Claims:1. A composition comprising concentrated bulk algal culture and cryoprotectant.
2. The composition as claimed in claim 1, wherein biomass concentration of the concentrated bulk algal culture is ranging from about 10% to 15%; wherein the concentrated bulk algal culture is concentrated from a bulk algal culture having volume ranging from about 4KL to 80KL.
3. The composition as claimed in claim 2 wherein the bulk algal culture comprises algal culture selected from group comprising cultures of Nannochloropsis sps, Picochlorum sps, Chlorella, Nannochloris and Cyanobacterium sp., or any combination thereof.
4. The composition as claimed in claim 1, wherein the cryoprotectant is selected from a group comprising Dimethyl Sulfoxide (DMSO), Methanol, Polyethylene glycol (PEG), propylene glycol, dimethyl formamide and Glycerol or any combination thereof.
5. The composition as claimed in claim 1, wherein concentration of the concentrated bulk algal culture is ranging from about 90% (v/v) to 95% (v/v) and concentration of the cryoprotectant is ranging from about 5% (v/v) to 10% (v/v).
6. The composition as claimed in claim 1, wherein the composition is present in laminated foil bags; wherein the laminated foil bags are triple laminated aluminium foil bags; wherein the laminated foil bags are maintained at temperature ranging from about -75°C to --80°C.
7. The composition as claimed in claim 1, wherein the concentrated bulk algal culture of the composition is an outdoor adapted culture.
8. A process for bulk cryopreservation of algal culture to obtain the composition as claimed in claim 1, comprising-
concentrating the bulk algal culture;
mixing the concentrated bulk algal culture with cryoprotectant to obtain a mixture; and
incubating the mixture, followed by cooling
to obtain the composition.
9. The process as claimed in claim 8, wherein the bulk algal culture is concentrated by centrifugation.
10. The process as claimed in claim 8, wherein the mixture of concentrated bulk algal culture and cryoprotectant comprises about 90% to 95% of the concentrated bulk algal culture mixed with about 5% (v/v) to 10% (v/v) of the cryoprotectant.
11. The process as claimed in claim 8, wherein the mixture comprising the concentrated bulk algal culture and the cryoprotectant are incubated at a temperature ranging from about 23°C to 30°C for a duration ranging from about 30 minutes to 45 minutes, in low light condition.
12. The process as claimed in claim 8, wherein the process further comprises loading the mixture into laminated foil bags.
13. The process as claimed in claim 8, wherein the cooling is stepwise cooling, comprising:
• cooling to a temperature of about 4°C for a duration ranging from about 10 hours to 12 hours;
• cooling to a temperature of about 0°C for a duration ranging from about 10 hours to 12 hours; and
• cooling to a temperature of about -20°C for a duration ranging from about 10 hours to 12 hours.
14. The process as claimed in claim 8, wherein the composition is maintained at a temperature ranging from about -75°C to -80°C.
15. A process of revival of the composition obtained by the process as claimed in claim 8, comprising directly exposing the composition to waterbody to obtain revived algal culture.
16. The process as claimed in claim 15, wherein the composition is thawed prior to the exposure to the waterbody.
17. The process as claimed in claim 16, wherein the composition is thawed at temperature ranging from about 23°C to 30°C, preferably room temperature for about 1 hour to 1.5 hours, preferably about 1.5 hours.
18. The process as claimed in claim 15, wherein the waterbody is culture pond having area ranging from about 1m2 to 16m2; wherein the culture pond is an outdoor culture pond.
19. The process as claimed in claim 15, wherein the waterbody has culture media selected from a group comprising Bold's Basal Medium (BBM), F/2 medium, Artificial sea water nutrient medium III (ASN III medium), (Blue Green) BG11 medium, and Plymouth and erdschreiber medium (PE) or any combination thereof.
20. The process as claimed in 15, wherein the revival is carried out in darkness for about 10 to 12 hours followed by low light condition maintained for a duration of about 8 hours to 10 hours post which the water body is exposed to sunlight for about 1 hour to 2 hours.
21. The process as claimed in claim 15, wherein viability of cells of the revived algal culture ranges from about 90% to 95%.
, Description:TECHNICAL FIELD
The present disclosure relates to the field of cell biology, microbiology and biotechnology. More specifically, the present disclosure provides a composition comprising concentrated bulk algal culture and cryoprotectant; a process of bulk cryopreservation to obtain said composition and a process for revival of said composition. Said process of bulk cryopreservation is simple and economical and enables cryopreservation of bulk volumes of cultures which can be revived directly at large scale, for example in outdoor waterbodies, thus eliminating lab-scale revival and scale-up processes. The process ensures genetic stability of the bulk cryopreserved cultures.
BACKGROUND OF THE DISCLOSURE

Any industrial process which is dependent on biological systems for production has a basic requirement of stable function fulfilling master stock cultures. Maintenance of actively growing microbial strains over a long period of time tends to be costly and time consuming. Algal cultivation, particularly, is a skilful job, which involves establishment of cultures from lab scale to outdoors. Strain selection, adaptation and outdoor cultivation of algae requires considerable amount of time, capex and opex.
Long term phenotypic and genotypic stability is a fundamental prerequisite for the successful biotechnological exploitation of any microorganism, as without stable starter culture productivity cannot be guaranteed. Sub culturing is a traditional process which is employed to maintain the microorganism, but the process has its own advantages and disadvantages – for example: repeated sub culturing may lead to changes in genetic makeup. Thus, for long term preservation of micro flora such as algal cultures, various other processes such as preservation in mineral oil, lyophilization, vitrification and cryopreservation are employed to ensure revival of strains. Amongst all mentioned techniques cryopreservation is widely used for long term maintenance of algal strains. Cryopreservation is one of the long-term preservation processes which is widely used to ensure the preservation of genetic stability which has certain advantages over routine sub culturing. Conventional cryopreservation technique involves cryopreservation of commercially important strains at lab scale with establishment of revival process. As most of the cryopreservation studies are conducted at lab scale, comparatively smaller volumes of cultures are handled, which ensures the uninterrupted supply of the cultures. For production strains, conventional cryopreservation approach takes comparatively more retention time in lab and frequent scaling up while demanding for more laborious work also leads to change in genotypic composition. The cultures during scale up (Agar / Liquid) may get contaminated with bacteria, fungus and non-targeted organisms, which may lead to culture loss.
As potential strain adaptation and establishment at outdoors involves several challenges, adaptation of the strain which is performing at actuals on site needs to be preserved with same genetic composition. Performance of algal strains in culture depends on the culture environment either in close system or in open ponds. Conventional cryopreservation till date is performed at very small scale in cryovials which is scalable to indoor lab conditions Translatability of algal strains from laboratory to outdoors involve many unforeseen challenges such as physical and biological stresses which cause alteration in genetic composition due to adaptation of strain at different levels of cultivation. Owing to biotic and abiotic stresses the outdoor production strain faces ¬severe crashes during inoculum development process. The need of the hour is a process that allows long term storage of outdoor adapted algal cultures with their intrinsic characters and adaptations which the microorganism has endured through time in outdoors, to be arrested in the same stage in situ; a cryopreserved algal culture which eliminates the need for laboratory scale revival and a revival process, all of which together overcome the problem of loss of genetic stability associated with lab scale revival of the cryopreserved algal strains. The instant disclosure overcomes the shortcomings of prior art without compromising on the genetic stability of cultures.
SUMMARY OF THE DISCLOSURE
The object of the present disclosure is to provide a composition comprising bulk cryopreserved algal culture and a process of preparing the same, which allows successful cryopreservation of bulk volumes of algal culture while ensuring genetic stability and reproducibility of the bulk cryopreserved cultures on a large scale. The present disclosure further provides a process for revival of the bulk cryopreserved algal culture wherein the revival can be performed directly, on a large scale.
Accordingly, the present disclosure relates to a composition comprising concentrated bulk algal culture and cryoprotectant.
Further, the present disclosure relates to a process for bulk cryopreservation of algal culture to obtain the above described composition comprising,
concentrating the bulk algal culture;
mixing the concentrated bulk algal culture with cryoprotectant to obtain a mixture; and
incubating the mixture, followed by cooling
to obtain the composition.
Further, the present disclosure relates to a process of revival of the composition obtained by the process described above, comprising directly exposing the thawed composition to waterbody to obtain revived algal culture.
BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
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, in accordance with the present disclosure where:

