FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
ALGAE CULTIVATION SYSTEM AND METHOD
APPLICANT
RELIANCE INDUSTRIES LIMITED
An Indian Company Of
3rd Floor, Maker Chamber-IV, 222, Nariman Point, Mumbai-400 021, Maharashtra, India
The following specification particularly describes the invention and the manner in which it is to be
performed.
1
FIELD OF THE INVENTION
The present invention relates to the field of algal cultivation and production of high value
biomolecules and products thereof. The present disclosure relates to an algal cultivation system
and method comprising multiple cultivation apparatus for production of high-density biomass,
biomolecules, feedstock and phytonutrients.
BACKGROUND 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. While algae are
sometimes produced on land that generally wouldn't be accessible for food production or various
other purposes, they are a particularly enticing source. However, there are nevertheless
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.
Basis the purpose or application of the algae, 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 pumps were tried.
2
One of them is a raceway pond which is a specific kind of artificially constructed pond used
essentially for the growth of microorganisms like microalgae. The raceway pond also includes
some pumping instrument(s), such as a paddle wheel, pumps, and the like, that generates the flow
movement required for the development of microorganisms, typically algae.
The functioning concentration of the algae and specific growth rate are estimated to produce the
mass productivity of algae as it being photosynthetic microorganisms. Utilizing light
advantageously contributes to both the overall large-scale output and the specific growth rate. To
increase overall productivity, light must therefore be used effectively. By reducing the optical path
and improving light use efficiency, the shallow pond conception constitutes considerable
advantageous features. Evaporation is, on the other hand, a serious issue that causes problems
with the efficient functioning of shallow-depth systems. Algae have experienced biological stress
as a result of evaporation's quick changes in the salinity and temperature of the operational
environment. Eventually, the rate of culture growth is interrupted.
Additionally, if the flow movement is not maintained in algae cultivation systems, flow
components settle to the bottom surface of the passageway and more necessities and issues arise.
For instance, reduction in the efficiency of the cultivation process in terms of low productivity,
contamination etc. These drawbacks ultimately lead to the need for repeated processes to de-settle
the waste material that has accumulated in the pathways, however due to which the cost to execute
for one or more of the processes mentioned above increases. For better homogenization of the
biomass and nutrient uniformity, vertical mixing is crucial. However, because there is little to no
vertical mixing in the raceway pond, the flow completely stratifies, which impairs the mass transfer
of high-density particles.
However, the available methods/technologies suffer from one or more drawbacks or limitations
such as low biomass density, time consuming, huge water requirement, nutrient wastage and many
more. Thus, there still remains a need to develop improved technologies or methods to address
the challenges associated with the aforesaid prior art approaches, to ensure reduction in water
usage, harvesting chemicals, cost, time, reduction in the grazers and contamination level, and
increase in biomass production which is more important in open cultivation system.
3
OBJECTIVES OF THE INVENTION
The primary object of the present invention is to provide a system [15] or method for high density
biomass production and biomolecules induction of microalgae.
Another object of the present invention is to provide a system [15] or method of microalgae
cultivation that reduces water usage, harvesting chemicals, cost, time, the grazers, and
contamination level.
Another object of the present disclosure is to provide a system [15] for cultivating microalgae that
includes combinatorial approach of one or more cultivation apparatus.
Another object of the present disclosure is to provide a system [15] for cultivating microalgae that
includes transferring about 10%-30% of the microalgal culture of the first cultivation apparatus
[1] to second cultivation apparatus [2] for high density biomass production of microalgae.
Another important object of the present invention is to provide a method that facilitates sustainable
high density biomass production and biomolecules induction of microalgae.
In particular, the object of the present invention is to provide a method for cultivating microalgae
using the system [15] for high density biomass production and biomolecules induction of
microalgae.
Yet another object of the present invention is to provide a system [15] and method for cultivating
microalgae that prevents unwanted microbial growth as well as contamination.
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.
4
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.
In an aspect, the present invention provides a novel approach for cultivation of microalgae
involving a combination approach of two or more cultivation apparatus wherein the second
cultivation apparatus [2] is of the lower depth than the first cultivation apparatus [1].
In an aspect, the present invention provides a novel approach for cultivation of microalgae
involving a combination approach of deep and shallow cultivation apparatus wherein the first
cultivation apparatus [1] is deep apparatus, and the second cultivation apparatus [2] is shallow
apparatus optionally in controlled fluid communication with each other.
In an aspect, the present invention provides an improved integrated microalgae cultivation system
[15], said system comprising a first cultivation apparatus [1] and a second cultivation apparatus
[2] which are deep and shallow respectively, one or more circulating means, one or more inlets
and one or more outlets.
In another aspect, the said system provides transferring of about 10-30% of the microalgal culture
of the first cultivation apparatus [1] to the second cultivation apparatus [2].
In another aspect, the present invention provides an improved method for microalgae cultivation
comprising,
(a) Adding microalgal inoculum to be cultured in a first cultivation apparatus [1];
(b) Supplying nutrient(s) to the microalgal culture I in said first cultivation apparatus [1]
in a controlled manner;
(c) Harvesting a portion of said microalgal culture I from the first cultivation apparatus
[1] periodically;
5
(d) Transferring the harvested microalgal culture I of step (c) into a second cultivation
apparatus [2] and allowing the microalgae to grow to obtain microalgal culture II;
(e) Harvesting the microalgal culture II of step (d) from the second cultivation
apparatus [2].
In another aspect, the present invention provides use of the microalgae cultivation system for
producing microalgae.
In another aspect, the present invention provides use of the microalgae cultivation method for
producing microalgae.
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 THE ACCOMPANYING DRAWINGS
The drawings described herein are for illustrative purposes only of selected embodiments and not
all possible implementations and are not intended to limit the scope of the present disclosure. The
invention itself, however, both as to organization and method of operation, may best be understood
by reference to the detailed description which follows taken in conjunction with the accompanying
drawings in which:
Figure 1 illustrates an overview of the proposed system in accordance with the present invention.
Figure 2 illustrates an overview of another embodiment of the proposed system in accordance
with the present invention.
Figure 3 illustrates results of biomass areal productivity of microalgae using the system and
method of the present invention.
6
Figure 4 shows comparison of the biomass areal productivity of microalgae between the present
system, present system volume based and single shallow cultivation system.
DETAILED DESCRIPTION OF THE INVENTION
At the very outset, 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, without
intending to imply any limitation on the scope of this invention. Accordingly, the description and
examples are to be understood as exemplary embodiments for teaching the invention and not
intended to be taken restrictively.
The details of one or more embodiments of the invention are set forth in the accompanying
description below including specific details of the best mode contemplated by the inventors for
carrying out the invention, by way of example. It will be apparent to one skilled in the art that the
present invention may be practiced without limitation to these specific details.
