FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
"A hybrid cultivation system, and methods thereof
APPLICANT (S) Reliance Industries limited.
3rd Floor, Maker Chamber-IV, 222, Nariman Point, Mumbai-400021.
Maharashtra, India
The following specification particularly describes the invention and the manner in which it is to be performed.

A HYBRID CULTIVATION SYSTEM, AND METHODS THEREOF
FIELD OF INVENTION
The present invention belongs to the field of microalgae biotechnology. The present invention provides a hybrid cultivation system for aquatic algae that allows high-density light autotrophy, high yield and high-level efficiency and cultivation. Further, the present invention also provides methods for production of high-density biomass.
BACKGROUND AND PRIOR ART OF INVENTION
Algae are classified into several categories, each having thousands of species. Microalgae is one of those algae that has the potential to be used as a substitute for conventional sources of energy and other biomolecules such as biofuel, animal feed, pharmaceuticals, nutraceuticals, polyunsaturated fatty acids, phytonutrients, minerals, vitamins, and many other commodities. Microalgae are rudimentary plants that endure in water-based environments. Microalgae are unicellular photosynthetic microorganisms that depend on sunlight energy in combination with water and carbon dioxide to form biomass containing considerable amounts of molecules comparable to those found in vegetable oils. Microalgae reproduce predominantly by asexual cell division, although sexual reproduction can occur under certain growth conditions.
Microalgae have been used in aquaculture as a food supplement and in the production of chemical compounds (Raja et al, 2008), and more recently they have been proposed as an energy source for fuel production, offering several advantages over traditional cultures, such as high photosynthetic efficiency, high lipid content, continuous production of biomass and fast growth (Moo-Younga and Chisti, 1994; Sanchez et al, 2003; Miao and Wu, 2006), and also because they are a renewable source with low emissions of pollutants into the atmosphere (C02).
Cells of micro-algae are rich in various bioactivity substances such as proteins, amino acids, carbohydrates, vitamins, antibiotics, highly unsaturated fatty acids, polysaccharides, and colorants. This makes algae great resources with high economic value. Some algae possess abilities to produce hydrocarbons and oil lipids, and thus have promising application in field of renewable energy production. For example, algae lipids can be processed into biodiesel and/or jet-fuel (third generation feedstock for Biofuel), sugar and hydrocarbons into ethanol, and potentially into hydrogen, methanol and bio-power, while the residual biomass (such as

proteins, pigments, etc.) can be used for pharmaceuticals, nutraceuticals, cosmetics, fishmeal, biochar, or other applications.
Algae can double in volume overnight and can be continuously harvested on a daily basis. Algae need sunlight, carbon-dioxide, water, nutrients and temperature for their growth. The algal cells fix carbon-dioxide through photosynthesis and carbon usually comprises more than half of its dry weight. Therefore, sufficient carbon source and sunlight are needed during algae cultivation.
While algae are sometimes produced on land naturally that generally wouldn't be accessible for food production or various other purposes. However, there are numerous obstacles to overcome when manufacturing these products from algae, including choosing an appropriate alga, creating ideal growth conditions for maximum biomass yield, and avoiding contamination from undesirable algal species. All of these obstacles become more serious when algal growth is attempted on a large scale outdoors where weather and pollution are continual dangers. Accordingly, there is a considerable need for innovative algal production technology.
It is required to produce the algae using specific cultivation systems and extensive research efforts around the world have been made to understand different designs and strategies for biomass enhancement of the algae. Conventionally, to enhance algal biomass, different light intensities, nutrients, temperature, CO2 concentrations, pH, pond designs, depths, orientations, seasons, mixing with paddle wheel or motor were tried.
While the biological bases of the microalgae culture are widely developed on a small scale, they lack culturing capacity on a large scale to produce biomass at a low cost. For intensive production of microalgae, two culture systems are mainly used, open systems or Raceway ponds and closed systems or photobioreactors (PBRs).
In open systems, cultures are exposed to the atmosphere in a type of channel of large dimensions and are constantly stirred by a paddlewheel. Open systems can be categorized into natural waters (lakes, lagoons, ponds) and artificial ponds or containers. The most commonly used systems include shallow big ponds, tanks, circular ponds, raceway ponds and high-rate ponds (HRP). One advantage of open ponds is that they are easier and cheaper to construct and operate than most closed systems. However, limitations in open ponds include poor light utilization by cells, evaporative losses, diffusion of C02to the atmosphere, and requirement of large areas of land. Furthermore, contamination by viruses, fungi, predators and other fast-