Figure 1 depicts bulk cryopreserved concentrated algal culture tubes for revival after 15 days, 1 month, 3 months, 6 months and 1 year of bulk cryopreservation.
Figure 2 depicts the step of loading the mixture comprising the concentrated bulk algal culture cryoprotectant into laminated foil bags for subjecting to the bulk cryopreservation protocol of the instant invention
Figure 3 depicts revival of samples after 1 month of bulk cryopreservation. Revival is observed for samples containing 5% and 8% DMSO. But no revival is observed for samples containing 25% and 50% DMSO.
Figure 4 depicts the study on revival of concentrated algal culture under winter simulation conditions in environmental photobioreactor after bulk cryopreservation.
Figure 5 depicts variation in OD observed at 750nm under winter simulation conditions in the ePBR in turbidostat mode of operation during revival of the algal culture after 15 days of bulk cryopreservation.
Figure 6 depicts variation in OD observed at 750nm under winter simulation conditions in the ePBR in turbidostat mode of operation during revival of the algal culture after 1 month of bulk cryopreservation.
Figure 7 depicts revival of the algal culture after 6 months of bulk cryopreservation. Revival is observed for samples containing 5%, 8% and 50% DMSO. However, the sample containing 50% DMSO did not yield healthy culture. No revival is observed for samples containing 25% DMSO.
Figure 8 depicts revival of the algal culture after 9 months of bulk cryopreservation. Revival is observed for samples containing 5% and 8% DMSO. But no revival is observed for samples containing 25% and 50% DMSO.
Figure 9 depicts algal culture revived after one-month of bulk cryopreservation, wherein the bulk cryopreservation is initiated in monsoon condition; (a) depicts microphotograph of the revived culture and (b) depicts the revived culture in a 1m2 pond.
Figure 10 depicts OD jump of algal culture revived after 3-months of bulk cryopreservation, wherein the bulk cryopreservation is initiated in monsoon condition
Figure 11 depicts algal culture revived after 3-months of bulk cryopreservation, wherein the bulk cryopreservation is initiated in monsoon condition; (a) depicts the revived culture in a 1m2 pond and (b) depicts scale up of the revived culture in two 5m2 ponds.
Figure 12 depicts a graph reflecting minimum and maximum temperatures by the algal cultures during revival in a 1 m2 pond.
Figure 13 depicts growth performance of the algal culture during revival of the culture after 6 months of bulk cryopreservation, wherein the bulk cryopreservation was initiated during monsoon season.
Figure 14 depicts growth performance of the algal culture during revival of the culture after 3 months of bulk cryopreservation; wherein the bulk cryopreservation was initiated during winter season.
Figure 15 depicts study of the algal culture at turbidostat mode of operation for seven days to study under daily dilution mode, post revival of the culture after 6 months of bulk cryopreservation, wherein the bulk cryopreservation was initiated during monsoon season.
Figure 16 depicts study of the algal culture at turbidostat mode of operation for seven days to study under daily dilution mode, post revival of the culture after 3 months of bulk cryopreservation, wherein the bulk cryopreservation was initiated during winter season.
Figure 17 (a) depicts revival of bulk cryopreserved algal culture, wherein the bulk cryopreservation was initiated during in the monsoon season, after 6 months of bulk cryopreservation in 1m2 pond (b) depicts revival of bulk cryopreserved algal culture, wherein the bulk cryopreservation was initiated during the winter season, after 3 months of bulk cryopreservation, in a 1m2 pond (c) depicts microphotographs of bulk cryopreserved algal culture, wherein the bulk cryopreservation was initiated during the monsoon season, after 6 months of bulk cryopreservation and microphotographs of bulk cryopreserved algal culture wherein the bulk cryopreservation was initiated during the winter season, after 3 months of bulk cryopreservation, in a 1m2 pond.
Figure 18 (a) depicts revival of bulk cryopreserved algal culture, wherein the bulk cryopreservation was initiated during the monsoon season, after 9 months of bulk cryopreservation, in a 1m2 pond (b) depicts revival of bulk cryopreserved algal culture, wherein the bulk cryopreservation was initiated during the winter season, after 6 months of cryopreservation, in a 1m2 pond.
Figure 19 depicts OD jump of algal culture revived after 9 months of bulk cryopreservation wherein the bulk cryopreservation is initiated in monsoon condition and algal culture revived after 6 months of bulk cryopreservation wherein the bulk cryopreservation is initiated in winter condition.
Figure 20 depicts revival of algal culture after 18 months of bulk cryopreservation, in a 1 m2 pond.
Figure 21 depicts turbidostat study of algal culture revived after 18 months of bulk cryopreservation, in a 1 m2 pond.
Figure 22 depicts turbidostat study of algal culture revived after 24 months of bulk cryopreservation, in a 1m2 pond.
Figure 23 depicts molecular analysis data to confirm presence of production strain Picochlorum in pond sample, mixture before bulk cryopreservation, cultures revived after 18 months and 24 months of bulk cryopreservation.
Figure 24 (a) depicts revival of algal culture prepared from bulk cryopreservation of centrifuged slurry, in 1m2 ponds (b) depicts revival of algal culture prepared from bulk cryopreservation of diluted DAF slurry, in 1m2 ponds.
Figure 25 (a) depicts microphotograph of revived algal culture prepared from bulk cryopreservation of centrifuged slurry (b) depicts microphotograph of revived algal culture prepared from bulk cryopreservation of diluted DAF slurry.
Figure 26 depicts revival of Picochlorum sp bulk cryopreserved culture after 3 months of bulk cryopreservation in a 5m2 pond, wherein bulk cryopreservation was initiated during winter season.
Figure 27 (a) depicts growth performance of revived algal culture after 3 months of bulk cryopreservation of membrane filtered slurry, in 5m2 ponds (b) depicts growth performance of revived algal culture after bulk cryopreservation of centrifuge slurry, in 5m2 ponds.
Figure 28 depicts revival of Picochlorum sp winter bulk cryopreserved culture in 16m2 pond after 12 months of bulk cryopreservation.
Figure 29 (a) depicts turbidostat profile of bulk cryopreserved Picochlorum clone I revived after one year of bulk cryopreservation (b) depicts turbidostat profile of bulk cryopreserved Nannochloropsis I revived after one year of bulk cryopreservation (c) depicts turbidostat profile of bulk cryopreserved Picochlorum clone II revived after one year of bulk cryopreservation.
Figure 30 depicts outdoor revival of bulk cryopreserved algal culture, in a 2.6 m2 pond.
Figure 31 depicts microphotographs of revived cultures of Nannochloropsis clone I, Picochlorum clone I and Picochlorum clone II after bulk cryopreservation, respectively.
Figure 32 depicts molecular analysis of revived algal cultures after bulk cryopreservation; Legend for each of the Lanes: M- 1Kb ladder, D- Nannochloropsis clone I, E- Picochlorum clone I, F- Picochlorum clone II, P-Positive, N-Negative (a) depicts presence of gDNA of Nannochloropsis clone I, Picochlorum clone I and Piclochlorum clone II (b) depicts the presence of genes specific to 18S specific primer in Nannochloropsis clone I, Picochlorum clone I and Picochlorum clone II, respectively (c) depicts the presence of Nannochloropsis clone I upon subjecting the algal cultures to molecular analysis with Nannochloropsis specific primer (d) depicts the presence of Picochlorum sp (clone I and clone II) upon subjecting the algal culture to molecular analysis with Picochlorum specific primer.
Figure 33 depicts comparison between conventional inoculum development and inoculum development using the process of the present dislcosure.
Figure 34 depicts scaled-up bulk cryopreserved cultures revived after 1 month and 3 months of being subjected to bulk cryopreservation, in two 5m2 and 500m2 culture ponds respectively.

