Claims:We Claim:
1. A method for inducing lipid production in algae, said method comprising exposing algae to infra-red radiation.
2. The method of claim 1, wherein the algae is microalgae selected from a group comprising green algae, diatoms, red algae, brown algae, gold algae, yellow-green algae, cyanobacteria and combinations thereof; and wherein the lipid is neutral lipid, total lipid, or a combination thereof.
3. The method of any of the preceding claims, wherein the green algae is selected from a group comprising Picochlorum, Nannochloris, Chlorella, Cyclotella, Navicula and combinations thereof; and the cyanobacteria is selected from a group comprising Cyanobacterium aponinum, Synechococcus elongatus and a combination thereof.
4. The method of any of the preceding claims, wherein the algae is exposed to infra-red radiation of a wavelength ranging from about 700 nm to 1200 nm, preferably about 1000 nm to 1100 nm.
5. The method of any of the preceding claims, wherein the algae is exposed to infra-red radiation at a wavelength of about 1100 nm.
6. The method of any of the preceding claims, wherein the algae is exposed to infra-red radiation for a time-period ranging from about 1 second to 60 minutes.
7. The method of any of the preceding claims, wherein the algae is exposed to infra-red radiation for a time-period ranging from about 5 minutes to 30 minutes, preferably about from 5 minutes to about 15 minutes; and wherein maximum neutral lipid induction occurs in about 5 minutes.
8. The method of any of the preceding claims, wherein infra-red radiation source is employed to generate the infra-red radiation, and wherein the distance between the infra-red radiation source and the algae ranges from about 1 cm to 100 cm.
9. The method of any of the preceding claims, wherein the distance between the infra-red radiation source and the algae ranges from about 10 cm to 30 cm.
10. The method of any of the preceding claims, wherein the exposure is carried out in absence of light other than infra-red radiation light to obtain irradiated algae.
11. The method of any of the preceding claims, wherein the method is carried out in presence of carbon dioxide (CO2) concentration ranging from about 0 % to 10 %, preferably 0 %.
12. The method of any of the preceding claims, wherein said method is maintained at a temperature ranging from about 30 °C to 35 °C for enhanced neutral lipid production in algae.
13. The method of any of the preceding claims, wherein said temperature is maintained by heat decoupling using a liquid medium, preferably by cold water circulation.
14. The method of any of the preceding claims, wherein the method for inducing lipid production in algae comprising exposing algae to infra-red radiation, comprises steps of:
a. exposing a culture of algal cells to infra-red radiation to obtain irradiated algae;
b. harvesting the irradiated algae from the culture to obtain harvested algae;
c. drying the harvested algae to obtain powdered algae; and
d. extracting the powdered algae with a solvent to obtain lipid.
15. The method of any of the preceding claims, wherein the steps of exposing, harvesting, drying, comminuting and extracting comprises technique selected from a group comprising shaking, centrifugation, heating, grinding, drying, stirring, sonication, decoupling of heat using a liquid medium and combinations thereof.
16. The method of any of the preceding claims, wherein the method for inducing lipid production in algae comprising exposing algae to infra-red radiation, comprises steps of:
a. exposing culture of algal cells to infra-red radiation at a wavelength of about 1000 nm to 1100 nm for a time-period ranging from about 5 minutes to 30 minutes to obtain irradiated algae, wherein the distance between the IR source and the algal cells is about 10 cm to 30 cm;
b. harvesting the irradiated algae from the culture to obtain harvested algae;
c. drying the harvested algae to obtain powdered algae; and
d. extracting the powdered algae with a solvent to obtain lipid.
17. The method of any of the preceding claims, wherein the exposure is carried out after the algal culture reaches stationary phase.
18. The method of any of the preceding claims, wherein said method enhances neutral lipid yield in algae compared to the neutral lipid yield when algae is not exposed to infra-red radiation.
19. The method of any of the preceding claims, wherein the enhancement of neutral lipid yield is about 30 % to 100 %.
20. The method of any of the preceding claims, wherein said method enhances total lipid content in algae compared to the total lipid yield when algae is not exposed to infra-red radiation.
21. The method of any of the preceding claims, wherein the enhancement of total lipid yield is about 1% to 5%
22. The method of any of the preceding claims, wherein the method is performed in a cultivation apparatus selected from a group comprising a cultivation pond, a raceway type cultivation apparatus, a tubular type cultivation apparatus, a liquid membrane-forming cultivation apparatus, photobioreactor (PBR) system and combinations thereof.
23. The method of any of the preceding claims, wherein said method is combined with any neutral lipid induction method.
24. The method of any of the preceding claims, wherein the method is combined with stress inducing method selected from a group comprising nutrient stress, pH, salinity, high density, high light, temperature stress and combinations thereof; and wherein the nutrient stress is selected from a group comprising nitrogen stress, phosphorous stress, potassium stress and combinations thereof. , Description:TECHNICAL FIELD
[001]. The instant disclosure relates to the field of algal cultivation and biofuels. Particularly, the present disclosure relates to a method of inducing neutral lipids during algal culturing. In an exemplary embodiment, the present disclosure relates to a method of enhancing neutral lipid production by exposing algae to infra-red radiation.