growing heterotrophs have applied some restriction on the commercial production of algae in open culture systems to essentially only those organisms that can grow under extreme conditions. Also, due to low-efficient stirring mechanisms in open cultivation systems, their mass transfer rates may be very poor resulting in low biomass productivity.
Algal cultivation outdoors has long been established in ponds of different sizes and generally in elongated ellipsoidal shapes with separators inside the geometry to provide for channels where the culture would be in continuous flow. The continuous flow of the culture in the channels, essential for efficient mixing and availability of nutrients and CO2, is ensured by an external agency, the most conventional method is using Paddle wheels. Different operation methodologies at depths ranging from 15 cm to 20 cm using paddle wheel have been established. However, the major challenge of raceway ponds using paddle wheels is that they cannot be effectively run at heights below 15 cm. The optimum depth at which the ponds fitted with paddle wheels are to be run has been ascertained over the years to be between 15-20 centimeters. It has been observed that at this operational depth, cultivation is constrained by lower light penetration (~ 5 cm), higher sedimentation, lower maximum possible algal density at harvest and higher downstream volume to be processed.
To address these shortcomings of the conventional paddle wheel operated ponds, the inventors of the present invention have strived to develop and optimize a novel hybrid cultivation system which would provide better light availability, higher possible harvest density and consequently lower downstream processing volume. Thus, to provide a solution to the above-mentioned problem and further better the output-to-input ratio in terms of energy to biomass production, the inventors of the present invention have come up with a modification in the existing conventional paddle wheel ponds to enable running them at shallow depths and using paddle wheels itself.
Objectives of the Invention
The primary object of the present invention is to provide a hybrid cultivation system for high density biomass production and biomolecules from algal species.
Yet another important object of the present disclosure is to provide a method for production of biomass from microalgae.
Another object of the present invention is to provide a system and/or method of aquatic algal cultivation that reduces water usage, harvesting chemicals, cost, time,

Yet another important object of the present invention is to provide a hybrid cultivation system and/or method that facilitates sustainable high density biomass production and biomolecules induction of algae.
Yet another important object of the present invention is to provide use of the hybrid cultivation system for cultivating microalgae for high density biomass production and biomolecules induction of algae.
Additional objects, advantages, and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention.
SUMMARY OF THE INVENTION
The following summary is provided to facilitate an understanding of some of the innovative features unique to the disclosed embodiments and is not intended to the full description of the invention. A full appreciation of the various aspects of the preferred embodiments disclosed herein can be gained by taking the entire specification, claims, drawings, and abstract as a whole.
The present invention provides a hybrid cultivation system for aquatic organisms that allow high-density light autotrophy, high yield and high-level efficiency and cultivation. The present invention provides a hybrid cultivation system comprising a shallow channel; a deep local channel containing the paddle wheel, and wherein there exists a level difference between the depth of the shallow channel and the localised channel. Further, the present invention also provides methods for production of high-density biomass, from aquatic algae and the use of the system/methods for the cultivation of biomass.
Further areas of applicability of the present invention will become apparent from the detailed description provided hereinafter. It should be understood that the detailed description and specific examples while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
BRIEF DESCRIPTION OF DRAWINGS:
The accompanying drawings illustrate some of the embodiments of the present invention and together with the descriptions, serve to explain the invention. These drawings have been provided by way of illustration and not by way of limitation. The components in the