DETAILED DESCRIPTION
As used throughout the present disclosure, ranges are a shorthand for describing each and every value within the range. Any value within the range can be selected as the terminus of the range.
As used herein, the phrase ‘bulk culture’ or ‘bulk algal culture’ refers to algal culture prepared from bulk amount of the culture of interest. Said ‘bulk culture’ or ‘bulk algal culture’ may be any algal culture obtained from a large-scale culture or culture pond or directly from a waterbody including but is not limited to pool, pond, river, sea and ocean, in large volumes. The bulk amount of algal culture also referred to herein as ‘pond sample’ is centrifuged to prepare concentrated bulk algal culture. Said concentrated bulk algal culture is cryopreserved with cryoprotectant to form a ‘bulk cryopreserved composition; interchangeably referred to as ‘bulk cryopreserved algal culture’ or ‘bulk cryopreserved culture’ which is fit for direct outdoor or large-scale revival.

As used herein, the terms ‘slurry’ or ‘concentrated bulk algal culture’ used interchangeably are primarily used in reference to the slurry resulting from the centrifugation of the bulk algal culture which is subjected to the bulk cryopreservation process of the present disclosure.

As used herein, the phrases ‘outdoor adapted culture’ and ‘outdoor adapted algal culture’ refer to microbial or specifically, algal cultures sourced or procured from outdoor waterbodies with their entire biome, wherein when subjected to bulk cryopreservation, all cells growing in same environment get arrested and preserved at sub-zero temperatures. Said cultures provide the advantage allowing revival directly on a large scale, for example in outdoor culture ponds, thereby eliminating the genotypic changes generally observed during revival and/or scale-up of conventionally cryopreserved cultures.

As used herein, the phrase ‘low light condition’ refers to conditions of darkness or conditions where light intensity is ranging from about 50 microeinstein (mE) to 100mE. In the context of the experiment being performed outdoors, the term ‘darkness’ refers to conditions of darkness achieved at night when the sun is naturally absent or when said condition is recreated artificially. The phrase ‘low light condition’ refers to evening when there is low light naturally, or when said low light conditions similar to evening are re-created artificially, for example, by covering of culture ponds by shade net cloth.

As used herein, the term ‘production strain’ refers to algal strains having industrial applicability such as but not limited to Picochlorum sp., Nannochloropsis sp, Chlorella spp., and Cyanobacerium sp.

As used herein, the term ‘production pond’ refers to culture facilities such as but not limiting to culture pond have area ranging from about 1m2 to 16m2, in the indoors or outdoors; preferably in the outdoors.
The present disclosure relates to a composition comprising concentrated bulk algal culture, and cryoprotectant.
In an embodiment of the present disclosure, the bulk algal culture is derived from a large-scale culture or culture ponds or directly from a waterbody including but is not limited to pool, pond, river, sea and ocean. In an embodiment of the present disclosure, the bulk algal culture is derived from a culture pond. Said bulk algal culture is concentrated to prepare the concentrated bulk algal culture. In a non-limiting embodiment, the bulk algal culture is concentrated by centrifugation to prepare the concentrated bulk algal culture.
In an embodiment, the bulk algal culture is selected from a group comprising cultures of Nannochloropsis sp, Picochlorum sp, Chlorella sp and Cyanobacerium sp or any combination thereof.
In an embodiment, the concentrated bulk algal culture has biomass concentration ranging from about 10% to 15%.
In another embodiment, the concentrated bulk algal culture has ash free dry weight (AFDW) ranging from about 70gm/L to 140gm/L and Optical Density (OD) at 750nm ranging from about 200 to 250.
Further, the cryoprotectant in the above described composition is a penetrating or non-penetrating cryoprotectant. In an embodiment, the cryoprotectant is a penetrating cryoprotectant selected from a group comprising Dimethyl Sulfoxide (DMSO), Methanol, Polyethylene glycol (PEG), propylene glycol, dimethyl formamide and Glycerol or any combination thereof.
In another embodiment, the cryoprotectant is Dimethyl Sulfoxide (DMSO).
In an embodiment of the disclosure, the composition comprises about 90% to 95% of the concentrated bulk algal culture and about 5% (v/v) to 10% (v/v) of cryoprotectant.
In an embodiment, the Figure 3 shows that the composition comprising concentration of DMSO exceeding 25% cannot be successfully revived.
In an embodiment, the composition is present in laminated foil bags.
In an embodiment, the laminated foil bags are composed of material selected from a group comprising aluminium, low density polyethylene (LDPE) and polypropylene or any combination thereof.
In another embodiment, the laminated foil bag has multiple layers of lamination. In an embodiment, the number of layers of lamination is ranging from about 1-3 layers of lamination. In a preferred embodiment, the laminated foil bags are triple laminated aluminium foil bags, wherein said laminated foil bags have volume ranging from about 125ml to 3000ml. Said laminated foil bags are capable of containing bulk volume of algal culture and facilitate reduction of operational costs.
In an embodiment, the algal culture in the composition is resistant to stress such as abiotic stress and biotic stress due to the bulk volume of the algal culture. As result, said composition, when subjected to revival in the direct outdoor condition is not susceptible to crashing out due to high density of the biomass. Thus, said composition of the present disclosure is superior when compared to the compositions obtained by conventional cryopreservation techniques, which are prone to crashing when subjected to revival under direct outdoor conditions.
In an embodiment, the algal culture in the composition is resistant to external contamination when subjected to revival under direct outdoor conditions. Said resistance of the algal culture of the composition is due to the higher biomass concentration in the composition.
In an embodiment, the composition of the present disclosure eliminates the need of scaling-up during revival, which is followed when the cultures are subjected to conventional cryopreservation technique.
In an embodiment, the composition of the present disclosure is capable of being directly revived in a water body selected from a group comprising pool, pond, river, sea and ocean.
Further, the present disclosure provides a process for bulk cryopreservation of algal culture for obtaining the above defined composition.