BACKGROUND
[002]. In the present scenario, the energy from renewable sources are interesting as they are the main raw sources for replacing the limited supply of fossil fuels. Among all, algae is more attractive potential source for biofuel production since it has high energy neutral lipids. However, the current methods provide algal lipid densities/quantities which are not competing with traditional fuels. Thus, breakthrough strategies or methods should be developed for increased neutral lipid production.
[003]. There have been intense research efforts aimed at increasing and modifying the accumulation of neutral lipids in algae through physical, biochemical and genetic means. The biochemical approach entails controlling cultivation conditions (e.g., nutritional content, salinity, temperature, and pH). For example, the elimination of nitrogen, phosphorus or silicon from algal culture medium to induce stress conditions in the cells, increasing or decreasing temperature, salinity, pH during culturing thereby causing generation and accumulation of high-energy lipids, but at the cost of reduced algal replication. In physical approach, UV irradiation has been used for lipid induction. However, such physical approaches suffer from limitations including lack of penetration and heat energy. The genetic approach further involves over or under-expressing genes involved in lipid biosynthesis pathways. Thus, the aforesaid approaches are complicated and suffer from various drawbacks.
[004]. Therefore, there is a need to develop simple, cost-effective and more efficient process for inducing neutral lipid production in algae without compromising biomass. The present disclosure addresses said need.

SUMMARY OF THE DISCLOSURE
[005]. The present disclosure relates to a method of enhancing lipid production in algae.
[006]. In an embodiment, the present disclosure relates to a method of neutral lipid induction in algae.
[007]. In yet another embodiment, the present disclosure relates to a method of increasing total lipids in algae.
[008]. In an embodiment of the present disclosure, the present method comprises exposing algae to infra-red rays.
[009]. In another embodiment of the present disclosure, the algae is microalgae selected from a group comprising green algae, diatoms, red algae, brown algae, gold algae, yellow-green algae, cyanobacteria and combinations thereof.
[0010]. In yet another embodiment of the present disclosure, the algae is exposed to infra-red radiation for a time-period ranging from about 1 second to 60 minutes.
[0011]. In still another embodiment, the distance between the infra-red radiation source and the algae ranges from about 1 cm to 100 cm.
[0012]. In still another embodiment of the present disclosure, the method is combined with known lipid inducing method(s).

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
[0013]. In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, where:

[0014]. Figure 1 depicts the comparison of neutral lipid production in algae under IR, UV and Yellow light.
[0015]. Figure 2 depicts the study of neutral lipid production with respect to time of IR exposure.
[0016]. Figure 3 depicts the study of neutral lipid production with respect to distance of IR exposure.
[0017]. Figure 4 depicts the study of neutral lipid production with respect to time of IR exposure combined with heat decoupling mechanism.
[0018]. Figures 5 and 8 depict the study of neutral lipid production in cyanobacteria with respect to IR exposure.
[0019]. Figure 6 depicts the effect of IR exposure for short duration on neutral lipid production.
[0020]. Figure 7 depicts the study of combined IR exposure and Nutrient stress on neutral lipid production.
[0021]. Figure 9 depicts the induction of neutral and total lipids in algae cultivated in outdoor pond and subsequently subjected to IR exposure.