Unless otherwise defined, scientific and technical terms used herein 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.
Abbreviations used:
OD- Optical density
CO2- Carbon Dioxide
Definitions:
The use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”,
“include”, “includes”, and “including” are not intended to be limiting. It is to be understood that
both the foregoing general description and this detailed description are exemplary and explanatory
only and are not restrictive.
7
The term “cultivation system” as used herein refers to a system which is dedicated to the
development or the farming of algae species such as phytoplankton, microphytes, or planktonic
algae.
The term “algal culture” or “microalgal culture” as used herein refers to the form of aquaculture
that involves the farming of various species of algae.
The term “optical density” as used herein refers to estimate the growth and metabolic activity of
the microalgae cells.
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.
In an important embodiment of the present invention, a microalgae cultivation system and method
is set out/organized for high density biomass production and biomolecules induction of microalgae
culture.
In one of the embodiments of the present invention, the microalgae cultivation system is
configured as a combinatorial approach comprising: one or more cultivation apparatuses and one
or more circulating means positioned in the first cultivation apparatus [1] and the second
cultivation apparatus [2] to enable circulation of the microalgal culture.
In one of the embodiments of the present invention, the microalgae cultivation system is
configured as a combinatorial approach comprising: one or more cultivation apparatuses; one or
more circulating means positioned in the first cultivation apparatus [1] and the second cultivation
apparatus [2] to enable circulation of the microalgal culture; one or more inlets; and one or more
outlets.
8
In one of the embodiments of the present invention, the microalgae cultivation system is
configured as a combinatorial approach comprising: one or more cultivation apparatuses; one or
more circulating means positioned in the first cultivation apparatus [1] and the second cultivation
apparatus [2] to enable circulation of the micro algal culture; one or more inlets; and one or more
outlets.
In one of the embodiments of the present invention, the microalgae cultivation system is
configured as a combinatorial approach comprising: one or more cultivation apparatuses; one or
more circulating means positioned in the cultivation apparatuses to enable circulation of the micro
algal culture; one or more inlets in the cultivation apparatuses; and one or more outlets in the
cultivation apparatuses; wherein a second cultivation apparatus [2] is of lower depth than a first
cultivation apparatus [1].
In one of the embodiments of the present invention, the microalgae cultivation system is
configured as a combinatorial approach comprising: one or more cultivation apparatuses; one or
more circulating means positioned in the cultivation apparatuses to enable circulation of the micro
algal culture; one or more inlets in the cultivation apparatuses; and one or more outlets in the
cultivation apparatuses; wherein about 10%-30% of the microalgal culture is transferred from one
of the cultivation apparatus to another cultivation apparatus.
In one of the embodiments of the present invention, the microalgae cultivation system is
configured as a combinatorial approach comprising: one or more cultivation apparatuses; one or
more circulating means positioned in the cultivation apparatuses to enable circulation of the micro
algal culture; one or more inlets in the cultivation apparatuses; and one or more outlets in the
cultivation apparatuses; wherein a second cultivation apparatus [2] is of lower depth than a first
cultivation apparatus [1] and wherein about 10%-30% of the microalgal culture is transferred from
one of the high depth cultivation apparatus to another lower depth cultivation apparatus.
In one of the embodiments, the present invention provides a microalgae cultivation system [15]
comprising: a first cultivation apparatus [1] for cultivation of microalgae; a second cultivation
apparatus [2] for cultivation of microalgae; one or more circulating means positioned in each of
9
the first cultivation apparatus [1] and the second cultivation apparatus [2] to enable circulation of
microalgal culture.
In one of the embodiments, the present invention provides a microalgae cultivation system [15]
comprising: a first cultivation apparatus [1] for cultivation of microalgae; a second cultivation
apparatus [2] for cultivation of microalgae; one or more circulating means positioned in each of
the first cultivation apparatus [1] and the second cultivation apparatus [2] to enable circulation of
microalgal culture; wherein the second cultivation apparatus [2] is of lower depth than the first
cultivation apparatus [1].
In one of the embodiments, the present invention provides a microalgae cultivation system [15]
comprising: a first cultivation apparatus [1] for cultivation of microalgae; a second cultivation
apparatus [2] for cultivation of microalgae; one or more circulating means positioned in each of
the first cultivation apparatus [1] and the second cultivation apparatus [2] to enable circulation of
microalgal culture; wherein the second cultivation apparatus [2] is of lower depth than the first
cultivation apparatus [1] and wherein about 10%-30% of the microalgal culture of the first
cultivation apparatus [1] is transferred to the second cultivation apparatus [2] once every 24 hours.
In one of the embodiments of the system of the present invention, the first cultivation apparatus
[1] is a high depth apparatus, and the second cultivation apparatus [2] is a shallow depth apparatus.
In one of the embodiments of the system of the present invention, the depth of the first cultivation
apparatus [1] is 20-30 cm and the depth of the second cultivation apparatus [2] is 5-10 cm.
In one of the embodiments of the system of the present invention, the first cultivation apparatus
[1] has a depth ranging from about 20-30 cm, 20-28 cm, 20-26 cm, 20-24 cm, or 20-22 cm.
In one of the embodiments of the system of the present invention, the first cultivation apparatus
[1] has a depth of about 20 cm.
10
In one of the embodiments of the system of the present invention, the second cultivation apparatus
[2] has a depth ranging from about 5-10 cm, 5-9 cm, 5-8 cm, 5-7 cm or 5-6 cm.
In one of the embodiments of the system of the present invention, the second cultivation apparatus
[2] according to the present invention has a depth of about 5 cm.
In one of the embodiments of the system of the present invention, the first cultivation apparatus
[1] and the second cultivation apparatus [2] are optionally connected by a connecting means.
In one of the embodiments of the system of the present invention, the first cultivation apparatus
[1] and the second cultivation apparatus [2] are optionally in controlled fluid communication with
each other.
In one of the embodiments of the system of the present invention, the circulating means located in
the first cultivation apparatus [1] is a paddle wheel assembly [3].
In one of the embodiments of the system of the present invention, the circulating means located in
the second cultivation apparatus [1] is a pump assembly [4] to facilitate the increase in high density
biomass concentration.
In one of the embodiments of the system of the present invention, the first cultivation apparatus
[1] and the second cultivation apparatus [2] comprise at least one or more inlets and at least one
or more outlets.
In one of the embodiments of the system of the present invention, the one or more inlets in the first
cultivation apparatus [1] and second cultivation apparatus [2] comprise at least one or more water
inlets [5,6], one or more algal culture inlets [7], one or more nutrient inlets [8], one or more CO2
inlets [9], one or more pH probe inlets [11] and one or more temperature probe inlets [11].
11
In one of the embodiments of the system of the present invention, the one or more outlets in the
first cultivation apparatus [1] and second cultivation apparatus [2] comprise at least one or more
culture harvest outlets [12] and at least one or more discharge outlets [13].