The present disclosure provides a process for bulk cryopreservation of algal culture to obtain a composition comprising concentrated bulk algal culture, wherein said process comprises
concentrating the bulk algal culture;
mixing the concentrated bulk algal culture with cryoprotectant to obtain a mixture; and
incubating the mixture, followed by cooling
to obtain the composition.

In an embodiment, bulk algal culture is concentrated by centrifugation to achieve maximum solid concentration, to prepare the concentrated bulk algal culture.
The bulk algal culture is subjected to centrifugation at about 8000rpm to 9000rpm, for about 30 minutes to 60 minutes, at temperature of about 30°C to 35°C, to prepare the concentrated bulk algal culture.
In an embodiment, centrifugation of the algal cultures is performed till biomass concentration of about 10% to 15% is achieved.
In an embodiment, the concentrated bulk algal culture has OD at 750nm ranging from about 200 to 250. Further, the concentrated bulk algal culture has a biomass concentration of about 50 gm wt. to 150 gm wt., preferably about 100gm wt.
Post centrifugation, the concentrated bulk algal culture is mixed with a cryoprotectant to obtain a mixture.
The mixture of concentrated bulk algal culture and cryoprotectant is processed at low light condition, wherein the mixture further is mixed by continuous stirring.
The cryoprotectant is a penetrating or non-penetrating cryoprotectant. Preferably, the cryoprotectant is a non-penetrating cryoprotectant selected from a group comprising Dimethyl Sulfoxide (DMSO), Methanol, Polyethylene glycol (PEG) and Glycerol or any combination thereof. In a preferred embodiment, the cryoprotectant is Dimethyl Sulfoxide (DMSO).
In a further embodiment, concentration of the cryoprotectant in the composition is ranging from about 5 % to 10 % (v/v), preferably about 8% to 10% (v/v). The resultant mixture of concentrated algal culture and cryoprotectant is stirred with the help of mixing rods and subsequently subjected to incubation in low light condition of about 50mE to 100mE at a temperature of about 23°C to 30°C, for about 30 minutes to 45 minutes, preferably for about 45 minutes.
In an embodiment, after incubation in low light condition, the process further comprises loading the mixture comprising the concentrated algal culture and the cryoprotectant into laminated foil bags. In an embodiment, Figure 2 shows the step of loading the mixture into laminated foil bags for subjecting to the bulk cryopreservation.
In a non-limiting embodiment, the laminated foil bags are of material selected from a group comprising aluminium foil, low density polyethylene and polypropylene or any combination thereof.
Said laminated foil bags have multiple layers of lamination. In an embodiment, the number of layers of lamination is ranging from about 1-3 layers of lamination. In a preferred embodiment, the laminated foil bags are triple laminated aluminium foil bags; wherein said laminated foil bags have volume ranging from about 125ml to 3000ml.
In an embodiment, the laminated foil bags comprising the incubated mixture of concentrated bulk algal culture and cryoprotectant are sealed and subjected to cooling wherein the cooling is stepwise cooling, comprising:
cooling to a temperature of about 4°C for a duration ranging from about 10 hours to 12 hours;
cooling to a temperature of about 0°C for a duration ranging from about 10 hours to 12 hours; and
cooling to a temperature of about -20°C for a duration ranging from about 10 hours to 12 hours.
Post cooling at about -20 degrees for about 10 to 12 hours, the sealed laminated foil bags containing the mixture of concentrated bulk algal culture and cryoprotectant are transferred to a freezer and maintained at a temperature of about --75°C to -80°C, preferably about -80°C.
The resultant bulk cryopreserved composition of algal culture may be cryopreserved for periods up to 24 months. In an embodiment, the composition of the present disclosure may be prepared at any time throughout the year, irrespective of the season.
In an embodiment, process for bulk cryopreservation of algal culture allows long term preservation of bulk amounts of culture having high biomass concentration. Said high biomass concentration of the bulk cryopreserved algal cultures yielded by the above described process confers to the algal culture resistance to external contamination when subjected to direct revival in outdoor conditions.
Further, the process, in view of the use of laminated foil bags instead of cryovials used in conventional cryopreservation techniques, the bulk cryopreservation process of the present disclosure is more cost effective as compared to conventional cryopreservation techniques while also allowing cryopreservation of larger volumes of culture. The bulk cryopreservation process of the present disclosure thus reduces the operational costs generally associated with cryopreservation.
In an embodiment, the process of the present disclosure yields a composition such that the need of repeated scale-up is eliminated, which is generally required when cultures are subjected to conventional cryopreservation technique that allows cryopreservation of small quantities of culture. The bulk cryopreservation process allows long term storage of outdoor adapted algal culture with its intrinsic characters and adaptations which the strain has endured through time in outdoors to be arrested in the same stage in situ. The bulk cryopreservation process of the present disclosure facilitates cryopreservation of outdoor adapted cultures which can be directly revived on a large scale, for example, in outdoor culture ponds, thus eliminating the requirement for lab scale revival and scale-up thus allowing reduction of scale up time, capex and opex compared to conventional inoculum development process. The composition prepared by the bulk cryopreservation process of the present disclosure allows revival at site with the same genetic composition as before cryopreservation.
The present disclosure further provides a process for revival of the composition comprising concentrated bulk algal culture described above.
The present disclosure provides a process for revival of the composition of concentrated bulk algal culture, wherein said process comprises directly exposing the composition to waterbody to obtain revived algal culture.
In an embodiment, the composition is subjected to thawing prior to the exposure to the waterbody.
In an embodiment, the composition is thawed at temperature ranging from about 23°C to 30°C, preferably room temperature for about 1 hour to 1.5 hours, preferably about 1.5 hours.
In an embodiment, the waterbody is a culture pond have area ranging from about 1m2 to 16m2. In an embodiment, the culture pond is an outdoor culture pond. In another the embodiment, the composition is directly exposed the waterbody by direct inoculation.
Said waterbody that the culture is directly exposed to contains culture media selected from a group comprising Bold's Basal Medium (BBM), F/2 medium, Artificial sea water nutrient medium III (ASN III medium), (Blue Green) BG11 medium, and Plymouth and erdschreiber medium (PE) or any combination thereof.
The revival process is carried out in darkness and then low light condition followed by direct exposure to light.
In an embodiment, the revival is carried out in the evening to mimic dark incubation in the laboratory at the time of revival. Post said dark incubation, the pond is exposed to low light condition wherein the pond is covered with shade net fabric to maintain low light condition in the pond. After said exposure to low light condition, the cloth is removed to expose the culture to direct sunlight. In an embodiment, the revival is carried out in batch mode of cultivation.
In an embodiment, the dark incubation is carried out for about 10 hours to 12 hours, post which the pond is exposed to low light condition for about 8 hours to 10 hours. After said exposure to low light condition, the pond is exposed to direct sunlight for about 1 hour to 2 hours. In an embodiment, light intensity in low light condition is ranging from about 50mE to 100mE and light intensity during exposure to direct sunlight is ranging from about 600mE to 1500mE.
In a non-limiting embodiment, the dark incubation is carried out for about 10 hours, about 10.5 hours, about 11 hours, about 11.5 hours or about 12 hours, post which the pond is exposed to low light condition for about 8 hours, about 8.5 hours, about 9 hours, about 9.5 hours or about 10 hours. In another non-limiting embodiment, after said exposure to low light condition, the pond is exposed to direct sunlight for about 1 hour, about 1.5 hours or about 2 hours.
In a further non-limiting embodiment, light intensity in low light condition is about 50mE, about 55mE, about 60mE, about 70mE, about 80mE, about 90mE or about 100mE and light intensity during exposure to direct sunlight is about 600mE, about 700mE, about 800mE, about 900mE, about 1000mE, about 1100mE, about 1200mE, about 1300mE, about 1400mE or about 1500mE.
In an embodiment, temperature of the culture during revival is ranging from about 15°C to 35°C.
The revival may be performed anytime throughout the year, irrespective of the season or climatic conditions. In an embodiment, the revival of the bulk cryopreserved algal culture contained in the composition of the present disclosure can be performed at intervals of up to 24 months after bulk cryopreservation.
In another embodiment, revival of the bulk cryopreserved algal culture is performed at time intervals of about 1 month, about 3 months, about 6 months, about 9 months, about 12 months, about 24 months etc after bulk cryopreservation. In a preferred embodiment, said revival process may be performed at time intervals of about 15 days, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months or about 24 months after bulk cryopreservation.
Revival at said time intervals after bulk cryopreservation is checked by molecular analysis techniques. In an embodiment of the present disclosure, successful revival is confirmed by molecular analysis of cultures revived after about 15 days, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months or about 24 months after bulk cryopreservation.
Molecular analysis techniques to analyse the revived samples are selected from a group comprising Polymerase Chain Reaction (PCR), gel electrophoresis and genomic DNA extraction.
In a further embodiment, growth of culture during the revival process is assessed by measuring absorbance of the culture. In a preferred embodiment, absorbance of the revived culture is assessed by measuring OD at 750 nm on a spectrophotometer.
In an embodiment of the present disclosure, revival of the bulk cryopreserved composition of the present disclosure is observed within about 3 days to 5 days of inoculation in the culture pond. Further, the revived cultures show OD jump of about 0.1 to 0.3 per day. In an embodiment, the revived cultures have biomass concentration ranging from about 350 mg/L to 450 mg/L and ash free dry weight (AFDW) ranging from about 200 mg/L to 400mg/L.
Further, the revived cultures show a productivity of about 15 gm/m2/day to 20 gm/m2/day. In a non-limiting embodiment, the revived cultures show a productivity of about 15 gm/m2/day, about 16 gm/m2/day, about 17 gm/m2/day, about 18 gm/m2/day, about 19 gm/m2/day or about 20 gm/m2/day.
The bulk cryopreservation and revival process of the instant disclosure ensures successful revival of algal cultures derived from bulk cultures, on a large scale, for example in outdoor culture ponds, despite environmental stresses such as unfavourable temperatures, rain etc. Further, in view of the bulk cryopreserved composition comprising concentrated bulk algal cultures which are outdoor adapted, the revived cultures have minimal or no alteration in genetic composition due to adaptation of strain at different conditions of cultivation.
Post revival, the revived cultures may be subjected to further scale up.
Taken together, the bulk cryopreserved composition comprising the concentrated bulk algal culture, the process of arriving at the same and the process of revival of said bulk cryopreserved composition ensures that the entire ecosystem of a large scale culture or culture pond that the concentrated bulk algal culture is derived from is preserved, thus maintaining the genetic stability and reproducibility of the bulk cryopreserved strain while substantially reducing the scale up time. In a non-limiting embodiment, as shown in Figure 33, the process of the present disclosure is able to show revival within a time period ranging from about 3 days to 5 days as opposed to conventional techniques that take about 2 months or more for revival.
The processes of the present disclosure yield algal cultures with high density in open ponds which subsequently reduces risk of crashing of cultures due to biotic and abiotic stresses. The processes of the present disclosure allow upscaling of cultures at higher densities in open pond with minimal operational cost. In an embodiment of the present disclosure, Figure 34 depicts scaled-up bulk cryopreserved cultures revived after 1 month and 3 months of being subjected to bulk cryopreservation respectively.
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
EXAMPLE 1: Bulk Cryopreservation of the algal culture and revival of the composition obtained after bulk cryopreservation -
Bulk Cryopreservation-
About 10 litre culture of Picochlorum sp was used for bulk cryopreservation of production strain.
Biomass and OD at 750nm were measured before cryopreservation. 0.4 g/lit biomass (ash free dry weight (AFDW)) with OD 1.19 was estimated for the Picochlorum sp stock culture. 10 litres of the stock culture was centrifuged at 7000 rpm for about 20 min to concentrate the culture. The pellet was re-suspended in about 500 ml of commercial media having final OD of 20 at 750 nm and biomass concentration of about 40 gm weight (wet weight). 6 experiments were designed with about 500 ml of concentrated culture – wherein four experiments were with concentrated culture having 20 OD, each comprising 100 ml culture and remaining 100 ml concentrated culture was diluted 1:1 with F/2 culture media to get 10 OD for two experiments (table 1). For each experiment 5 tubes of about 20 ml concentrated culture were prepared to check the revival after 15 days, 1 month, 6 months, 9 months and 1 year (figure 1).