STATMENT OF THE DISCLOSURE
[0022]. The present disclosure relates to a method of inducing lipid production in algae, said method comprising exposing algae to infra-red rays.

DESCRIPTION OF THE DISCLOSURE
[0023]. To address the limitations as stated in the background, the present disclosure provides an simple and efficient process for enhancing lipid production in algae.
[0024]. In an embodiment, the present disclosure relates to a method of neutral lipid induction in algae.
[0025]. As used herein, ‘neutral lipids’ refer to hydrophobic molecules (lipid moieties) lacking charged groups. Triacylglycerols (TAGs), steryl esters (SEs) and wax esters (WEs) majorly form the group of neutral lipids.
[0026]. In yet another embodiment, the present disclosure relates to a method of increasing total lipid content in algae.
[0027]. As used herein, ‘total lipids’ consists of neutral lipids and polar lipids.
[0028]. In another embodiment, the present disclosure relates to a method of enhancing lipid production in algae by infra-red (IR) radiation exposure.
[0029]. In another embodiment, the algae is microalgae selected from a group comprising green algae, diatoms, red algae, brown algae, gold algae, yellow-green algae, cyanobacteria and combinations thereof.
[0030]. In a preferred embodiment of the present disclosure, the algae is green algae including but not limiting to Picochlorum, Nannochloris, Chlorella, Cyclotella, Navicula and combinations thereof.
[0031]. In a preferred embodiment of the present disclosure, the algae is cyanobacteria including but not limiting to Cyanobacterium aponinum, Synechococcus elongatus and a combination thereof.
[0032]. In an embodiment, the method comprises exposing algae to infra-red (IR) radiation at a wavelength ranging from about 700 nm to 1200 nm for increasing neutral lipid production in algae.
[0033]. In a preferred embodiment of the present disclosure, the algae is exposed to IR radiation at a wavelength ranging from about 1000 nm to 1100 nm.
[0034]. In a more preferred embodiment, the algae is exposed to IR radiation at a wavelength of about 1100 nm.
[0035]. In an embodiment of the present disclosure, the algae is exposed to IR radiation for a time-period ranging from about 1 second to 60 minutes.
[0036]. In a preferred embodiment, the algae is exposed to IR radiation for a time-period ranging from about 1 second to 30 minutes.
[0037]. In a preferred embodiment, the algae is exposed to IR radiation for a time-period ranging from about 1 second to 60 minutes.
[0038]. In a more preferred embodiment, the algae is exposed to IR radiation for a time-period ranging from about 30 seconds to 15 minutes.
[0039]. In another preferred embodiment, the algae is exposed to IR radiation for a time-period ranging from about 5 minutes to 15 minutes.
[0040]. In an exemplary embodiment, the algae is exposed to IR radiation for about 5 minutes.
[0041]. In an embodiment of the present disclosure, the distance between the IR radiation source and the algae ranges from about 1 cm to 100 cm.
[0042]. In a preferred embodiment, the distance between the IR radiation source and the algae ranges from about 1 cm to 30 cm.
[0043]. In another preferred embodiment, the distance between the IR radiation source and the algae ranges from about 10 cm to 30 cm.
[0044]. In an embodiment of the present disclosure, the IR exposure is carried out in absence of light other than IR radiation to increase neutral lipid production in algae.
[0045]. In another embodiment of the present disclosure, the method is carried out in presence or absence of carbon dioxide (CO2).
[0046]. In an embodiment, the CO2 concentration ranges from about 0 % to10 %.
[0047]. In another embodiment of the present disclosure, the method is maintained at a temperature ranging from about 30 °C to 35 °C for enhanced neutral lipid production in algae.
[0048]. In a preferred embodiment, the method is maintained at the temperature of about 30 °C to 35 °C by heat decoupling mechanism.
[0049]. In another preferred embodiment, the method is maintained at the temperature of about 30 °C to 35 °C by heat decoupling mechanism using a liquid medium.
[0050]. In an exemplary embodiment, the method is maintained at the temperature of about 30 °C to 35 °C by heat decoupling mechanism using cold water circulation.
[0051]. The method of the present disclosure for enhancing lipid production in algae by IR exposure comprises acts of:
(i) exposing algal cells to IR radiation to obtain irradiated algae;
(ii) harvesting the irradiated algae;
(iii) drying the harvested algae to obtain powdered algae;
(iv) extracting the powdered algae with a solvent to extract neutral lipid.
[0052]. In an embodiment, the above described steps of exposing, harvesting, drying, and extracting comprises techniques selected from shaking, sonication, centrifugation, heating, grinding, drying, stirring, or decoupling of heat using a liquid medium, or any combination thereof.
[0053]. In another embodiment, the algal cells are in a culture and IR exposure is carried out after the culture reaches stationary phase.