In one of the embodiments of the system of the present invention, the pH of the first [1] and second
[2] cultivation apparatus is maintained between 6.8-7.2.
In one of the embodiments of the system of the present invention, the pH of the first [1] and second
[2] cultivation apparatus is maintained in the range of about 6.8-7, 7-7.2, 6.8-6.9, 6.9-7, 7-7.1 or
7.1-7.2.
In one of embodiments of the system of the present invention, the pH is maintained using CO2
using pH controller.
In one of the embodiments of the system of the present invention, the temperature of the first
cultivation apparatus [1] ranges from about 20-36 °C, 20-34 °C, 20-32 °C, 20-30 °C, 20-28 °C,
20-26 °C, 20-24 °C, and 20-22 °C.
In one of the embodiments of the system of the present invention, the temperature of the second
cultivation apparatus [2] ranges from about 20-43 °C, 20-41 °C, 20-39 °C, 20-37 °C, 20-35 °C,
20-33 °C, 20-31 °C, 20-29 °C, 20-27 °C, 20-25 °C, 20-23 °C or 20-21 °C.
In one of the embodiments of the system of the present invention, at least one mid-wall [14] is
optionally disposed in the first cultivation apparatus [1] and the second cultivation apparatus [2]
for improving the fluid circulation.
In one of the embodiments of the system of the present invention, the cultivation apparatuses are
connected via connecting means for transferring the microalgal culture. Accordingly, the
movement of culture from the first cultivation apparatus [1] to the second cultivation apparatus [2]
helps in minimizing the cross contamination and improving the growth of target microalgae
population to more than 99% to maintain the quality of biomass for algal biorefinery.
12
In one of the embodiments of the system of the present invention, the paddle wheel [3] of the first
cultivation apparatus [1] facilitates the semi-continuous mode of cultivation.
In one of the embodiments of the system of the present invention, the algal culture inlet [7]
provides a passage to the culture to enter in the first cultivation apparatus [1] followed by mixing
of the microalgae culture by the circulating means using the paddle wheel assembly [3].
In one of the embodiments of the system of the present invention, the culture harvest outlet [8]
provides a passage to the culture ensuring harvesting of the algal culture.
In one of the embodiments of the system of the present invention, the second cultivation apparatus
[2] facilitates the full harvest of the microalgal culture.
In one of the embodiments of the system of the present invention, the microalgae strain is selected
from but not limited to unicellular green microalgae, blue-green microalgae, diatoms and
combinations thereof.
In one of the embodiments of the system of the present invention, the unicellular green microalgae
strain is selected from but not limited to Chlorella, Nannochloris, Nannochloropsis, Picochlorum
and combinations thereof.
In one of the embodiments of the system of the present invention, the blue-green microalgae strain
is selected from but not limited to Synechococcus, Chroococcus and combinations thereof.
In one of the embodiments of the system of the present invention, the microalgae are cultured in a
nutrient media selected from but not limited to micro and macro nutrients containing carbon,
nitrogen, phosphorus, and trace metals.
In one of embodiments of the system of the present invention, the source of nitrogen and
phosphorous is urea and phosphoric acid respectively.
13
One of the embodiments of the present invention is to provide a method for cultivation of
microalgae for selectively cultivating a target microalgal culture in high density.
In one of embodiments of the present invention, a method for cultivation of microalgae is provided,
the method comprising:
(a) Adding microalgal inoculum to be cultured in a first cultivation apparatus [1];
(b) Supplying nutrient(s) to the microalgal culture I in said first cultivation apparatus [1]
in a controlled manner;
(c) Harvesting a portion of said microalgal culture I from the first cultivation apparatus
[1] periodically;
(d) Transferring the harvested microalgal culture I of step (c) into a second cultivation
apparatus [2] and allowing the microalgae to grow to obtain microalgal culture II;
(e) Harvesting the microalgal culture II of step (d) from the second cultivation apparatus
[2].
In another embodiment of the method of the present invention, during the operation of the
microalgae cultivation system, the configuration of the first cultivation apparatus and the operation
of the flow diverter device causes mixing of the microalgal culture under semi-continuous mode
of cultivation throughout the first cultivation apparatus followed by transferring of 10%-30% of
the total culture of the first cultivation apparatus to the second cultivation apparatus for culturing
in semi-continuous turbidostatic dilution mode.
In one of embodiments of the present invention, a method for cultivation of microalgae is provided,
the method comprising:
(a) Adding microalgal inoculum to be cultured in a first cultivation apparatus [1], wherein the
pH of the first cultivation apparatus [1] is maintained in the range of about 6.8-7.2
(b) Supplying nutrient(s) to the microalgal culture I in said first cultivation apparatus [1] in a
controlled manner, wherein the nutrients are nitrogen and phosphorous in the ratio of 16:1
(N:P);
14
(c) Harvesting a portion of said microalgal culture I from the first cultivation apparatus [1]
periodically, wherein the portion of microalgae is in the range of about 10%-30% of the
microalgal culture I;
(d) Transferring the harvested microalgal culture I of step (c) into a second cultivation apparatus
[2] and allowing the microalgae to grow to obtain microalgal culture II;
(e) Harvesting the microalgal culture II of step (d) from the second cultivation apparatus [2].
In one of the embodiments of the method of the present invention, the first cultivation apparatus
[1] is a high depth apparatus, and the second cultivation apparatus [2] is a shallow depth apparatus.
In one of the embodiments of the method of the present invention, the depth of the first cultivation
apparatus [1] is 20-30 cm and the depth of the second cultivation apparatus [2] is 5-10 cm.
In one of the embodiments of the method of the present invention, the first cultivation apparatus
[1] has a depth ranging from about 20-30 cm, 20-28 cm, 20-26 cm, 20-24 cm, or 20-22 cm.
In one of the embodiments of the method of the present invention, the first cultivation apparatus
[1] has a depth of about 20 cm.
In one of the embodiments of the method of the present invention, the second cultivation apparatus
[2] has a depth ranging from about 5-10 cm, 5-9 cm, 5-8 cm, 5-7 cm or 5-6 cm.
In one of the embodiments of the method of the present invention, the second cultivation apparatus
[2] according to the present invention has a depth of about 5 cm.
In one of the embodiments of the method of the present invention, the first cultivation apparatus
[1] and the second cultivation apparatus [2] are optionally connected by a connecting means.
In one of the embodiments of the method of the present invention, the first cultivation apparatus
[1] and the second cultivation apparatus [2] are optionally in controlled fluid communication with
each other.
15
In one of the embodiments of the method of the present invention, the circulating means located
in the first cultivation apparatus [1] is a paddle wheel assembly [3].