Final biomass in experiments 1, 2, 5, and 6 for 20 ml concentrated culture was about 1.6 gm/lit wt. weight and for experiments 3 and 4 was about 0.8 gm/lit wt. weight. After addition of cryoprotectant as per the concentration provided in table 1, the culture was incubated for about 15 min to 20 min at room temperature. Slow cooling process was applied for cryopreservation of bulk pond samples, wherein the samples were stored at a temperature of about 4°C for about 12 hours, followed by about 0°C for about 12 hours, followed by about -20°C for about 12 hours. Said samples were stored at about -80°C for further use.

Table 1

Experiment No Final OD of culture % cryoprotectant (DMSO)
1 20 5
2 20 8
3 10 5
4 10 8
5 20 50
6 20 25

Revival
Revival after 15 days and 1 month of bulk cryopreservation-

For each experiment, the culture tube having about 20 ml of concentrated culture was thawed at room temperature for about 1.5 hours. After thawing, the culture was inoculated in about 250 ml F/2 medium having final OD of about 0.8 at 750nm, at the time of revival in culture bottles. The culture was kept in dark for about 10 to 12 hours. After about 10 to 12 hours of dark incubation, the culture was kept at low light in culture bottles on a growth rack.
All the experimental replicates for experiments 1, 2, 3 and 4 were revived after 15 days and 1 month of cryopreservation (figure 3). Experimental replicates for experiments 5 and 6 did not show any revival after 15 days and 1 month (figure 3 depicts revival at one month). Experiments 1, 3 and 4 showed low levels of revival when compared with Experiment 2.
Experiment 2 having culture OD 20 at 750nm at the time of cryopreservation showed a promising revival after about 3 to 4 days of incubation and was further studied and inoculated in environmental photobioreactor (ePBR). After revival for about 3 to 4 days, the culture from the growth rack was transferred to ePBR under winter simulation conditions to study turbidostat mode of operation of culture (figure 4). Culture conditions in ePBR were: temperature -14 °C to 25 °C, light-dark cycle of 12 hours, light intensity 1100 µE in sinusoidal mode, CO2-2%. Picochlorum sp culture of experiment 2 showed OD jump of about 0.25 at 750nm under winter simulation conditions in the ePBR in turbidostat mode of operation revived after 15 days and 1 month (figures 5 and 6 respectively).
Revival after 6 months of bulk cryopreservation-