[0054]. In yet another embodiment, the present method enhances neutral lipid yield in algae by about 30% to 100% compared to the neutral lipid yield when algae are not exposed to infra-red radiation.
[0055]. In still another embodiment, the present method enhances neutral lipid yield in algae by about 0.1 % to 50 % compared to the neutral lipid yield when algae are not exposed to infra-red radiation.
[0056]. In yet another embodiment, the present method increases the total lipid content in algae by about 1 % to 5% compared to the total lipid content when algae is not exposed to infra-red radiation.
[0057]. In another embodiment of the present disclosure, the method of enhancing lipid production in algae comprises acts of:
(i) exposing algal cells to IR radiation at a wavelength of about 700 nm to 1200 nm for a time-period ranging from about 30 seconds to 60 minutes to obtain irradiated algae, wherein the distance between the IR source and the algal cells is about 1 cm to 100 cm;
(ii) harvesting the irradiated algae;
(iii) drying the harvested algae to obtain powdered algae; and
(iv) extracting the powdered algae with a solvent to obtain lipid.
[0058]. In a preferred embodiment of the present disclosure, the method of enhancing lipid production in algae comprises acts of:
(i) exposing algal cells to IR radiation at a wavelength of about 1000 nm to 1100 nm for a time-period ranging from about 1 second to 30 minutes to obtain irradiated algae, wherein the distance between the IR source and the algal cells is about 1 cm to 30 cm;
(ii) harvesting the irradiated algae;
(iii) drying the harvested algae to obtain powdered algae; and
(iv) extracting the powdered algae with a solvent to obtain lipid.
[0059]. In another preferred embodiment of the present disclosure, the method of enhancing lipid production in algae comprises acts of:
(i) exposing algal cells to IR radiation at a wavelength of about 1100 nm for a time-period of about 1 second to 5 minutes to obtain irradiated algae, wherein the distance between the IR source and the algal cells is about 1 cm to 30 cm;
(ii) harvesting the irradiated algae;
(iii) drying the harvested algae to obtain powdered algae; and
(iv) extracting the powdered algae with a solvent to obtain lipid.
[0060]. In yet another preferred embodiment of the present disclosure, the method of enhancing lipid production in algae comprises acts of:
(i) exposing algal cells to IR radiation at a wavelength of about 1000 nm to 1100 nm for a time-period ranging from about 30 second to 30 minutes to obtain irradiated algae, wherein the distance between the IR source and the algal cells is about 1 cm to 30 cm;
(ii) harvesting the irradiated algae;
(iii) drying and the harvested algae to obtain powdered algae; and
(iv) extracting the powdered algae with a solvent to obtain lipid.
[0061]. In yet another preferred embodiment of the present disclosure, the method of enhancing lipid production in algae comprises acts of:
(i) exposing algal cells to IR radiation at a wavelength of about 1000 nm to 1100 nm for a time-period of about 5 minutes to obtain irradiated algae, wherein the distance between the IR source and the algal cells is about 1 cm to 30 cm;
(ii) harvesting the irradiated algae;
(iii) drying and the harvested algae to obtain powdered algae; and
(iv) extracting the powdered algae with a solvent to obtain lipid.
[0062]. In an embodiment, harvesting of irradiated algae is carried out by centrifugation, flocculation, coagulation, electrocoagulation, filtration or any combination thereof.
[0063]. In another embodiment, drying the harvested algae is carried out by oven/sunlight drying, lyophilization/freeze drying, or any combination thereof.
[0064]. In yet another embodiment, extracting the powdered algae is carried out by solvent extraction with or without sonication. The solvents which can be used include chloroform, methanol, toluene, hexane, isopropanol or any combination thereof.
[0065]. In an embodiment of the present method, culturing and inducing neutral lipid production in algae is carried out in a cultivation apparatus selected from a group comprising a cultivation pond, a raceway type cultivation apparatus, a tubular type cultivation apparatus, a liquid membrane-forming cultivation apparatus, photobioreactor (PBR) system and combinations thereof.
[0066]. The present disclosure further relates to a method of enhancing neutral lipid production in algae by combining IR exposure with known neutral lipid inducing methods.
[0067]. In an embodiment, the neutral lipid inducing method includes but is not limited to stress inducing method selected from a group comprising nutrient stress, salinity, pH, high density, high light, temperature stress, or any combination thereof.
[0068]. In another embodiment, nutrient stress is selected from a group comprising nitrogen stress, phosphorous stress, potassium stress and combinations thereof.
[0069]. Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well-known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating the above described embodiments, and in order to illustrate the embodiments of the present disclosure certain aspects have been employed. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein.