In one of the embodiments of the method of the present invention, the circulating means located
in the second cultivation apparatus [1] is a pump assembly [4] to facilitate the increase in high
density biomass concentration.
In one of the embodiments of the method of the present invention, the first cultivation apparatus
[1] and the second cultivation apparatus [2] comprise at least one or more inlets and at least one
or more outlets.
In one of the embodiments of the method of the present invention, the one or more inlets in the
first cultivation apparatus [1] and second cultivation apparatus [2] comprise at least one or more
water inlets [5,6], one or more algal culture inlets [7], one or more nutrient inlets [8], one or more
CO2 inlets [9], one or more pH probe inlets [11] and one or more temperature probe inlets [11].
In one of the embodiments of the method of the present invention, the one or more outlets in the
first cultivation apparatus [1] and second cultivation apparatus [2] comprise at least one or more
culture harvest outlets [12] and at least one or more discharge outlets [13].
In one of the embodiments of the method of the present invention, the pH of the first [1] and second
[2] cultivation apparatus is maintained between 6.8-7.2.
In one of embodiments of the method of the present invention, the pH of the first cultivation
apparatus [1] is maintained in the range of about 6.8-7, 7-7.2, 6.8-6.9, 6.9-7, 7-7.1 or 7.1-7.2.
In one of embodiments of the method of the present invention, the pH is maintained using CO2
using pH controller.
16
In one of the embodiments of the method of the present invention, the temperature of the first
cultivation apparatus [1] ranges from about 20-36 °C, 20-34 °C, 20-32 °C, 20-30 °C, 20-28 °C,
20-26 °C, 20-24 °C, and 20-22 °C.
In one of the embodiments of the method of the present invention, the temperature of the second
cultivation apparatus [2] ranges from about 20-43 °C, 20-41 °C, 20-39 °C, 20-37 °C, 20-35 °C,
20-33 °C, 20-31 °C, 20-29 °C, 20-27 °C, 20-25 °C, 20-23 °C or 20-21 °C.
In one of embodiments of the method of the present invention, the harvesting performed in step
(e) is the complete harvest of everyday.
In one of the embodiments of the present invention, the method of the harvesting of the microalgae
culture in step (e) is performed manually, physical methods, gravity settling or pumping means.
In one of embodiments of the method of the present invention, the culture is allowed to grow in
the first cultivation apparatus [1] with lower density using paddle wheel assembly [3] at 750nm
till the optical density (OD) is increased to 2.0.
In one of the embodiments of the method of the present invention, at least one mid-wall [14] is
optionally disposed in the first cultivation apparatus [1] and the second cultivation apparatus [2]
for improving the fluid circulation.
In one of the embodiments of the method of the present invention, the transferring of the microalgal
culture is facilitated via a connecting means between the first cultivation apparatus [1] and the
second cultivation apparatus [2]. In accordance with this, the movement of culture from the first
cultivation apparatus [1] to the second cultivation apparatus [2] helps in minimizing the cross
contamination and improving the growth of target microalgae population to more than 99% to
maintain the quality of biomass for algal biorefinery.
In one of the embodiments of the method of the present invention, the paddle wheel [3] of the first
cultivation apparatus [1] facilitates the semi-continuous mode of cultivation.
17
In one of the embodiments of the method of the present invention, the algal culture inlet [7]
provides a passage to the culture to enter in the first cultivation apparatus [1] followed by mixing
of the microalgae culture by the circulating means using the paddle wheel assembly [3].
In one of the embodiments of the method of the present invention, the CO2 inlet [9] provides
passage of CO2 gas in first cultivation apparatus [1] or second cultivation apparatus [2] as and
when required.
In one of the embodiments of the method of the present invention, the nutrient inlet [8] provides
passage of nutrients in first cultivation apparatus [1] or second cultivation apparatus [2] as and
when required.
In one of the embodiments of the method of the present invention, the pH probe inlet [11] and one
or more temperature probe inlet [11] provides passage for respective probes for measuring of the
pH and temperature respectively of the first cultivation apparatus [1] or second cultivation
apparatus [2].
In one of the embodiments of the method of the present invention, the culture harvest outlet [8]
provides a passage to the culture ensuring harvesting of the algal culture.
In one of the embodiments of the method of the present invention, the discharge outlet [13]
provides water discharge from first cultivation apparatus [1] or second cultivation apparatus [2].
In one of the embodiments of the method of the present invention, the second cultivation apparatus
[2] facilitates the full harvest of the microalgal culture.
In one of the embodiments of the method of the present invention, the microalgae strain is selected
from but not limited to unicellular green microalgae, blue-green microalgae, diatoms and
combinations thereof.
18
In one of the embodiments of the method of the present invention, the unicellular green microalgae
strain is selected from but not limited to Chlorella, Nannochloris, Nannochloropsis, Picochlorum
and combinations thereof.
In one of the embodiments of the method of the present invention, the blue-green microalgae strain
is selected from but not limited to Synechococcus, Chroococcus and combinations thereof.
In one of the embodiments of the method of the present invention, the microalgae are cultured in
a nutrient media selected from but not limited to micro and macro nutrients containing carbon,
nitrogen, phosphorus, and trace metals.
In one of embodiments of the method of the present invention, the source of nitrogen and
phosphorous is urea and phosphoric acid.
One of embodiments of the present invention, relates to a product produced from microalgae
obtained using the system or the method, wherein the product comprises high-density biomass,
biomolecules, feedstock, and phytonutrients.
One of embodiments of the present invention, relates to use of the micro-algae cultivation system
[15] for producing microalgae.
One of embodiments of the present invention relates to use of the method for producing
microalgae.
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.
EXAMPLES
The present invention is further explained in the form of the following examples. The following
examples particularly describe the manner in which the invention is to be performed. However, it
19
is to be understood that the following examples are merely illustrative and are not to be taken as
limitations upon the scope of the invention.
Example 1: Inoculation and cultivation
Example 1A:
In first cultivation apparatus [1], 30,000 L of Picochlorum sp inoculum, collected from Karanja,
Maharashtra, was mixed with 70,000 L of UPA medium, which was prepared using 0.2μm filtered
sea water. After inoculation, the culture pH was maintained between 6.8-7.2 using CO2 (99%
purity) using pH controller. In the experimental cultivation apparatus [1] 30 ppm of total nitrogen
was maintained (urea as nitrogen source and phosphoric acid used as phosphorus source) by
keeping 16:1 ratio of N:P. Nutrients were toped up based on its consumption.
The algal culture was then allowed to reach maximum of growth in terms of OD at 750nm. Once
the algal culture OD increased to 2.0, cultivation was initiated. During the cultivation, every day
at fixed volume (20%) of the total culture was harvested from the first cultivation apparatus while
the density of culture was maintained around 2.0 OD at the time of harvest. Every day in the
morning at 7:00 hrs 20,000 L of culture was harvested and replenished with fresh medium. Prior
to harvest the culture nutrients level was maintained at 16:1 ratio of Nitrogen: Phosphorus.