drawings are not necessarily drawn to scale, emphasis instead being placed upon clearly illustrating the principles of the aspects of the embodiments.
Figure 1: Top view of the algal cultivation system as embodied by the present invention.
Figure 2: Side view of the algal cultivation system as embodied by the present invention.
Figure 3: Areal Productivity Comparison of conventional cultivation system with hybrid cultivation system.
DETAIL DESCRIPTION OF THE INVENTION
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 an exemplary embodiment, and without intending to imply any limitation on the scope of this invention. Accordingly, the description is to be understood as an exemplary embodiment and teaching of invention and not intended to be taken restrictively.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
As used herein, biotechnological terms have their conventional meaning as illustrated by the following illustrative definitions:
The terms "algae", "algal cell", "algal strain" used interchangeably refer to eukaryotic aquatic organisms belonging to the kingdom Protista, that have the ability to conduct photosynthesis.
The term "biomass" as used herein, refers in general to organic matter produced by a biological cell. The renewable biological resource can include microbial materials (including algal materials), or materials produced biologically. In certain embodiments, the biomass is algal biomass. The algal biomass can be dry, substantially dry, or wet. "Biomass" should be understood to include proteins, lipids, and polysaccharides, whether retained within a biological cell or excreted from a biological cell, in addition to other molecules synthesized by a biological cell.
Detailed description of Figures:

Figure 1 shows the top view of the hybrid cultivation pond as embodied by the present invention. The cultivation apparatus (1) comprises of a shallow channel [2], a deep localised channel [3] and, a paddle wheel [4]. The shallow channel [2] and the localised channel [3] are connected to each other via two inclined planes [5,6], and the said paddle wheel [4] is placed between the two inclined planes [5,6] within the deep localized channel [3].
The system also comprises at least one or more water inlets [7], one or more inoculum inlets [8], one or more nutrient inlets [9], one or more algal culture inlets [10], one or more CO2 inlets [11], at least one or more culture harvest outlet [12], at least one or more discharge outlet [13] and one or more sensors [14],[15].
Figure 2 shows the sideview of the hybrid cultivation pond embodied by the present invention. It shows the inclination plane [5] after the paddle wheel [4] connecting the localized channel [3] with the shallow channel [2]. The arrows represent the continued fluid flow from the localized channel [3] with the shallow channel [2] due to the controlled movement of the submerged paddle wheel [4].
The inventors of the present invention conceptualized that the shallow culture height of the hybrid system would provide better light availability, higher possible harvest density and consequently lower downstream processing volume. The inventors understood that the modification can be made to the raceway pond by altering the depth and/or width of the raceway pond channels. For example, the depth and/or width of the raceway ponds may be non-uniform in order to modify the flow rate and mix in the raceway pond. The variable depth can also be used to alter the light penetration of different wavelengths of light, thus altering the light intensity and/or light wavelength ratio experienced by the algae.
Thus, the inventors designed the novel design of the hybrid cultivation system. Moreover, the novel design of the pond provides a better the output-to-input ratio in terms of energy to biomass production. The inventors of the present invention have come up with a modification to the existing conventional paddle wheel system to enable running them at shallow depths and using paddle wheels itself.
The inventors have developed a novel hybrid cultivation system wherein the design of the raceway ponds involves inclination planes on either side of the deep localized channel where the angle of ascend and the factors on which the angle of ascend is dependent on the pressure exerted by the paddle wheel as well as the structural features of the cultivation system. Accordingly, the angle of ascend is determined by striking a balance between the pressure

criteria and the constructional limitation and specifications. Angle of ascend on either side of the cultivation system would depend on the following factors:
F actors favouring lower ascend angles:
a. The angle of ascend or the gradient affects the back pressure exerted on the paddle
wheel: higher the angle of ascend, greater will be the pressure on the paddle wheel
which results in higher operational stresses on the wheel and even power consumption.
This would suggest that the lower the gradient better the design.
b. Steeper gradient increases the chances of deposition of biomass as the larger/accreted
algal cells might settle facing steep gradients. This also would suggest gentler ascend
angles.
F actors favouring higher ascend angles:
1. Construction limitations: Civil constructions with very low gradients become increasingly difficult and impractical and without providing much value. This makes higher angles more attractive from the construction point of view.
2. Effective depth in the region of the ascend is the other factor which supports higher gradients. Lower the gradient higher will be the effective depth in that portion of the pond so as to maintain a depth of 10 cm in the major portion of the pond.
Accordingly, depending on the above-mentioned competing factors, a range for angle for the system slopes can be designed. The inventors have deduced to design a hybrid cultivation system with an angle in the range of 6°-10° preferably 8°.
Further, the hybrid cultivation system comprises various inlets such as:
a. Inoculum inlet: This inlet brings in the initial culture to the pond which is to be
further stabilized and scaled up into the SOC volume.
b. Sea-Water inlet: This is meant for topping up the culture with the required sea water
to make-up for the volume which is harvest on a regular basis.
c. Fresh-water inlet: This line provides for freshwater injection if and when required.
This line ensures salinity control if need be.

