DESC:FIELD OF THE INVENTION
The present invention relates to a process of growing algae of different class in a co-culture.

BACKGROUND OF THE INVENTION
Microalgae find application in the area of food, feed, cosmetics, pharmacy, agri-nutrients and in biofuel making. Microalgae are generally cultivated in open raceway ponds, under greenhouse tanks, and bioreactors and fermenters. The growth of the organism is regulated by pH meter, temperature, BOD and COD meters and agitators. Thereafter, the microalgae are harvested.

The microalgae are typically grown as single strain entities. If they grow or with a purpose are grown or cultivated together as groups of two or more microalgae along with the yeasts and bacteria, they are considered to be of not much commercial use and are generally discarded as contaminants. Mainly because combined biomass of two or more algae growing or grown together in open or covered raceway ponds along with the yeasts and bacteria normally adds-up. And thus it provides a consortium of biomass rather than a single strain biomass, and which is as per FDA norms and regulations not found suitable and advisable for usage especially in the food, cosmetics and pharmacy usages for mankind mainly due to their non-consistent but mainly due to ever variable biochemical composition as one part and the combined algae cum yeast and bacillus bacteria biomass consortium is not legally allowed in food and cosmetics beyond a permissible limits as it may create undesirable effects and impacts on the health and skin of mankind due to presence of multiple array of many a bio-nutrients and associated chemical entities.

Technologies existing in area of algae based food, cosmetics; pharmacy and feed sector presently encompass processes which are employed to grow two algae of different families and classes with useful fermentative yeast and probiotic bacillus bacteria in independent individual single growth open unit or closed bioreactors.

WO2013088407 discloses a process for producing an algal biomass comprising inoculating in an aqueous cultivation medium at least a first algal species and at least a first protozoan species obtaining a symbiotic co-culture, the at least first protozoan species being suitable to generate symbiosis with the at least first algal species, cultivating the symbiotic coculture obtaining an algal biomass and harvesting at least a portion of the algal biomass.

CN108587916A discloses process of co-cultivation single needle algae rapid flocculation in neutral conditions, where to cultivate two plants of different single needle algae respectively first, it is to be grown to exponential phase later stage collection frustule, it is cocultured,
for adjustment algae solution pH value to 7.0.

CN103215212B discloses Spirulina platensis mixed culture process. During the growth process of Spirulina platensis, the illumination intensity is controlled in stages, and glucose is fed as a heterotrophic carbon source, so that the technical problems that the Spirulina platensis is limited by light intensity, concentration of an organic carbon source and the like during the culture process are solved, an optimal feeding way with most appropriate light intensity and carbon source in the mixed culture process is found, and high efficient carbon fixation and fast growth of the Spirulina platensis are realized.

Angelis et al., studied the co-culture of microalgae, cyanobacteria, and macromycetes for exopolysaccharides production. In the study, the biomass and exopolysaccharides (EPS) production in co-cultures of microalgae/cyanobacteria and macromycetes as a technology for
producing new polysaccharides for medical and/or industrial application. The study presents an innovative technological process of EPS production through cyanobacteria, microalgae, and Basidiomycetes co-culture in submerged fermentation. It is also concluded that when cultivated in co-culture, EPS production was favoured, and time of fermentation was significantly reduced.

Iasimone et al., studied combined yeast and microalgal cultivation to enhance lipid and biomass production from urban wastewaters. The microalgal polyculture was obtained from a native wastewater biomass. Microalgae and yeast were both added to the cultivation water pond at time zero and their growth was monitored for 14 days. The findings suggest that lipid accumulation, for the tested experimental conditions, could be enhanced by adding the yeast inoculum during the microalgal exponential growth and increasing the easily assimilated organic substrates in urban wastewater or using dairy wastewater.

The pH adjustments of these individually grown bacterial units are mostly done with manual or automated CO2 gas or acids. Addition of CO2 is mostly possible in the autotrophic growing phase of the algae and not so advisable and suitable in the mixotrophic growth phases of an algae and the addition of acids may lead to a rapid change in the pH of the algal culture plus the add-on of not so required excess of Chloride or Sulphate or Nitrate ions etc imparted by acids can alter the growth culture and its ionic conditions to jeopardise the algae biomass growth mechanism and its overall working and biomass delivery economics.