Example 1B:
The 20,000 L of harvested culture from first cultivation apparatus [1] was transferred into the
second cultivation apparatus [2] and the initial culture depth was kept 5 cm at 7:00 hrs. The algal
culture pH was maintained similar in both the cultivation apparatuses. The culture growth was
monitored on hourly basis by measuring OD at 750nm. Once the culture density reached to desired
level, semi-continuous turbidostatic dilution was initiated in both the cultivation apparatuses by
increasing depth, by dilution of culture, by maintaining desired density and, end of the day at 17:30
hrs the entire culture was taken for harvesting. The same kind of practices were continued
throughout the experimental design of 52 days.
20
Analytical methods
Example 2: Determination of ash-free dry biomass
The ash free dry weight of Picochlorum sp. was measured by gravimetric method. 50 ml of sample
was withdrawn from the experimental cultivation apparatus on daily basis and filtered through
1.2μm GF/C filter paper and washed thrice with 50 mL of 4.0% ammonium bicarbonate to remove
the excess salts (Zhu and Lee, 1997). Then, the washed sample was placed in hot air oven and its
final dry weight was recorded after drying at 105 C overnight, dry weight was calculated and
recorded. Then the same filter was placed in pre-weighed glass crucible and ashed at 550 C for 1
hour in a muffle furnace, cooled, weighed and ash-free dry biomass (AFDW g/L) was calculated.
Areal biomass productivity was calculated as:
Areal productivity
=
Biomass concentration at the time of harvest (g L − 1) × harvested volume (L)
Cultivation apparatus area (m2)
Where, cultivation apparatus area is footprint area of an cultivation apparatus.
Example 3: Measurement of the Culture pH, temperature, salinity
The temperature and pH of the first [1] and second [2] cultivation apparatus having the culture was
monitored through pH probe [10] wherein the pH probe [10] is Rosemount analytical sensor and
recorded on hourly basis using online SCADA system. The culture salinity was measured using
ATAGO pocket refractometer (PAL-03S model).
Example 4: Microscopy and PAM (Fv/Fm) analysis
Everyday apparatus samples were observed under light microscope (Olympus) to check the algal
health as well as contamination level. In addition, the maximum quantum yield of dark- adapted
samples (Fv/Fm) were measured using an AquaPen-C portable fluorometer (Photon Systems
Instruments, Czech Republic). Fv/Fm values assessed the reclamation potential of photosynthesis
21
after samples were dark adapted for 30 min. The estimated maximum quantum yield of PSII
photochemistry is regularly used as an indicator of plant stress
Cultivation results
In first cultivation apparatus [1], initially the algal culture was harvested in amounts of 22,360 L
and 22,800 L on 1st and 2nd day, respectively, for culture stabilization, and once the culture was
stabilized in the apparatus a fixed volume of 20,000 L (20%) was harvested on daily basis
throughout the experiment and culture was maintained in exponential phase. In overall the culture
showed an average of 1.81 and 2.19 OD as initial and final density at 9:30 and 17:30h respectively.
Based on the OD data, 0.38 OD increment/jump per day was observed, which was 21% growth
per day in apparatus. Hence, 20% of the total volume of the apparatus (100,000L) was daily
harvested.
In second cultivation apparatus [2] the dilution set OD was desired at 3.0, which was chosen as an
optimum density based on the multiple trials carried out in the apparatus i.e. depth vs density. The
set OD was reached between 12:30 and 1:30h in the noon. The average initial density at 9:30h was
2.33 OD with culture volume 25,000 L and depth was 5 cm in second cultivation apparatus.
During turbidostatic dilution the maximum culture volume was increased up to 35,500 L on 7th
day and the increment was 42%, when compared to the initial volume of culture. The maximum
of 1.33 OD jump per day was recorded on 22nd day, whereas the minimum of 0.13 OD was
recorded on 10th day. There was no harvest on 9th day and culture density was 3.12 OD, and same
culture was continued on 10th day for regular cultivation and the growth was less which was less
than 5% on 10th day, when compared to rests of the days.
This result substantiates to the trials (3.0 OD was optimum at 5 cm operational depth in 500 m2
cultivation apparatus) that the light is limiting factor of photosynthesis, when the culture density
was 3.0 or above OD in the apparatus. However, in this example an average of 0.64 OD increment
per day was recorded for 52 days of cultivation.
22
The percentage of average growth in the apparatus was 27.60% per day, over average initial OD.
In second cultivation apparatus [2], dilution set OD was changed between 2.60-2.75 instead of 3.0
between 40- 46th days due to fluctuation in the daily growth. The possible reason for fluctuated
growth might be constant high culture temperature (average 42 °C) and light intensity (average
PAR 1164 μmol m-2 s-1).
Example 5: Biomass concentration and areal productivity
In first cultivation apparatus [1], the culture density was observed in the range between 0.276 and
0.504 g/L with an overall average biomass density of 0.375 g/L. Areal biomass productivity was
observed in the range between 12-20 g m-2 d-1 with an average productivity of 13.83 g m-2 d-1 in
first cultivation apparatus [1].
The experiment was carried out in second cultivation apparatus [2] at low depth cultivation (5 cm)
to increase biomass density in pump driven apparatus. The average initial inoculum density was
observed 0.529 g/L at 9:30h, and at end of the cultivation at the time of harvest at 17:30 h on the
same day the average culture density was recorded 0.677 g/L and maximum biomass density
reached up to 0.908 g/L. The average of 28% growth per day was recorded and maximum of 57%
was observed per day in the experimental second cultivation apparatus [2], and the average
biomass density was 1.8 times higher, when compared to first cultivation apparatus [1].
The results of the present invention demonstrated that the advantage of pump driven low depth
cultivation with high density culture in dual apparatus gives 28-30% higher productivity compared
to high cultivation depth. Further, low depth cultivation apparatus helps to reduce the water usage,
harvesting chemicals, cost, time and moreover pump driven apparatus helps to reduce the grazers
and contamination level, which is more important in open cultivation system. Importantly, high
density biomass can be used as inoculum for first cultivation apparatus [1] which is deep
cultivation apparatus. The maximum areal productivity was recorded 26 g m-2 d-1 and minimum
of 13 g m-2 d-1 on 5th and 2nd day, respectively. However, the overall average areal productivity
was 18.0 g m-2 d-1, which was 30.20% higher than first cultivation apparatus [1].