d. CO2 inlet: CO2 is sparged through this line into the culture. The CO2 line is
controlled by a feedback loop from the pH sensor. The pH is to be maintained in
the range of 6.8 - 7.2. The CO2 valve cuts off below 6.8 and opens at 7.2.
e. The pond enclosure includes a paddle wheel assembly. This assembly mounted in
the deep localized channel.
f The embodiment is also provided with the harvest outlet for the regular culture harvesting.
g. Sensors like pH and temperature are online and feeds to the centralised system.
Many algae may be cultured using the methods of the invention. Thus, the algae may be cultured in any type of water. In specific embodiments, the algae are cultured in seawater, hypersaline water, desalination brine, brackish water, wastewater or freshwater. The term "water" encompasses all types of water. The term "fresh water" (as opposed to freshwater) is used to mean water that has not previously been used as an algal culture medium in the methods of the invention, and encompasses fresh seawater, hypersaline water, desalination brine, brackish water, wastewater or freshwater.
In the hybrid cultivation system embodied by the present invention, the algae are cultured in raceway ponds, which may be completely open to the ambient environment. Further modifications may be made to the ponds to alter the growth environment of the algae in order recreate environmental conditions experienced by algae during natural blooms, including the light intensity and color, the mixing regimes, variability and availability of nutrients, water temperature, algal cell density, availability and variability of dissolved gases and other conditions that stimulate a bloom-forming environment. The rate and timing of the addition of fresh water can be controlled, and in particular fresh deep seawater, to replicate the temperature and nutrient fluctuations experienced by natural algae as they are mixed or migrated between the cold, nutrient-replete deep waters and the nutrient-depleted surface waters.
Accordingly, an embodiment of the present invention relates to a hybrid algal cultivation system [1] comprising:
a shallow channel [2],
a deep localised channel [3] and,

a paddle wheel [4],
wherein the shallow channel [2] and the localised channel [3] are connected to each other via two inclined planes [5,6], and the said paddle wheel [4] is placed between the two inclined planes [5,6] within the deep localized channel [3] and the shallow channel and deep channel are separated;
and
wherein the cultivation system [1] enhances algal production in the shallow channel [2] by continued fluid flow due to the controlled movement of the paddle wheel [4].
Another embodiment of the present invention provides that the system further comprises at least one or more water inlets [7], one or more inoculum inlets [8], one or more nutrient inlets [9], one or more algal culture inlets [10], one or more CO2 inlets [11], at least one or more culture harvest outlet [12], at least one or more discharge outlet [13] and one or more sensors [14],[15].
Another embodiment of the present invention provides the system, wherein the localized channel [2] is a reservoir.
Another embodiment of the present invention provides the system, wherein the paddle wheel [4] is mounted in the reservoir.
Another embodiment of the present invention provides that the system, wherein the controlled movement of the paddle wheel leads to continued mixing of the culture in the localized channel.
Another embodiment of the present invention provides the system, wherein the level difference between the shallow channel and the deep localised channel is at least 20 cm.
Another embodiment of the present invention provides the system, wherein the depth of the shallow channel ranges from 5-12 cm and is uniform.
Another embodiment of the present invention provides that the system, wherein the depth in the localised channel is up to 35 cm.
Another embodiment of the present invention provides that the system, wherein the fluid transition is the continuous flow from the localized channel [3] to shallow channel [2] and back to the localized channel [3], and wherein with the completion of each lap of the fluid the kinetic