For each experiment, a culture tube having about 20 ml of concentrated culture was thawed at room temperature for 1.5 hours. After thawing, the culture was inoculated in 500ml F/2 medium to achieve final OD of about 0.8. At the time of revival in culture bottles, the culture was kept in dark for 24 hours.
After about 10 to 12 hours of dark incubation, the culture was transferred to low light on growth rack. The experimental replicates for experiments 1, 2, 3, 4 and 6 were revived after 6 months of cryopreservation (figure 7). In experiment 6 however, the revived culture was found to be not healthy. Experimental replicates for experiment 5 did not show any revival after 6 months which was in consistent with the earlier results (15 days, and 1-month revival studies).
Revival after 9 months of bulk cryopreservation-

For each experiment, a culture tube having about 20 ml of concentrated culture was thawed at room temperature. After thawing, the culture was inoculated in commercial media having final OD of about 0.8 at the time of revival in culture bottles. The culture was incubated in dark for about 10 to 12 hours. After dark incubation, culture was transferred to low light on growth rack.
The experimental replicates for experiments 1, 2, 3, 4 were revived after 9 months (figure 8). Experimental replicates for experiments 5 and 6 did not show any revival after 9 months which is consistent with the earlier results (15 days, 1 and 6-months revival studies). The revival results were consistent for 0.5, 1, 6 and 9 months. Hence the experiment was terminated after checking the revival for 9 months.

Example 2: Bulk Cryopreservation of the algal culture (production strain) and revival of the composition obtained after bulk cryopreservation
Bulk cryopreservation-

Bulk cryopreservation was carried out in two batches- Batch I where the bulk cryopreservation was initiated in the monsoon season and Batch II where the bulk cryopreservation was initiated in the winter season. The disk stack centrifuge algal biomass of was obtained from about 3.5-4KL of Picochlorum sp culture for each batch and the final volume was made to a volume of 7- 8 litres.
The solids content of the slurry was also measured and it was found to be 10-12 %. OD at 750nm was found to be 230.
The culture was put in a dark coloured tank and about 8% of cryoprotectant was added on volume basis. This was mixed with the help of plastic rods and continuous stirring was done, there after incubated for about 40 minutes in dark. The resultant slurry was distributed in 5-7 triple laminated aluminium foil bags (Table 2). The bags were labelled with the details of the cryopreservation i.e. date, sample details etc. They were sealed with the help of sealing machine. The bags were gradually cooled by storage at temperatures of about 4°C for 12 hours, followed by about 0°C for about 12 hours, followed by about -20°C for about 12 hours. The cryopreserved bags were finally placed at about -80°C in the freezer.
Table 2
Bag Number Batch I (gm) Batch II (gm)
1 1400.86 1270
2 1415.36 1320
3 1389.77 1420
4 1459.55 1300
5 1442.05 1420
6 1622.53 -
7 1369.10 -

Revival:
The cryopreserved bags were removed from the -80°C freezer and kept for thawing at room temperature for about 1.5 hours. Once the bags were completely thawed then they were inoculated in about 200 L F/2 medium in 1m2 ponds. Initial Optical density was set between 0.5 to 0.7 for study for batch mode and at 1 for turbidostat mode of cultivation.
The ponds were covered with shade net cloth for about 12 hours to mimic the dark conditions in the outdoors. After dark incubation, the ponds were exposed to low light condition for about 8 hours to 10 hours. The cultures were then exposed to direct sunlight for 2 hours. The cultures were observed for microscopy and OD.

Revival of batch I bulk cryopreserved composition after one month of bulk cryopreservation
Revival was seen within about 72 hours of inoculation in 1m2 pond (figure 9). Initial OD was about 0.6 at the time of revival. Due to sudden rain the culture was exposed to salinity drop from about 4% to 1.1%. Despite the salinity drop, after the scale-up, culture showed OD jump of 0.3 per day.

Revival of batch I bulk cryopreserved composition after 3 months of bulk cryopreservation
Revival was achieved within 72 hours of inoculation in 1m2 pond (figure 11a). Initial OD was 0.6 at the time of revival. The culture was scaled up in two 5m2 ponds within a week of revival (figure 11b). After the scale up, culture showed OD jump of about 0.3 per day.
Revival of batch I bulk cryopreserved composition after 6 months of bulk cryopreservation and revival of batch II bulk cryopreserved composition after 3 months of bulk cryopreservation
Cultures of Batch I and Batch II were inoculated at the volume of 200 litres in duplicate. During revival in a 1m2 pond, the culture observed minimum culture temperature as about 17.4 °C and maximum temperature as about 30 °C as shown in figure 12. The average light intensity at the site of experiment during experimental tenure was recorded as 790 µE m-2s-1 as per weather station data.
The cultures from batches (I and II) got revived after about 72 hours of inoculation, the cultures were studied for batch mode of operation during revival. After revival, the cultures were studied for turbidostat mode of operation till seven days.
In Batch I culture which was cryopreserved in monsoon season, successful revival after 6 months of cryopreservation was observed (figure 13). The initial OD at the time of revival was set as 0.5. the average OD jump observed was about 0.37 daily up to about 72 hours of revival in batch mode of cultivation (figure 13).
Cell count was measured by using haemocytometer chamber manually to study the cell numbers in culture. It was observed that the cell count also increased by about double as compared with 0 hour of inoculation and after about 72 hours of inoculation of culture in pond at time of revival (figure 13).
Flow cam analysis of the sample (Batch I) was conducted to continuously monitor for identifying contaminants, calculating size distribution and understanding the overall health of the culture in real-time.
Estimation of ash free biomass (AFDW) was also conducted for culture of Batch I. It is observed that at 0 hours the standing biomass was about 200 mg/lit in inoculated pond which increased up to about 360 mg/lit at about 72 hours of revival in batch mode (figure 13).
Batch II culture which was cryopreserved in winter season was successfully revived after 3 months of cryopreservation (figure 14). The initial OD at the time of revival was set as 0.7. The average OD jump observed was about 0.25 daily up to 72 hours of revival in batch mode of cultivation. (figure 14).
Cell count was measured by using haemocytometer chamber manually to study the cell numbers in culture. It was observed that the cell count also doubled as compared to 0 hour of inoculation after about 72 hours of inoculation of culture in pond at time of revival (figure 14).
Flow cam analysis of the sample (Batch II) was conducted for continuous monitoring of identifying contaminants, calculating size distribution and understanding the overall health of the culture in real-time.
Estimation of ash free biomass (AFDW) was also conducted for culture of Batch II. It was observed that at 0 hours the standing biomass was about 200 mg/lit in inoculated pond which increased up to about 337 mg/lit at about 72 hours of revival in batch mode (figure 14).
After revival in batch mode cultivation, the cultures were studied at turbidostat mode of operations for seven days to study under daily dilution mode. It was observed that both the revived cultures showed OD jump of about 0.25 to about 0.30 under daily dilution mode of cultivation (figures 15 and 16).
Microscopy of revived cultures showed dominance of Picochlorum sp as it was showing before cryopreservation and which was confirmed by molecular analysis (figure 17 (c)).
Revival of batch I bulk cryopreserved composition after 9 months of bulk cryopreservation and revival of batch II bulk cryopreserved composition after 6 months of bulk cryopreservation
Both the batches showed successful revival in 1m2 pond (figure 18).
Both the batches were studied in turbidostat mode after revival for a period of about 8 days. 9 month revived culture of batch I showed an average productivity of about 20 g/m2/day and the 6 month revived culture of batch II showed an average productivity of about 16 g/m2/day. Both the batches showed an OD jump of about 0.2 to 0.3 OD per day (figure 19).
Revival of batch I bulk cryopreserved composition after 12 months of bulk cryopreservation and revival of batch II bulk cryopreserved composition after 9 months of bulk cryopreservation