23
Example 6: Cultivation apparatus parameters and environmental conditions
In first cultivation apparatus [1], maximum culture temperature reached up to 37˚C and minimum
of 20˚C was recorded during cultivation. The mean of minimum and maximum culture temperature
was 24 ºC and 34 ºC, respectively. The maximum culture temperature was observed below 35˚C
up to 30th day of cultivation and during this period the average biomass productivity was 13.0 g
m-2 d-1. Thereafter, the culture temperature increased from 35 ºC to 37 ºC up to 52nd day and the
average biomass productivity was 16.0 g m-2 d-1. The biomass productivity was increased up to
23.0% when the culture temperature increased from ~30 ºC to 37 ºC. The results suggested that
the culture has higher temperature tolerance potential with average biomass productivity range
between 13-16 g m-2 d-1.
In second cultivation apparatus [2], maximum culture temperature reached up to 43 ºC (between
20-39th days) and minimum of 20 ºC was recorded throughout cultivation period. However,
significant temperature fluctuations were observed throughout the day, which was expected in
open cultivation apparatus. When culture temperature was high (~42 ºC) between 42nd and 52nd
days, the biomass productivity was observed in range between 14-22 g m-2 d-1 with an average
biomass productivity of 18 g m-2 d-1.
However, the strain was productive and shows temperature tolerance level up to 43 C in open
cultivation apparatus at low depth cultivation, between 37-40 C of culture temperature the average
biomass productivity was 19 g m-2 d-1 and between 40-43 C the average biomass productivity was
19 g m-2 d-1. Hence, there was no significant reduction observed in biomass productivity even at
higher culture temperature in this experiment.
With respect to the light intensity, maximum PAR of 1233 μmol m-2 s-1 and minimum of 992
μmol m-2 s-1 was observed during the experiment. Initially from 1st to 9th day the average PAR was
1053 μmol m-2 s-1
. Thereafter, the intensity of PAR was increased to ~1200 μmol m-2 s-1. In second
cultivation apparatus both culture OD and biomass productivity were observed and some
fluctuations from 41st to 50th day, but those days the PAR value observed was between 1136-1233
μmol m-2 s-1 and the variations was less than 10%. Whereas in first cultivation apparatus [1], the
24
productivity was 15% higher, due to higher cultivation depth which counteracted the temperature
effect but favoured the light penetration.
In respect to the culture salinity, in first cultivation apparatus [1] it was observed in the range
between 4.4% and 4.8%, whereas in second cultivation apparatus [2] it was between 4.6% and
5.1%. In second cultivation apparatus [2] it was observed little higher salinity range due to higher
fractional evaporation loss because of lower cultivation depth, which was obviously expected but
usage of fresh water was avoided to maintain the salinity. However, the culture shows the potential
to tolerate higher salinity in second cultivation apparatus.
In the present invention it is important to note that Picochlorum sp. has potential to tolerate wide
range of salinity, temperature and light intensity in the open cultivation apparatus at varying
depths.
The mean quantum yield of PSII photochemistry (Fv/Fm) was measured on daily basis in both the
cultivation apparatuses. However, first cultivation apparatus [1] having paddle wheel showed
values ranging between 0.58 and 0.66 with mean value of 0.62 for dark adapted samples of Fv/Fm.
In contrast, the pump driven second cultivation apparatus [2] showed lower value of Fv/Fm when
compared to the paddle wheeled first cultivation apparatus [1] with values ranging between 0.43
and 0.64 with mean value of 0.56.
Example 7: Average values of algae cultivation in the present algal cultivation system [15]
The method of cultivating microalgae of the present disclosure was performed in the area of 500
m2 with the arrangements of first cultivation apparatus [1] and second cultivation apparatus [2].
For optimal vegetative growth of the Picochlorum/ algae culture which served as the starting
inoculum or seed culture for actual experiment under following suitable conditions:
25
Table- A
S.No. Parameters/ Condition for
algae culture growth
First Cultivation apparatus
[1]
(Paddle wheel driven)
Second Cultivation
apparatus [2]
(Pump driven)
1 Apparatus area (m2) 500
2 Cultivation mode Daily turbidostatic dilution Daily full harvest
3 Cultivation depth (cm) 20 5
4 Working volume (L) 100,000 L 25,000 L
5 Daily harvest volume (L) 20,000 L 25,000 L
6 Culture OD750 at 9:30 h 1.81 ± 0.15 2.33 ± 0.25
7 Culture OD750 at 17:30 h 2.19 ± 0.17 2.97 ± 0.28
8 OD jump per day 0.38 ± 0.16 0.64 ± 0.30
9 biomass ash free dry weight
(g/L) at 9:30h
0.31 ± 0.02 0.53 ± 0.06
10 biomass ash free dry weight
(g/L) at 17:30h
0.38 ± 0.05 0.68 ± 0.09
11 Areal productivity (g/m2/d)
ash free basis
13.83 ± 4.57 18.10 ± 4.55
12 Volumetric productivity
(g/L/d) ash free basis
0.066 ± 0.05 0.16 ± 0.08
13 Harvest mode Daily semi-turbidostatic
harvest
Daily full harvest
14 Fv/Fm value (fluorescence) 0.62 ± 0.02 0.56 ± 0.06
15 Salinity range (%) 4.20 - 4.80 4.40 - 5.10
16 Culture temperature (˚C) 24 - 34 27 - 40
17 Total Nitrogen (TN)
consumption per day (ppm)
6.21 ± 3.14 16.35 ± 6.05
18 Total phosphate (TP)
consumption per day (ppm)
0.58 ± 0.28 1.03 ± 0.53
19 PAR range (μE) 992 - 1233
26
Experiments were performed at outdoor environment with Picochlorum species of algae. Total
25,000 L of algae culture was harvested by transferring 10-30% of the total culture from the first
cultivation apparatus [1] to the second cultivation apparatus [2]. Throughout the experiment run,
pH of the culture, temperature and salinity was monitored. Once OD increased to +2.0, cultivation
was initiated. During the cultivation, every day at fixed volume (20%) of the total culture was
harvested from the first cultivation apparatus while maintaining the density of culture around 2.0
OD at the time of harvest and transferred to second cultivation apparatus [2]. Every day in the
evening at 17:30 hrs 25,000 L (i.e., 100%) of culture was harvested from the second cultivation
apparatus [2] and replenished with the inoculum from the first cultivation apparatus [1] with fresh
medium. The above data of table- A shows enhanced production of algal biomass when the algae
were subjected to about 8 hours to 12 hours of incubation. Hence, the present invention has
enhanced technical effect as demonstrated by the examples and data of Table A.
The microalgae culture can be maintained in both nutrient rich and nutrient depleted conditions as
per requirement. For example, nutrient rich culture can be used for protein production, whereas
nutrient depleted culture can be used for biomolecules and pigment induction. When the algae
culture is maintained in nutrient rich conditions, as in the above example, both biomass and protein
production can be maintained as long as the experiment continues. However, when the culture is
introduced into nutrient depleted conditions, biomass can be maintained for few days, i.e., later on
the algal growth is arrested (cell multiplication) and significant amounts of biomolecules are
induced in the algal cells / biomass (lipids, fatty acids, pigments and carbohydrates) due to nutrient
stress /nutrient depleted conditions.