energy of the fluid is increased thereby facilitating the traversal of the fluid through the remainder of the system.
Another embodiment of the present invention provides that the system, wherein the inclined plane ascends to an angle of 6-10°.
Another embodiment of the present invention provides the system, wherein the fins of the paddle wheel are submerged by at least 2 cm below the surface of water.
Another embodiment of the present invention provides the system, wherein the paddle wheel has controlled movement and operated at a speed of 12-18 rpm.
Yet another important embodiment of the present invention provides a method for cultivation of algae, the method comprising:
a. Adding algal inoculum through the inoculum inlet [8] along with the fresh water
and sea water through the respective inlets [7a, 7b] to be cultured in the deep
localized channel [3] of the cultivation system [1];
b. Supplying nutrient(s) to the algal culture in said deep localized channel [3] of
the cultivation system [1] in a controlled manner;
c. allowing by continuous flow of fluid from the deep localized channel to a
shallow channel [2] in the cultivation system [1] through the controlled
movement of the submerged paddle wheel [4];
d. cultivating the algae in the shallow channel [2] of the cultivation system [1];
e. Harvesting the algal culture obtained in step (d) from the shallow channel [2]
from the harvesting outlet [12] in the cultivation apparatus.
Yet another important embodiment of the present invention provides a method for enhanced biomass production comprising the steps of:
a. inoculating algal culture in water, nutrients and CO2 in the deep localized
channel of the hybrid cultivation system as claimed in claim 1,
b. powering the paddle wheel to allow fluid transition from the deep localized
channel to the shallow channel at a uniform velocity,
c. cultivating the algae in the shallow channel maintaining the daily temperature
and pH of the cultivation system for optimum growth of the biomass from algae;

d. running the paddle wheel of step (b) continuously to allow steady flow of fluid
and allow continuous biomass production,
e. harvesting the biomass obtained in step (d) from the shallow channel.
Yet another embodiment of the present invention provides a by-product obtained from the system and/or the method as embodied by the present invention, and wherein the product comprises high-density biomass.
Another embodiment of the present invention provides the hybrid cultivation system and/or the method for enhanced biomass production, as and when used for cultivating aquatic algae.
Yet another important embodiment of the present invention provides use of the hybrid cultivation system for producing algae.
Without limiting the scope of the present invention as described above in any way, the present invention has been further explained through the examples provided below.
Experimental Data:
Illustrative Example 1:
The operational philosophy of the hybrid cultivation system as embodied by the present invention is in line with the conventional pond operation in respect of inoculation, stabilization, harvesting, CO2 and nutrient dosing, monitoring and crop control.
Inoculum is always maintained in an exponential phase of growth. While inoculating any production pond the density of inoculum will decide the volume of inoculum. The inventors of the present invention have deduced that 20-30% inoculum will be used to start pond. Nutrient which will be used in production pond, same will be used while inoculum development. The inventors have demonstrated that the inoculum harvested at the end of the day should be dominated by about 90-95 % by targeted algae with 5-10% of contamination in open pond being under permissible limit.
By using the above formula, the calculated nutrients can be added to maintain residual nutrient at optimum concentration as per cultivation SOC. Further, the actual initial OD after inoculation is measured and the reading is recorded as "Day 0". The culture is allowed to grow till it reaches to desired OD or saturation. Thus, the culture gets stabilized over a number of days.