The Algal growth processes presently available, grow mostly a single species of algae. The process designed to grow single algae does not offer microbial biomass yields using fermentative yeast and useful bacillus microbes. The cost incurred using the present growth process is more as it can sustain and grow only one type of algae culture at a time, and thus only one type algal biomass is generated in given unit area and in given unit of time and the cost of employment of manpower and input of utilities like the electric power turns out to be much higher thus making the manufacturing cost of the single algae biomass go up considerably.

The process presently available to regulate pH in the algal growth units utilizes carbon dioxide, or acid or the alkali base.

SUMMARY OF THE INVENTION
The present invention provides a process for co-culturing algae of different class. The process comprises the steps of inoculating two algae in a medium at a pH growing first algae and maintaining second algae in said medium at the first pH, regulating the pH of the medium by adding an organic or inorganic carbon source and mixotrophically growing the second algae and maintaining the first algae in said medium at the second pH and harvesting the first and second algae together or individually where the pH of the medium is regulated such that both the algae sustain and grow simultaneously in the said medium.
The two algae co-cultured can be selected from the group comprising Cyanophyceae and Chlorophyceae. Algae from Cyanophyceae can be Spirulina species. Spirulina can be selected from Spirulina platensis. Algae from Chlorophyceae can be selected from Chlorella species. Chlorella can be selected from Chlorella vulgaris.
The first algae cocultured according to the process of the present invention can be Spirulina species or Chlorella species. The second alga cocultured according to the present invention can be Chlorella species or Spirulina species. The Spirulina can be grown in pH 8.2 to 9.2. The Chlorella can be grown in pH 6.8 to 7.5.
The present invention provides a process of coculturing spirulina and chlorella. The process comprises inoculating Spirulina and Chlorella in a medium at a pH 8.2 to 9.2, growing Spirulina algae and maintaining Chlorella in said medium at pH 8.2 to 9.2 . The pH of the medium can be regulated to pH 6.8 to 7.5 by adding an organic or inorganic carbon source and mixotrophically growing the Chlorella and maintaining the Spirulina in said medium at pH 6.8-7.5. The pH of the medium can be regulated between an alkaline pH and a neutral pH such that Spirulina and Chlorella sustain and grow simultaneously in the medium.
The present invention also provides a process of coculturing Chlorella and Spirulina. The process comprises inoculating spirulina and chlorella in a medium at a pH 6.8 to 7.5, growing Chlorella algae and maintaining Spirulina in said medium at pH 6.8 to 7.5; regulating the pH of the medium to pH 8.2 to 9.2 by adding an organic or inorganic carbon source and mixotrophically growing the Spirulina and maintaining the Chlorella in said medium at pH 8.2 to 9.2; and wherein the pH of the medium is regulated between a neutral pH and an alkaline pH such that Spirulina and Chlorella sustain and grow simultaneously in the medium.
The process of the present invention further comprises co-culturing a microorganism with said two algae wherein the microorganism is inoculated in the said medium containing two algae at a regulated pH for growing and sustaining said microorganism along with the algae. The microorganism can be selected from yeast and bacteria. The process of co-culturing algae of different class and yeast is provided by inoculating yeast in the medium containing Spirulina and Chlorella at a pH of 6.8 – 7.5. The process of co-culturing algae of different class and bacteria is provided by inoculating in the medium containing Spirulina and Chlorella at a pH of 6.8 – 7.5. The included microbe generate and provide inbuilt lactic acid and acetic acid that helps to lower pH. The yeast can be Saccharomyces cerevisiae. The bacteria can be Lactobacillus plantarum, Bacillus subtilis, Lactobacillus lactis.
The process of the present invention comprises harvesting the two algae and microorganisms. The algae and the microorganisms can be harvested together or individually.
The organic carbon source can be glycerol. The inorganic carbon source can be selected from sodium bicarbonate or urea.
DESCRIPTION OF THE INVENTION
The present invention provides a process of growing algae that allows growth of two different classes of algae in a co-culture. The growth of algae can be optionally along with useful microbes. The growth of the algae can be regulated by pH using inorganic and organic carbon in a single unit. The pH of the growth medium can be regulated with precise and timely inputs of the inorganic bicarbonate and organic carbon glycerol.