Cultures of Batch I (after 12 months of cryopreservation) and Batch II (after 9 months of cryopreservation) were inoculated at the volume of 200 litres in duplicate.
At the time of outdoor trials of experiment, the culture was put through diverse environmental conditions such as heavy rains. The sudden dilution shock of salinity leading to salinity drop of from about 4% to 1.1% was also experienced by the culture.
The cultures from both batches I and II were revived after about 72 hours of inoculation; the cultures were studied for batch mode of operation during revival.
Batch I culture which was cryopreserved in monsoon season was successfully revived after one year of bulk cryopreservation. The initial OD at the time of revival was set at about 0.9, the average OD jump observed was about 0.25 to 0.3 daily up to about 72 hours of revival in batch mode of cultivation.
Batch II culture which was cryopreserved in winter season was successfully revived after 9 months of bulk cryopreservation. The initial OD at the time of revival was set at about 0.9. The average OD jump observed was about 0.25 daily up to about 72 hours. of revival in batch mode of cultivation.

Revival of bulk cryopreserved composition after 18 months of bulk cryopreservation
The Batch I production strain was successfully revived after 18 months of cryopreservation in a 1m2 pond.
During revival in ponds, culture observed minimum culture temperature as about 15 °C and maximum temperature as about 28 °C as it was winter conditions at the experiment site.
The cultures were revived after 72 hours of inoculation, the cultures were studied for batch mode of operation during revival (figure 20). The average OD jump observed was about 0.25 daily up (figure 21).
Microscopy of revived cultures showed dominance of Picochlorum sp as it was showing before cryopreservation and which was confirmed by molecular analysis (figure 23).

Revival of bulk cryopreserved composition after 24 months of bulk cryopreservation
The Batch I production strain was successfully revived after 24 months of cryopreservation in a 1m2 pond. The cultures were revived after 72 hours of inoculation, the cultures were studied for turbidostat mode of operation after revival. OD jump of about 0.228 is observed during turbidostat mode of operation with average about 23 % harvest on daily basis (figure 22).

Molecular analysis of bulk cryopreserved and revived production strain
Sample slurry used for cryopreservation and cultures revived after 18 months and 24 months of cryopreservation were processed. DNA was extracted of all the samples and PCR was set. Four primers were used for PCR namely 18STM and Picochlorum specific primers.
Presence of production strain Picochlorum was observed in all the samples – i.e. pond sample, slurry before cryopreservation, cultures revived after 18 months and 24 months of cryopreservation) (figure 23).

Example 3: Different volumes of revival
Scale of revival - 1m2 pond (200 liters)

3 experiments were carried out and their revival was checked after one month of cryopreservation.

Experiment 1 - Disk Stack slurry bulk cryopreservation

The Disk Stack centrifuge algal biomass was obtained from about 4KL of Picochlorum sp culture and the final volume was made to a volume of 8 litres. Microscopy of the culture was done. The OD of the slurry was measured and it was found to be about 229.83 at 750nm.
The culture was put in a dark coloured tank and about 10% of cryoprotectant was added on volume basis. This was mixed with the help of plastic rods; continuous stirring was done. This was incubated for about 40 minutes at about 23°C to 30°C. The slurry was distributed in 7 triple laminated aluminium foil bags. The bags were labelled with the details of the cryopreservation i.e. date, sample details etc. They were sealed with the help of sealing machine. The laminated aluminium foil bags comprising the culture was subjected to step-wise cooling process wherein the samples were stored at temperatures of about 4°C for about 12 hours, followed by about 0°C for about 12 hours, followed by about -20°C for about 12 hours. The cryopreserved bags were finally placed at about -80°C in the freezer.
Experiment 2: Direct DAF slurry bulk cryopreservation
About 40 ml of 16% slurry was taken and directly 10% of cryoprotectant was added volume by volume basis. It was mixed by pipetting and incubated for 20 minutes. Then 10 ml of the resultant slurry was added in 4 falcon tubes and they were subjected to step-wise cooling. The falcon tubes were labelled with the details of cryopreservation like date, sample no etc. Finally, they were transferred to a temperature of about -80°C freezer.
Experiment 3: Diluted DAF slurry cryopreservation
About 900 ml of 16% DAF slurry was taken and about 100 ml media was added to make the consistency of the slurry suitable for handling for cryopreservation. 100ml of cryoprotectant was added volume by volume basis. This was incubated for about 40 minutes at about 23°C - 30°C. This was distributed in 4 triple laminated aluminium foil bags. The bags were labelled with the details of the cryopreservation i.e. date, sample details etc. They were sealed with the help of sealing machine. The laminated aluminium foil bags comprising the culture were subjected to step-wise cooling process wherein the bags were stored at temperatures of about 4°C for about 12 hours, followed by about 0°C for about 12 hours, followed by about -20°C for about 12 hours. Finally, the bags were placed at about -80°C in the freezer.
Revival:
The bags of experiments 1 and 3 were removed from the -80°C freezer and kept for thawing for about 1.5 hours. Once the bags were completely thawed then they were inoculated in about 200L commercial media in 1m2 ponds (figure 24). The ponds were covered with greenhouse net cloth and kept in the dark for about 12 hours. After 12 hours, the cultures were exposed to low or diffused light for about 8 to 10 hours followed by exposure to direct sunlight for 2 hours.
Experiments 1 and 3 showed revival after 3 days of inoculation in 1m2 pond (figures 24 and 25). Initial OD was about 0.6 at the time of revival. Due to sudden rain the culture was exposed to salinity drop from 4% to 1.1%. The culture was then taken for scale up to higher volumes. After the scale up culture showed OD jump of about 0.3 per day.
On comparison, centrifuge slurry was found to be better than DAF for revival since microscopic observation revealed clumps and contamination in DAF slurry revival. Centrifuge slurry revival was healthy without clump and contamination. Successful revival at 200L was achieved.

Scale of revival - 5m2 pond (1000 liters)- Picochlorum sp winter bulk cryopreserved culture revival

Two experiments were carried out for the cryopreservation of slurry from a 2.5 acre pond culture of Picochlorum sp for bulk cryopreservation.