EXAMPLES
[0070]. The following algae were employed in the present examples -
Picochlorum Sp sourced from Karanja, Maharashtra; Nannochloris Sp sourced from Morva, Bhayander, Maharashtra and Cyanobacterium aponinum sourced from Gagva, Jamnagar, Gujarat, and Synechococcus elongatus 7942 is sourced from Pasteur culture collection of Cyanobacteria (PCC), Paris [accession number - PCC 7942]. Further details of the algae are provided below (Table 1):
Table 1: Details of Algae employed in the Examples
S. No. Location Geographical Location Identity by PCR and DNA Sequencing
1 Karanja, Maharashtra, N18.844663, E72.946313 Picochlorum
2 Morva, Bhayander, Maharashtra N19.295811, E72.807436 Nannochloris
3 Gagva, Jamnagar, Gujarat N 22.389406, E69.812691 Cyanobacterium aponinum

EXAMPLE 1:
Preparing Algal Cultures
[0071]. Experiments were performed in biological as well as technical replicates. Two different algal strains Picochlorum sp. and Nannochloris sp. were used for the study. Initially, 0.1 OD culture was used as a startup of experiment. All setups were kept at under controlled environment with shaking. The culture was grown in commercially available media such as f/2 media or BG11 media using 1 liter conical flasks with the working volume of 0.5 L. Stationary phase or semiturbidostat whole culture was exposed to predefined light conditions and time under controlled environment as described in the below examples. Before exposing to the respective light, the culture beakers were completely covered by aluminum for preventing the entry of external light.

EXAMPLE 2:
Comparison of neutral lipid production under IR, UV and Yellow light
[0072]. Passing of UV and IR light to the algal culture as obtained in Example 1 was done for 30 minutes individually, whereas the algae culture was exposed to yellow light for 24 hours. Algal culture exposed to yellow light for 30 minutes do not induce significant lipid production. Along with all individual test runs with respective light sources, different controls were maintained under normal light [Figure 1a]. At the end of experiment, algal cells were harvested by pelleting using centrifugation. Once the pellet was collected, it was kept for drying in oven and dried at around 60 oC. Finally, the dried pellet was powdered and used for neutral lipid extraction by employing solvent extraction protocol and transesterification process. Said neutral lipid extraction was performed using the well-known National Renewable Energy Laboratory (NREL) protocol. The different light sources employed in the experiments were - 254 nm UV Light Source, Infrared (1100 nm, shortwave IR-A, 240V, 150W Osram Theratherm Deluxe Par) and 590 nm Yellow light source.

The above experiment was conducted with Nannochloris. Subsequent experiments were conducted with technical and biological replicates in Picochlorum, Cyanobacterium aponinum and Synechococcus elongatus 7942 respectively.

Results:
Compared to control conditions, enhancement in the neutral lipid content was noticed in IR, UV and yellow light induced studies in both strains Nannochloris and Picochlorum respectively.
Neutral Lipid induction in Nannochloris -
Nannochloris culture exposed to control light for 30 minutes resulted in 19.3% neutral lipids/100 mg dry algal biomass, IR exposure for 30 minutes gave 32.9% neutral lipids /100 mg dry algal biomass (i.e. 70% more over control) and UV exposure for 30 minutes yielded 27.7% neutral lipids /100 mg dry algal biomass (i.e. 43% more compared to control). Further, control light exposure (24 hours) yielded 20.3% neutral lipids /100 mg dry algal biomass whereas exposure to yellow light (at a time-period as high as 24 hours) provided 33.2% neutral lipids/100 mg dry algal biomass only (i.e. 63% more compared to control) [Figure 1b].