In an embodiment, the present invention is directed to a process of coculturing algae of different class which has been designed according to nutrient culture pH change. The pH of the medium is allowed to fluctuate from alkaline to neutral pH or from neutral pH to alkaline pH depending upon the algae chosen to grow first.
The process comprises the step of inoculating two algae of different class in a medium at a first pH growing first algae and maintaining second algae in said medium in said first pH. The first pH of the medium can be chosen depending upon the pH at which the chosen first algae grows, and the second algae could sustain and not die such that the second algae is maintained in the medium.
The first pH is then regulated to a second desired pH by adding and organic or inorganic carbon source and mixotrophically growing the second algae in the medium and maintaining the grown first algae in said medium in said second pH. After the desired growth of the second algae in the said medium, the algae can be harvested together and individually. The pH of the medium is regulated such that both the algae sustain and grow simultaneously in said medium.
The pH of the medium is preferably regulated using organic or inorganic carbon source. The organic carbon source can be selected from glycerol. The glycerol can be derived from varied vegetable oils or generated as waste from the biofuel making processes. The inorganic carbon source can be selected from sodium bicarbonate or urea.
In an embodiment, pH of the medium can be lowered by adding glycerol. In another embodiment, the pH of the medium can be raised by avoiding the addition of organic carbon source and/or by adding inorganic carbon source.
In a preferred embodiment, the algae can be cocultured in a medium comprising sodium bicarbonate, sodium/potassium nitrate, NPK, glycerol, Urea, Potassium hydrogen phosphate (K2HPO4), Potassium dihydrogen phosphate (KH2PO4), Magnesium sulphate (MgSO4), Calcium chloride (CaCl2), Ferrous sulphate (FeSO4). The remaining volume can be made up with water. The culture volume can preferably be 5000 litres. The pH of the algal growth medium can be regulated to fluctuate between neutral to alkaline or alkaline to neutral by adding or avoiding Glycerol, urea, Potassium hydrogen phosphate (K2HPO4), and/or sodium bicarbonate to obtain the growth of the desirable algae or microorganism or both.

The present invention provides a process for co-culturing algae of different class selected from Cyanophyceae and Chlorophyceae. In an embodiment, the present invention provides growing Cyanophyceae algae first. The process includes inoculating a fresh live culture of algae of the class Cyanophyceae and Chlorophyceae in a medium at alkaline pH of about 8.0 to 9.2. Regulating the pH of the medium to pH 6.8 to 7.5 by adding an organic or inorganic carbon source and mixotrophically growing the Chlorophyceae and maintaining the Cyanophyceae in said medium at pH 8.2 to 9.2; and wherein the pH of the medium is regulated between an alkaline pH and a neutral pH such that Cyanophyceae and Chlorophyceae sustain and grow simultaneously in the medium.

In another embodiment, the present invention provides a process for co-culturing Cyanophyceae and Chlorophyceae and growing Chlorophyceae algae first. The process comprises inoculating Cyanophyceae and Chlorophyceae algae in a medium at a pH 6.8 to 7.5, growing Chlorophyceae algae and maintaining Cyanophyceae algae in said medium at pH 6.8 to 7.5; regulating the pH of the medium to pH 8.2 to 9.2 by adding an organic or inorganic carbon source and mixotrophically growing the Cyanophyceae algae and maintaining the Chlorophyceae algae in said medium at pH 8.2 to 9.2; and wherein the pH of the medium is regulated between a neutral pH and an alkaline pH such that Cyanophyceae and Chlorophyceae sustain and grow simultaneously in the medium.