After stabilization, in the evening every day, required amount of sample is withdrawn and sent to laboratory for analysis of residual nutrient and other necessary parameters. At the end of the day, samples of required quantity from designated location(s) are withdrawn for OD750, and other required parameters and part of the sample is sent to laboratory for AFDW and other necessary analysis (microscopy and others - crop control). The paddle wheels operate at an RPM of 12-18.
Nutrients required to be dosed into pond are calculated to maintain desired residual concentration as per pond operation SOC and added to the pond. The above steps are repeated throughout the operation of the pond. Recording and monitoring pond operation parameter such as pH, culture height, CO2 sparging and consumption help maintaining culture health and pond productivity.
The pH is to be monitored using the pH probes put in place in the ponds at different locations and in varying numbers depending upon the size of the pond. CO2 is sparged through the solvocarbs® which controls the pH in the system in the range of 6.8-7.2. Culture height is maintained according to the SOC of the pond operation.
Experimental example 1: Construction of the hybrid cultivation system embodied by the present invention.
For the performance evaluation purpose, one 120m2 cultivation system was selected for modification. A shallow channel of culture, about the paddle wheel, is created by increasing the depth of the pond in the section below 20 cm. The depth of the culture where the paddle wheel runs is thus 30 cm. The total length of the cultivation system was approximately made to be 6 meters. Figures 1 and 2 show the top and side view of the cultivation pond as embodied by the present invention. Further, minor modification to the paddle wheel was carried out to ensure better localised mixing. The modification was made to the fin of the paddle wheel such that the fin is completely submerged of about 2 - 3 cm height of culture topples over the fin.
To evaluate the performance of the modified setup, the inventors prepared a conventional cultivation pond contrasted it with the conventional paddle wheel pond run at 20 cm. The velocities in the ponds as a result of extension of the paddles also have been compared and detailed below:
Table 1: Specifications for the cultivation system of the present invention and the control.

Description Present invention Control unit
Area 120.00 120.00 2
m
Method of Harvest Semi-Turbidostat Semi-Turbidostat
Time taken for one complete rotation (of paddle wheel) 5.50 5.70 seconds
Angular velocity 1.14 1.10 rad/s
Paddle fin length 50.00 30.00 cm
Culture height ( reservoir, vicinity of the paddle wheel) 30.00 20.00 cm
Culture height (rest of the pond) 10.00 20.00 cm
Clearance 3.00 5.00 cm
Representative radius 37.50 22.50 cm
Imparted radial velocity by the paddle wheel 42.84 24.80 cm/s
Observed surface velocity 37.00 20.00 cm/s
Velocity reduction 5.84 4.80 cm/s
Head loss 0.24 0.11 cm
Example 2: Cultivation of the algae in the hybrid cultivation system as embodied by the present invention.
The cultivation system designed in example 1 was tested for its operational capability as per the similar parameters as per a conventional pond operation. The inoculation of the algae, stabilization of the pond, harvesting the biomass, addition of CO2 and nutrient dosing, monitoring and crop control were maintained identical in the pond. The inoculum was maintained in exponential phase of growth based on the density of inoculum will decide the volume of inoculum. In the hybrid cultivation system, 20-30% inoculum was used to start pond. Further, the nutrient which will be used in production pond, was kept similar to the nutrient used in the inoculum development.
Inoculum should be dominated (90-95 %) by targeted algae with 5-10% of contamination in open pond being under permissible limit.
Volume of inoculum and TSW required to start batch at desired initial OD at desired operating volume was calculated using following formula:

• Harvest or dilution % = (Inoculum OD-Desired OD)/ Inoculum OD
• TSW required = Harvest or dilution % x Target operating volume
• Inoculum required = (Total volume-TSW required)
The calculated amount of the nutrients was added to maintain residual nutrient at optimum concentration as per cultivation SOC. Actual initial OD after inoculation is measured and the reading is recorded as 'Day 0". The paddle wheels were operated at an RPM of 12-18 and the culture was allowed to grow till it reaches the desired OD or saturation (stabilization).
Nutrients required to be dosed into hybrid cultivation system were calculated to maintain desired residual concentration as per pond operation SOC and added to the pond. The above steps are repeated throughout the operation of the pond.
The inventors then carried out recording and monitoring pond operation parameter such as pH, culture height, CO2 sparging and consumption help maintaining culture health and pond productivity. The pH was monitored using the pH probes put in place in the ponds at different locations and in varying numbers depending upon the size of the pond. CO2 is sparged through the solvocarbs® which controls the pH in the system in the range of 6.8-7.2. Culture height is maintained according to the SOC of the pond operation.
At the end of the day, after sufficient growth of the algae, the required amount of sample is withdrawn and was sent to laboratory for analysis of residual nutrient and other necessary parameters. At the end of the day, samples of required quantity from designated location(s) are withdrawn for OD750, and other required parameters and part of the sample is sent to laboratory for AFDW and other necessary analysis (microscopy and others - crop control).
Harvest % and respective volume as per post-harvest OD set point were calculated by using following formulas.
• Harvest % = (Evening OD-Post harvest OD)/ Evening OD
• Harvest height= Harvest % x Total operating height
• Harvest height = TSW top up height
• Harvest or top up volume = Harvest or top up height x volume per unit height specific to pond