Example 8: Comparative productivity of the present invention over the conventional system
Methodology: As discussed in the above examples, 20% of the microalgae culture I is harvested
from first cultivation apparatus which has the area of 500 m2 and is transferred to second
cultivation apparatus which has the area of 500 m2(System A). Considering the experiment in
volume based cultivation system, the harvested culture I (i.e., 20,000 L from first cultivation
apparatus of 500 m2 area) is transferred into 500 m2 area of second cultivation apparatus and
maintained at 5 cm depth of culture of second cultivation apparatus.
27
In another embodiment, the amount of harvested culture may be accommodated to 350 m2 area of
second cultivation apparatus with 5 cm depth and the calculated areal productivity is
approximately 28.82 g/m2/d (System B). The pH of the culture was maintained between pH 6.8-
7.2 throughout the study, culture temperature was observed between 25-35°C, salinity observed
between 4-5% and at the end of the day whole culture was harvested for biomass collection.
The following formula was used to calculate the biomass productivity:
Areal productivity = biomass density at the time of harvest (g/L) x volume of culture harvested (L)
apparatus area (m2)
Advantage: The advantage of volume based cultivation system (System B) expected is that the
30% of cultivation area is reduced simultaneously with ~15% and 37% increase in biomass
productivity when compared to system A and conventional system, respectively. The
corresponding data on the same is provided in the below table- B.
COMPARATIVE DATA
The Table-B below demonstrates the comparative productivity of the present invention over the
conventional system (Refer Figure 4).
Table- B: Areal productivity comparison of Present invention vs Conventional (shallow)
System
Type of algae
cultivation system
Present Invention Conventional System
System A System B
(Volume based)
Areal Productivity
(g/m2/d)
25.36 28.82 21
28
ADVANTAGES
The algal cultivation 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.
1. The microalage cultivation system [15] of the present invention helps to enhance the algal
biomass productivity to about ~30.87% when compared with single cultivation apparatus.
2. The combination of deep and shallow cultivation apparatus helps to reduce the water usage,
harvesting chemicals, cost, and time.
3. The pump driven cultivation apparatus helps to reduce the grazers and contamination level.
4. The cultivation area is reduced to 20-30% with increasing productivity of 30% compared
to shallow apparatus by daily harvest.
5. The microalgae culture can be maintained in both nutrient rich and nutrient depleted
conditions as per requirement. For example, nutrient rich culture can be used for protein
production, whereas nutrient depleted culture can be used for biomolecules and pigment
induction.
6. The biomass harvesting cost can be minimized due to high harvesting biomass density by
multiple cultivation apparatuses.
7. There is less contamination due to continuous transferring of the culture from the first
cultivation apparatus [1] to the second cultivation apparatus [2].
8. The micro algae cultivation system [15] of the present invention requires less water for
cultivation.
9. There is high light availability to the entire microalgae cultivation system [15].
10. There are seasonal benefits for scale-up and cultivation.
The foregoing description of the specific embodiments fully reveals the general nature of the
embodiments herein that others can, by applying current knowledge, readily modify and/or adapt
for various applications such specific embodiments without departing from the generic concept,
and, therefore, such adaptations and modifications should and are intended to be comprehended
within the meaning and range of equivalents of the disclosed embodiments. It is to be understood
that the phraseology or terminology employed herein is for the purpose of description and not of
29
limitation. Therefore, while the embodiments in this disclosure have been described in terms of
preferred embodiments, those skilled in the art will recognize that the embodiments herein can be
practiced with modification within the spirit and scope of the embodiments as described herein.
Throughout this specification, the word “comprise”, or variations such as “comprises” or
“comprising” wherever used, will be understood to imply the inclusion of a stated element, integer
or step, or group of elements, integers or steps, but not the exclusion of any other element, integer
or step, or group of elements, integers or steps. Similarly, terms such as “include” or “have” or
“contain” and all their variations are inclusive and will be understood to imply the inclusion of a
stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or steps.
As used herein, the term ‘comprising’ when placed before the recitation of steps in a method means
that the method encompasses one or more steps that are additional to those expressly recited, and
that the additional one or more steps may be performed before, between, and/or after the recited
steps. For example, a method comprising steps a, b, and c encompasses a method of steps a, b, x,
and c, a method of steps a, b, c, and x, as well as a method of steps x, a, b, and c. Furthermore, the
term “comprising” when placed before the recitation of steps in a method does not (although it
may) require sequential performance of the listed steps, unless the content clearly dictates
otherwise. For example, a method comprising steps a, b, and c encompasses, for example, a method
of performing steps in the order of steps a, c, and b, the order of steps c, b, and a, and the order of
steps c, a, and b, etc.
The terms “about” or “approximately” are used herein to mean approximately, in the region of,
roughly, or around. When the term “about” is used in conjunction with a numerical value/range, it
modifies that value/range by extending the boundaries above and below the numerical value(s) set
forth. In general, the term “about” is used herein to modify a numerical value(s) or a measurable
value(s) such as a parameter, an amount, a temporal duration, and the like, above and below the
stated value(s) by a variance of +/-20% or less, +/-10% or less, +/-5% or less, +/-1% or less, and
+/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform
in the disclosed invention, and achieves the desired results and/or advantages as disclosed in the
30
present disclosure. It is to be understood that the value to which the modifier “about” or
“approximately” refers is itself also specifically, and preferably, disclosed.
With respect to the use of substantially any plural and/or singular terms herein, those having skill
in the art can translate from the plural to the singular and/or from the singular to the plural as is
appropriate to the context and/or application. The various singular/plural permutations may be
expressly set forth herein for sake of clarity. The suffix ‘(s)’ at the end of any term in the present
disclosure envisages in scope both the singular and plural forms of said term.
As used in this specification and the appended claims, the singular forms “a,” “an” and “the”
includes both singular and plural references unless the content clearly dictates otherwise. The use
of the expression ‘at least’ or ‘at least one’ suggests the use of one or more elements or ingredients
or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the
desired objects or results. As such, the terms “a” (or “an”), “one or more”, and “at least one” can
be used interchangeably herein.
Numerical ranges stated in the form ‘from x to y’ include the values mentioned and those values
that lie within the range of the respective measurement accuracy as known to the skilled person. If
several preferred numerical ranges are stated in this form, of course, all the ranges formed by a
combination of the different end points are also included.
As regards the embodiments characterized in this specification, it is intended that each
embodiment be read independently as well as in combination with another embodiment. For
example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting
3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is to be
understood that the specification unambiguously discloses embodiments corresponding to
combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D,
G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I;
C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.