The above results conclude that IR exposure is most effective in inducing neutral lipid production in algae when compared to control (untreated), UV and yellow light exposure. It has to be also noted that only 63% increase in neutral lipid yield was observed with yellow light exposure for a long time-period of 24 hours, whereas IR exposure for only 30 minutes enhanced the neutral lipid yield up to 70%.

Neutral Lipid induction in Picochlorum:
In Picochlorum strain, 22.0% neutral lipids / 100 mg dry algal biomass with control under light for 30 minutes and 23.5 % neutral lipids / 100 mg dry algal biomass in control for 24 hours was observed. Neutral lipid productivity of 34.3%/100 mg dry algal biomass (45.95% more over control) was noticed with yellow light induction (at a time-period as high as 24 hours exposure), whereas 31.7 % neutral lipid production with IR induction for 30 minutes (44% more over control) and 30.7% with UV induction for 30 minutes (39% more over control) [Figure 1c].

The above results clearly conclude that IR exposure induces better enhancement and is most effective in neutral lipid production in algae when compared to control (untreated), UV and yellow light exposure. It has to be also noted that only 45.95% increase in neutral lipid yield was observed with yellow light exposure for a long time-period of 24 hours, whereas IR exposure for only 30 minutes enhanced the neutral lipid yield up to 44%.

EXAMPLE 3:
Studying Neutral lipid production with respect to time of IR exposure
[0073]. Experiments were planned to study neutral lipid yield from algae – Nannochloris under defined time intervals of IR exposure. Particularly, the time of IR passing to algae cells is varied from 5 minutes to 30 minutes - 0 minute (control – stationary phase), 5, 10, 20 and 30 minutes [Figure 2a]. Variation in time of IR exposure from minimum (5 minutes) to maximum (30 minutes) on algae cells provides better understanding on neutral lipid yield under stressed environment. Along with neutral lipid yields, the IR influence on biomass loss is also studied during the experimentation. The biological sets of experiments with IR were performed as in Example 1. At the end of experiments, the biomass was harvested and neutral lipid yields were estimated qualitatively (Nilered assay) and quantitatively (Gravimetric).

Results
Experiments were conducted to analyze neutral lipid yield from algae with respect to time of IR exposure. All the IR exposed algal cells yielded higher neutral lipid compared to control (19.9% per 100 mg dry algal biomass). Significantly higher percentage of average neutral lipid yield was observed in 5 minutes - 35.5 % (78% enhancement over control), 10 minutes - 32.5 % (63% enhancement over control), 30 minutes - 30.4 % (52 % enhancement over control) and 20 minutes - 30.2 % (51.7 enhancement over control) of IR exposed culture [Figure 2b]. Biomass was not reduced due to IR exposure for 5 minutes.

The above results show that IR exposure for different time-periods induces enhanced neutral lipid production in algae.

EXAMPLE 4:
Studying neutral lipid production with respect to distance of IR exposure
[0074]. The initial set of experiments prove that IR is more effective than other light sources including UV and yellow light in neutral lipid induction. Accordingly, further experiments were conducted on the basis of variation in distance (10 cm and 15 cm) and time (5 minutes and 15 minutes) of IR light exposure on the algal cells (Nannochloris) [Figure 3a]. Separate experiments were also conducted by varying the distance to 30 cm for time-period of 5 minutes. The experiment controls including all conditions were maintained separately. Further, cold water circulation was employed to reduce heat generation by IR radiation due to long time exposure. Said water circulation helps in decoupling heat and IR. CO2 sparging was also provided wherein CO2 was sparged at 0.2 LPM.

Results
The results by varying the distance of IR exposure to algal culture show influence on algal neutral lipid yield. Compared to respective controls - 23.7 (10 cm), 20.2 (15 cm), higher neutral lipid yields were observed in both these distances during 5 minutes - 33.8 % (10 cm) and 32.5 % (15 cm) and 15 minutes - 33.1 % (10 cm) of IR exposure [Figure 3b].