Example 3: Comparative assessment of efficiency of productivity and biomass output of the hybrid cultivation system
The hybrid cultivation system as exemplified in examples 1-2 and a convention cultivation system were prepared. Both the ponds were designed to have a surface area of 120 m2 each. Algal sp. Picochlorum (Karanja, Maharashtra) was inoculated in both of the cultivation systems and nutrient addition was done on residual concentration basis. Ash free dry weight (AFDW), Pulse Amplitude Modulation (PAM), and nutrient analysis were performed every day and microscopy for contamination profiling and crop protection was done as well.
The hourly temperature of the ponds during this time averaged between 22.0 °C to 37.5 °C while the daily temperature of the ponds during this time averaged between 25.9 °C to 31.0 °C. Further, the pH was maintained between 6.8 to 7.2 in both the ponds.
The salinity of the culture ranged between 3.6 to 4.4 during the experiment in both the ponds. PAM value for control pond ranged between 0.53 to 0.65 (average=0.59); for experimental system it ranged between 0.51 to 0.67 (average=0.60).
Contamination profile of the culture in both the systems was similar for the period of the study. The comparison of the pond performance was assessed which shows results in terms of productivity and biomass output. A graphical representation is shown in Figure 3. The results in this figure show a data comparison which demonstrates an 83% increase in terms of productivity in the Y01 pond (hybrid cultivation system) which has been operated at a shallow depth of 10 cm in contrast to 20 cm depth in Y02 (conventional system). The corresponding final biomass concentration also shows a 76 % higher value of Y01 as compared to Y02.
Thus, the inventors have experimentally proven that the particular design of the cultivation system as per the present invention has higher maximum attainable biomass concentration compared to 20 cm conventional cultivation practice. Accordingly, the present invention has the potential to be a game-changer in algal bio-refinery technology.
Advantages of the Invention:
The algal cultivation system and method represents an advancement over the existing methods to produce the algal culture in a minimum period to take the edge off the operational challenges in the production of high-density biomass from it. The advances are characterized by the following features.

• Lower operational depth ranging from 5 cm to 15 cm which ensures better photon/light availability to the algal cells.
• The algae cultivation system of the present invention helps to enhance the algal biomass productivity to about 76 % when compared with a conventional cultivation apparatus.
• The combination of deep and shallow depts of the hybrid cultivation system helps to reduce the water usage, harvesting chemicals, cost, and time.
• The wheel paddle driven cultivation system helps to reduce the grazers, contamination level and enables the use of fragile microorganisms in this system.
• The cultivation system increases productivity by about 83% compared to conventional paddle wheel systems.
• Algae culture can be maintained in both nutrient rich and nutrient depleted conditions as per requirement such as nutrient rich culture can be used for protein production, whereas nutrient depleted culture can be used for biomolecules and pigment induction.
• The biomass harvesting cost can be minimized due to high harvesting biomass density by multiple cultivation apparatus.
• The algae cultivation system of the present invention requires less water for cultivation as the plant incorporates a hybrid design in a single pond.
The foregoing broadly outlines the features and technical advantages of the present disclosure in order that the detailed description of the disclosure that follows may be better understood. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying the disclosed methods or for carrying out the same purposes of the present disclosure.
It must be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "an expression cassette" includes one or more of the expression cassettes disclosed herein and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but

not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or sometimes when used herein with the term "having".
When used herein "consisting of excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms.
Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present invention are generally performed according to conventional methods well-known in art. Generally, nomenclatures used in connection with techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.
The methods and techniques of the present invention are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e. g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001); Ausubel et al., Current Protocols in Molecular Biology, J, Greene Publishing Associates (1992, and Supplements to 2002); Handbook of Biochemistry: Section A Proteins, Vol 11976 CRC Press; Handbook of Biochemistry: Section A Proteins, Vol II 1976 CRC Press. The nomenclatures used in connection with, and the laboratory procedures and techniques of, molecular and cellular biology, protein biochemistry, enzymology and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