31
Throughout this specification, the term ‘a combination thereof’, ‘combinations thereof’ or ‘any
combination thereof’ or ‘any combinations thereof’ are used interchangeably and are intended to
have the same meaning, as regularly known in the field of patent disclosures.
Any discussion of documents, acts, materials, devices, articles and the like that has been included
in this specification is solely for the purpose of providing a context for the disclosure. It is not to
be taken as an admission that any or all of these matters form a part of the prior art base or were
common general knowledge in the field relevant to the disclosure as it existed anywhere before
the priority date of this application.
While considerable emphasis has been placed herein on the particular features of this disclosure,
it will be appreciated that various modifications can be made, and that many changes can be made
in the preferred embodiments without departing from the principles of the disclosure. These and
other modifications in the nature of the disclosure or the preferred embodiments will be apparent
to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that
the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not
as a limitation.
All references, articles, publications, general disclosures etc. cited herein are incorporated by
reference in their entirety for all purposes. However, mention of any reference, article, publication
etc. cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion
that they constitute valid prior art or form part of the common general knowledge in any country
in the world.
We Claim:
1. A microalgae cultivation system [15] comprising:
i. a first cultivation apparatus [1] for cultivation of microalgae;
ii. a second cultivation apparatus [2] for cultivation of microalgae;
iii. one or more circulating means positioned in each of the first cultivation
apparatus [1] and the second cultivation apparatus [2] to enable circulation of microalgal culture; wherein the second cultivation apparatus [2] is of lower depth than the first cultivation apparatus [1]; and
wherein about 10%-30% of the microalgal culture of the first cultivation apparatus [1] is transferred to the second cultivation apparatus [2] once every 24 hours.
2. The microalgae cultivation system [15] as claimed in claim 1, wherein the first cultivation apparatus [1] is a high depth apparatus, and the second cultivation apparatus [2] is a shallow depth apparatus.
3. The microalgae cultivation system [15] as claimed in claim 1, wherein the depth of the first cultivation apparatus [1] is 20-30 cm and the depth of the second cultivation apparatus [2] is 5-10 cm.
4. The microalgae cultivation system [15] as claimed in claim 1, wherein the first cultivation apparatus [1] and the second cultivation apparatus [2] are optionally connected by a connecting means.
5. The microalgae cultivation system [15] as claimed in claims 1-4, wherein the first cultivation apparatus [1] and the second cultivation apparatus [2] are optionally in controlled fluid communication with each other.
6. The microalgae cultivation system [15] as claimed in claim 1, wherein the circulating means located in the first cultivation apparatus [1] is a paddle wheel assembly [3].
7. The microalgae cultivation system [15] as claimed in claim 1, wherein the circulating means located in the second cultivation apparatus [1] is a pump assembly [4].
8. The microalgae cultivation system [15] as claimed in claim 1, wherein said first cultivation apparatus [1] and second cultivation apparatus [2] comprise at least one or more inlets and at least one or more outlets.
9. The microalgae cultivation system [15] as claimed in claim 8, wherein said one or more inlets in the first cultivation apparatus [1] and second cultivation apparatus [2] comprise at least one or more water inlets [5,6], one or more algal culture inlets [7], one or more

nutrient inlets [8], one or more CO2 inlets [9], one or more pH probe inlets [11] and one or more temperature probe inlets [11].
10. The microalgae cultivation system [15] as claimed in claim 8, wherein said one or more outlets in the first cultivation apparatus [1] and second cultivation apparatus [2] comprise at least one or more culture harvest outlets [12] and at least one or more discharge outlets [13].
11. The microalgae cultivation system [15] as claimed in claim 1, wherein at least one mid-wall [14] is optionally disposed in the first cultivation apparatus [1] and the second cultivation apparatus [2] for improving the fluid circulation.
12. A method for cultivation of microalgae, the method comprising:

(a) Adding microalgal inoculum to be cultured in a first cultivation apparatus [1];
(b) Supplying nutrient(s) to the microalgal culture I in said first cultivation apparatus [1] in a controlled manner;
(c) Harvesting a portion of said microalgal culture I from the first cultivation apparatus [1] periodically;
(d) Transferring the harvested microalgal culture I of step (c) into a second cultivation apparatus [2] and allowing the microalgae to grow to obtain microalgal culture II;
(e) Harvesting the microalgal culture II of step (d) from the second cultivation apparatus [2].

13. The method as claimed in claim 12, wherein the first cultivation apparatus [1] is a high depth apparatus, and the second cultivation apparatus [2] is a shallow depth apparatus.
14. The method as claimed in claim 12, wherein depth of the first cultivation apparatus [1] is 20-30 cm and depth of the second cultivation apparatus [2] is 5-10 cm.
15. The method as claimed in claim 12, wherein the culture is continuously mixed by using a circulating means, wherein the circulating means is paddle wheel assembly [3] in the first cultivation apparatus [1] and pump assembly [4] in the second cultivation apparatus [2].
16. The method as claimed in claim 12, wherein the harvested microalgal culture I is transferred from the first cultivation apparatus [1] to the second cultivation apparatus [2] once every 24 hours in a range of about 10%-30% of the total microalgal culture.
17. The method as claimed in claim 12, wherein the first cultivation apparatus [1] facilitates the growth of microalgae culture under semi-turbidostatic mode of cultivation.

18. The method as claimed in claim 12, wherein the first cultivation apparatus [1] and second cultivation apparatus [2] comprise at least one or more inlets and at least one or more outlets.
19. The method as claimed in claim 18, wherein the one or more inlets comprise at least one or more water inlets [5,6], one or more algal culture inlets [7], one or more nutrient inlets [8], one or more CO2 inlets [9], one or more pH probe inlets [11] and one or more temperature probe inlets [11].
20. The method as claimed in claim 18, wherein the one or more outlets comprise at least one or more culture harvest outlets [12] and at least one or more discharge outlets [13].
21. The method as claimed in claim 12-20, wherein the first cultivation apparatus [1] and the second cultivation apparatus [2] are optionally connected by a connecting means to facilitate controlled fluid communication with each other.
22. The method as claimed in claim 12-21, wherein at least one mid-wall [14] is optionally disposed in the first cultivation apparatus [1] and the second cultivation apparatus [2] for improving the fluid circulation.
23. A product produced from microalgal culture obtained using the system [15] of any of claims 1-11 or the method of any of claims 12-22, wherein the product comprises high-density biomass, biomolecules, feedstock, and/or phytonutrients.
24. A method of biomass enhancement using the microalgae cultivation system [15] as claimed in claims 1-11 or the method of cultivating microalgae as claimed in claims 12-22.
25. Use of the microalgae cultivation system [15] as claimed in claims 1-11, for producing microalgae.
26. Use of the method as claimed in claims 12-22, for producing microalgae.