The results of algal neutral lipid yield by varying the distance up to 30 cm for time-period of 5 minutes, 15 minutes, 30 minutes and 60 minutes is provided in Figure 3b and 3d. Compared to respective control – 18.5 %, higher neutral lipid yields (26.5 %) were observed for IR exposure at 30 cm for a duration of 5 minutes. Further, IR exposure at 10 cm and 15 cm distance for a duration of 5, 15 and 30 minutes also showed enhanced neutral lipid yields (Figure 3b).

Thus, the above results show improved neutral lipid yields by varying distance of IR exposure (such as 10 cm, 15 cm and 30 cm). The results also show that the efficacy of IR radiation to induce neutral lipids decreases with increase in distance. Further, no biomass reduction/loss is observed during 5 minutes IR exposure at varying distances [Figure 3c].

EXAMPLE 5:
Studying neutral lipid production with respect to time of IR exposure in combination with heat decoupling mechanism
[0075]. Experiments were performed with Nannochloris under controlled temperature with water bath under thermal control (cold water circulation). During the experiment, water bath temperature was maintained at 30 ºC. Due to maintenance, the algal culture was not heated up and the temperature did not increase above 35 ºC. After IR treatment for 5 minutes and 30 minutes respectively; a small amount of culture was inoculated in fresh growth medium and the rest of culture was used for the neutral lipid extraction. Cellular changes between IR treated cells and untreated (control) cells are indicated in Figure 4c and 4d respectively. There is an increase in the neutral lipid droplet count in the IR treated cells along with decrease in starch granule size and concentration.

Results:
The IR exposed algal cells yield higher neutral lipid compared to control (18.1% per 100 mg dry algal biomass in stationary phase – 0 minute). High percentage of neutral lipid yield was observed in 5 minutes - 31.1 % (78% enhancement over control) and 30 minutes – 26 % (52 % enhancement over control) of IR exposure with cold water circulation [Figure 4a].

Further, enhancement in OD was observed in 5 minutes IR treated culture which signifies that cells does not lose viability when they are exposed to IR for prescribed time [Figure 4b].
The above results show that IR exposure at different time-periods along with heat decoupling mechanism induces enhanced neutral lipid production in algae.

EXAMPLE 6:
Studying neutral lipid production in Cyanobacteria with respect to time of IR exposure
[0076]. (A) Marine cyanobacteria - Cyanobacterium aponinum culture was exposed to IR for a duration of 5 minutes with appropriate control (untreated).

Results:
Neutral lipid yield enhancement of at least 34.6 % was observed in cyanobacteria compared to control [Figure 5]. This shows the successful employment of IR exposure for increased neutral lipid production in cyanobacteria.

[0077]. (B) Fresh water cyanobacteria - Synechococcus elongatus 7942 culture was exposed to IR for a duration of 5 minutes with appropriate control (untreated).
Results:
Neutral lipid yield enhancement of at least 21.6 % was observed when compared to control [Figure 8]. This shows the successful employment of IR exposure for increased neutral lipid production in fresh water cyanobacteria such as Synechococcus elongatus.

EXAMPLE 7:
Effect exposure of IR for short duration on neutral lipid induction:
[0078]. Experiments were carried under controlled conditions for inducing the neutral lipid by IR for short duration of time. Based on the previous experiments, significantly improved neutral lipid yields were obtained at 5 min of IR exposure. Accordingly, in the present study, the algal culture (Nannochloris) was exposed from 30 seconds to 5 minutes to understand the neutral lipid yield under very short periods of exposure.

Results
Enhanced neutral lipid yield was observed at short durations of IR exposure wherein maximum neutral lipid yield was obtained at 5 minutes of exposure [Figure 6].

EXAMPLE 8:
Effect of combined IR exposure and Nutrient stress on neutral lipid production
[0079]. The method of IR induced neutral lipid production of the instant disclosure can be combined with known stress inducing methods such as nutrient stress selected from a group comprising nitrogen stress, phosphorous stress, potassium stress. A study was conducted to confirm the effect of IR exposure combined with nutrient stress in Nannochloris.