WE CLAIM:
1. A hybrid algal cultivation system [1] comprising:
a shallow channel [2],
a deep localised channel [3] and,
a paddle wheel [4],
wherein the shallow channel [2] and the localised channel [3] are connected to each
other via two inclined planes [5,6], and the said paddle wheel [4] is placed between the
two inclined planes [5,6] within the deep localized channel [3];
and
wherein the cultivation system [1] enhances algal production by continued fluid flow
due to the controlled movement of the paddle wheel [4].
2. The system as claimed in claim 1, wherein the system further comprises at least one or more water inlets [7], one or more inoculum inlets [8], one or more nutrient inlets [9], one or more algal culture inlets [10], one or more CO2 inlets [11], at least one or more culture harvest outlet [12], at least one or more discharge outlet [13] and one or more sensors [14], [15].
3. The system as claimed in claim 1, wherein the localized channel [2] is a reservoir.
4. The system as claimed in claim 1-3, wherein the paddle wheel [4] is mounted in the reservoir.
5. The system as claimed in claim 1, wherein the controlled movement of the paddle wheel leads to continued mixing of the culture in the localized channel.
6. The system as claimed in claim 1, wherein the level difference between the shallow channel and the localised channel is at least 20 cm.
7. The system as claimed in claim 1, wherein the depth of the shallow channel ranges from 5-12 cm and is uniform.
8. The system as claimed in claim 1, wherein the depth in the localised channel is up to 35 cm.
9. The system as claimed in claim 1, wherein the fluid transition is the continuous flow from the localized channel [3] to shallow channel [2] and back to the localized channel [3], and wherein with the completion of each lap of the fluid the kinetic energy of the fluid is increased thereby facilitating the traversal of the fluid through the remainder of the system.

10. The system as claimed in claim 1, wherein the inclined plane ascends to an angle of 6-10°.
11. The system as claimed in claim 1, wherein the fins of the paddle wheel are submerged by at least 2 cm below the surface of water.
12. The system as claimed in claim 1, wherein the paddle wheel has controlled movement and operated at a speed of 12-18 rpm.
13. A method for cultivation of algae, the method comprising:
a. Adding algal inoculum through the inoculum inlet [8] along with the fresh water
and sea water through the respective inlets [7a, 7b] to be cultured in the deep
localized channel [3] of the cultivation system [1];
b. Supplying nutrient(s) to the algal culture in said deep localized channel [3] of
the cultivation system [1] in a controlled manner;
c. allowing by continuous flow of fluid from the deep localized channel to a
shallow channel [2] in the cultivation system [1] through the controlled
movement of the paddle wheel [4];
d. cultivating the algae in the shallow channel [2] of the cultivation system [1];
e. Harvesting the algal culture obtained in step (d) from the shallow channel [2]
from the harvesting outlet [12] in the cultivation apparatus.
14. A method for enhanced biomass production by the method as claimed in claim 13, the
method comprising the steps of:
a. inoculating algal culture in water, nutrients and CO2 in the deep localized
channel of the hybrid cultivation system as claimed in claim 1,
b. powering the paddle wheel to allow fluid transition from the deep localized
channel to the shallow channel at a uniform velocity,
c. cultivating the algae in the shallow channel maintaining the daily temperature
and pH of the cultivation system for optimum growth of the biomass from algae;
d. running the paddle wheel of step (b) continuously to allow steady flow of fluid
and allow continuous biomass production,
e. harvesting the biomass obtained in step (d) from the shallow channel.
15. A by-product obtained from the system as claimed in claims 1-13 or the method as claimed in claims 13-14, and wherein the product comprises high-density biomass.
16. The hybrid cultivation system as claimed in claims 1-12 and the method as claimed in 13-14, as and when used for cultivating aquatic algae.

17. Use of the hybrid cultivation system as claimed in claims 1-12, for producing algae.