, Oscillatoria and Microcystis, preferably Chlorella sorokiniana UUIND6 (GenBank accession number: KY780616) for 4 days on Bold's Basal Medium (BBM);
b) maintaining Saccharomyces cerevisiae UUIND1 (GenBank accession number.: KY385556) in YEPD (Yeast Extract Peptone Dextrose- glucose, 10; peptone, 5; yeast extract, 3 g/1) media, supplemented with an antibiotic;
c) adding 2 ml of said YEPD broth media containing yeast cells to 98 ml of said BBM containing 4 days old algae, thereby making it in 2:98 ratio of yeast and microalgae; and
d) incubating for 10 days at 27°C, with a light- dark cycle and
—9 —1
irradiated with 6 white cool fluorescent tubes (200 umol m s ).
The process as claimed in claim 1, wherein said YEPD media is supplemented with 40 ul antibiotic ampicillin in 25 mg/ml concentration.
The process as claimed in claim 1, wherein said photoperiod of light- dark cycle is of 16 hours light: 8 hours dark.