Experiment 1: Membrane filtered slurry bulk cryopreservation
About 11 L of membrane filtered slurry with about 6% solid content was obtained. Microscopy of the culture was done.
The culture was put in a dark coloured tank and 8% of cryoprotectant was added on volume basis. This was mixed with the help of plastic rods continuous stirring was done. This was incubated at about 23°C to 30°C for about 45 minutes.
The slurry was distributed in 4 triple laminated aluminium foil bags. The bags were labelled with the details of the cryopreservation i.e. date, sample details etc. They were sealed with the help of sealing machine. The bags were gradually cooled by storage at temperatures of about 4°C for about 12 hours, followed by about 0°C for about 12 hours, followed by about -20°C for about 12 hours. The cryopreserved bags were finally placed at about -80°C in the freezer.
Table 3
Bag Number Membrane filtered slurry wet weight in each bag (kg)
1 2.94
2 2.76
3 2.8
4 3.08

Experiment 2: Centrifuge slurry bulk cryopreservation
14L of centrifuge slurry with 12% solid content was obtained. 8% of cryoprotectant was added volume by volume basis. This was incubated at 23°C- 30°C for 45 minutes. This was distributed in 5 triple laminated aluminium foil bags. The bags were labelled with the details of the cryopreservation i.e. date, sample details etc. They were sealed with the help of sealing machine. The bags were gradually cooled by storage at temperatures of about 4°C for about 12 hours, followed by about 0°C for about 12 hours, followed by about -20°C for about 12 hours. The cryopreserved bags were finally placed at about -80°C in the freezer.
Table 4
Bag Number Centrifuge Slurry wet weight in each bag (kg)
1 2.35
2 3.1
3 2.9
4 3.16
5 2.39
Revival:
Picochlorum sp bulk cryopreserved culture during the winter season was tried for revival after about 3 months of bulk cryopreservation. Two types of slurry were cryopreserved - membrane filtered slurry and centrifuge slurry (experiments 1 and 2). Once the bags were completely thawed at room temperature for 1.5 hours, they were inoculated in F/2 medium directly in 5m2 ponds (figure 26).
As the cultures were preserved during the winter season and the revival was tried during summer season wherein there was peak light intensity and high temperature, the cultures took an initial one week for acclimatization.
Also, revival at 5m2 scale directly was tried for the first time. The cultures were studied in turbidostat mode. The cultures showed an OD jump of about 0.2-0.3 OD jump per day (figure 27). The revival was successful in both the cases however comparatively membrane filtered slurry showed better revival.

Scale of revival - 16m2 pond (3200 liters) - Picochlorum sp winter bulk cryopreserved culture
The bags were removed from the -80°C freezer and kept for thawing at room temperature for 1.5 hours. Once the bags were completely thawed Picochlorum sp culture 12 months revival was done by inoculation of the contents of the thawed bags in F/2 media in 16 m2 ponds.
Total volume of culture was about 3200 litre in the 16 m2 pond. To obtain said 3200 litre culture, 10 kg of concentrated cryopreserved slurry in the bags was thawed and inoculated directly in pond.
Initial Optical density was set in between 1-1.5 to study the stability of revived cultures. The ponds were covered with greenhouse net cloth and kept in the dark for about 12 hours. After 12 hours, the cultures were exposed to low or diffused light for about 8 to 10 hours followed by exposure to direct sunlight. The cultures were observed for microscopy and OD. The culture was revived after about 72 hours of inoculation (figure 28).

Example 4: Cryopreservation and revival of different genera
Bulk cryopreservation protocol established on pilot scale included testing of generas like Nannochloropsis, chlorella and Picochlorum for all 3 seasons viz. Winter, summer and Monsoon with establishment of revival process.
Microscopy of each of the cultures was checked and OD at 750 nm was noted to be about 0.8.
The algal biomass was concentrated using Disk Stack centrifuge at maximum solid concentration of about 10% to 15%. In low light conditions the concentrated culture was transferred to a dark tank and 8% of CPA was added on volume basis. The contents of the dark tank were mixed with the help of plastic rods with continuous stirring. The mixed contents of the dark tank were incubated at about 23°C to 30°C for about 45 minutes.
The slurry was distributed in triple laminated aluminium foil bags. The bags were labelled with the details of the cryopreservation i.e. date, sample details etc. The bags were sealed with the help of sealing machine. The bags were subjected to step-wise cooling process at about 4°C for about 12 hours, followed by about 0°C for about 12 hours, about -20°C for about 12 hours and finally at a temperature of about -80°C. The cryopreserved bags were kept at about -80°C in the freezer.
Digital inventory was maintained for cryopreserved samples.
To establish bulk cryopreservation at large scale some optimizations and process troubleshooting were conducted at lab scale and pilot scale. Optimization was conducted in lab for concentration and incubation time required for cryoprotectant for this process. As bulk cryopreservation was conducted on larger scale, after various research and trying different materials at about -80°C low temperature complete replacement of cryovials with triple laminated aluminium foil bags were used for storing of algae slurry on large scale volume.

Revival:
The culture bags were removed from the freezer and kept for thawing at about 23°C - 30°C for about 1-1.5 hours. After thawing, the bags were directly opened in 1m2 pond thereby deleting the entire lab revival process. The revival was carried out in the evening to mimic the dark incubation in the laboratory at the time of revival.
The pond was covered with shade net cloth for about 10 to 12 hours to mimic the dark conditions in the outdoors. After dark incubation, the pond was exposed to low light condition for about 8 to 10 hours. After this cover was removed and pond was exposed to direct sunlight. Regular revival was checked up to one year.
During revival at pilot scale as the cultures were directly revived in 2.6 m2 pond the revival faced various biotic and abiotic stress such as during monsoon revival sudden drop in salinity is observed, during winter revival cultures were exposed to sudden temperature drop and during summer revival culture has to face high light and temperature. Despite all the stresses which culture has faced at outdoor all the strains from said generas are revived successfully after one year of cryopreservation.

Revival results:
Strains which were bulk cryopreserved in winter were revived in winter. All the strains revived successfully at outdoors after one year of cryopreservation (Figures 30 and 31). During winter studies revival of Nannochloropsis clone I, Picochlorum clone I and Picochlorum clone II showed average about 0.171, 0.199 and 0.251 OD jump/day with average about 17%, 21% and 19% harvest, respectively after one year of cryopreservation (Figure 29).
Microscopic analysis was done for samples after revival. The revival of the strains bulk cryopreserved in the winter season is also checked by using in house molecular techniques.

Molecular analysis:
Molecular analysis of revived samples was conducted using in-house molecular analysis technique. In-house primers were designed and samples analysed by the same primers. gDNA was extracted using standard protocol. The concentration of gDNA extracted was between 30-50 ng/ul. PCR was performed for all the samples using our different primers.
All the 3 strains viz.Nannochloropsis clone I, Picochlorum clone I & II were cryopreserved for all the 3 seasons. All the strains were revived after one year of cryopreservation
Molecular analysis after cryopreservation and revival using 18S specific primer, Nannochloropsis specific primer and Picochlorum specific primers showed the presence of same genus of production strain, wherein molecular analysis using the 18S specific primer yielded bands confirmed presence of genes specific to 18S specific primer in Nannochloropsis clone I, Picochlorum clone I and Picochlorum clone II (figure 32(b)), molecular analysis using the Nannochloropsis specific primer depicted the presence of Nannochloropsis clone I in the revived culture (figure 32(c)) and molecular analysis using the Picochlorum specific primer depicted the presence of Picochlorum sp (clone I and clone II) in the revived culture (figure 32(d)).