Inoculum Development
Inoculum was raised in flat panel glass PBR. Medium used for inoculum development comprised of 150 ppm ‘N’ (nitrogen), 9.5 ppm ‘P’ (phosphorous) and 1X F/2 ‘TM’ (trace metals) and pH of 7.2 was maintained through CO2 purging. Continuous light exposure of about 300 µmols/m2/s was provided on both sides of panel. Starting density of the culture was about 0.4 OD which reached to about 6 OD on 2nd day.

Experimental Conditions
The culture was exposed to 6 different conditions after concentrating it to 70 OD which is equivalent to 20 g/l of biomass [Figure 7a]. These conditions are:
1) Nitrogen (N), phosphorous (P), trace metals (T) added to the concentrated cells
2) N and T only added
3) only N added
4) Only P and T added
5) only P added
6) Nothing added

Cells were incubated at 300 micro mols/m2/d light intensity for maximum of 2 days.

Results: No nutrients were found in the medium on day 1, i.e. all the nutrients were taken up by the cells. Due to nutrient starvation, absolute neutral lipid content increased from 15% to 23% on day 1. Due to IR alone on day 0, neutral lipid content was increased to 23%. Combined effect of nutrient starvation and IR increased neutral lipid content to 35% from 15% in control (0 day sample).

The above results are shown in Figure 7b. Control condition is shown in red box giving 15% neutral lipid. This value was measured as soon as cells were concentrated, i.e. they were not nutrient starved. At this moment (day 0), one sample was exposed to IR. Neutral lipid increased to 23% from 15% in control (i.e. 53% increment with respect to control). This condition shows sole effect of IR on neutral lipid increase. In case, where no nutrients were added (shown in blue box), neutral lipid increased due to nutrient starvation alone up to 24% with respect to control (15%) which means that nutrient starvation alone can give similar outcome in 24 hours (as measurement was done every 24 h for 2 days) which IR can give in 5 minutes of exposure. Further, when nutrient starved culture was exposed to IR, neutral lipid content significantly increased up to 35% (shown in blue box) i.e. 133 % increment with respect to control.

The above results establish that IR alone has a significant effect on lipid induction in algae (both neutral and total lipids). Combined IR exposure and nutrient stress increases the lipid production further. Additionally, nitrogen limitation is most effective in enhancing lipid content.

EXAMPLE 9:
Neutral and total lipid induction in algae cultivated in outdoor pond:
Experiments were performed at outdoor environment with concentrated 2% slurry of Picochlorum species. Total 5 KL of culture was harvested by PAL filtration and concentrated to 50 L by centrifugation (0 hr Sample). Throughout the experiment run, pH of the culture, temperature and salinity was monitored. Once the pH of culture increased beyond 7 it was adjusted to 7 by sparging CO2 from external source. With all these operating conditions, culture was exposed to outdoor environment for 12/12 light (300 ± 20 µE) and dark conditions under nitrogen limitation. Sample was harvested once every 24 hours for lipid induction using IR light. Nitrogen starved culture was exposed to IR under laboratory condition (closed) and appropriate control (no IR treatment) was used. After the IR exposure, both the samples were centrifuged and culture pellet was collected for the lipid analysis.

The results show an increase in neutral lipids and total lipids induction upon IR exposure and that the method is capable of inducing both neutral and total lipids in algae cultivated in outdoor ponds as well [Figure 9].

ADVANTAGES
[0080]. The present disclosure enables enhanced lipid production in algae by a simple and efficient method using IR exposure.
[0081]. The present method shows enhanced neutral lipid production at a shorter time duration and without reduction of biomass.
[0082]. The present method also increases total lipid content in algae at a shorter time duration and without reduction of biomass.
[0083]. The enhanced neutral lipid production by the present method is immensely useful in producing high quantities of algal-derived biofuels.
[0084]. The present method is cost-effective in large scale cultivation. For example, solar radiation can be used in outdoor algal cultivation to induce neutral lipids by allowing only IR rays through selective solar filters.
[0085]. Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based on the description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well-known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein.
[0086]. 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 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.
[0087]. 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.
[0088]. 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.
[0089]. 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.
[0090]. 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.
[0091]. 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.