In an embodiment, Cyanophyceae algae can be selected from Spirulina species. Spirulina species can be selected from Spirulina platensis. The Chlorophyceae algae can be selected from Chlorella species. Chlorella species can be selected from Chlorella vulgaris.

In an embodiment, the present invention provides a process for co-culturing Spirulina and Chlorella. The Spirulina is selected from Spirulina platensis and Chlorella is selected from Chlorella vulgaris. In an aspect, Spirulina or Chlorella can be grown as the first algae. In another aspect, Spirulina or Chlorella can be grown as a second algae.

The Spirulina algae can be grown in a medium comprising sodium bicarbonate, sodium/potassium nitrate, NPK, KH2PO4, MgSO4, CaCl2.

The Chlorella can be grown in a medium comprising glycerol, urea, K2HPO4, MgSO4, FeSO4.

The present invention provides a process for co-culturing Spirulina and Chlorella where Spirulina is grown as a first algae and Chlorella is grown as a second algae in one growth system. The process comprises inoculating the first algae Spirulina and the second algae Chlorella in the medium with the rise in first pH to about 8.2-9.2. The pH is raised by adding sodium bicarbonate. Spirulina is grown to the desired dry biomass gain and optical density. Chlorella vulgaris does not grow in the first pH of 8.2-9.2. However, Chlorella vulgaris sustains in the pH of 8.2-9.2 of the medium. The pH of the medium is then lowered to the second pH of 6.8 to 7.5 to switch to mixotrophic growth of the second algae by adding organic or inorganic carbon source. In an aspect, the first pH is lowered to the second pH by adding organic carbon source selected from the group comprising glycerol, can be derived from varied vegetable oils or generated as waste from the biofuel making processes. Adding N,P,K, Ca, Mg AND Fe compounds and micronutrients salts intermittently enhance the growth of the algae. The Spirulina alga grows in bigger in size of about 50 microns or more and can be harvested by a nylon cloth screen with a suitable mesh size. The alga Chlorella was easily harvested as mostly all of it got auto flocculated and collected at the bottom of the growth unit. The harvested biomass can be collected and dried. The yield of Spirulina was about 0.15 – 0.18 g/l and Chlorella was about 0.2 – 0.25 g/l.

In another embodiment, the present invention provides a process for co-culturing Spirulina and Chlorella where Chlorella grown as a first algae and Spirulina grown as a second algae in one growth system. The process comprises inoculating the first algae Chlorella and the second algae Spirulina in the medium with the neutral first pH to about 6.8 to 7.5. Addition of organic or inorganic carbon source such as glycerol or urea could help to achieve the neutral first pH of 6.8 to 7.5. Chlorella is grown to the desired dry biomass gain. Spirulina does not grow in the neutral pH. However, Spirulina sustains in the neutral pH of the medium. The pH of the medium is then raised to the alkaline second pH of 8.2-9.2 to switch to mixotrophic growth of the second algae. The pH can be regulated from a neutral pH to alkaline pH by adding bicarbonate.

Adding N,P,K, Ca, Mg and Fe compounds and micronutrients salts intermittently enhance the growth of both the algae as co-culture. The Spirulina alga grows in bigger in size of about 50 microns or more and can be harvested by a nylon cloth screen with a suitable mesh size. The alga Chlorella was easily harvested as mostly all of it got auto flocculated and collected at the bottom of the growth unit. The harvested biomass can be collected and dried. The yield of Spirulina was about 0.15 – 0.18 g/l and Chlorella was about 0.2 – 0.25 g/l.

In an aspect of the present invention, optionally if needed yeast and other non-pathogenic bacteria can be grown along with said algae. The process comprises co-culturing a microorganism with said two algae in the said medium wherein the microorganism is inoculated in the said medium containing two algae at a regulated pH for growing and sustaining said microorganism. The microorganism can be selected from yeast and bacteria. The algae can be optionally grown along with the useful yeast and/or non-pathogenic probiotic bacillus bacteria. The yeast can be Saccharomyces cerevisiae. The bacteria can be Lactobacillus plantarum, Bacillus subtilis, Lactobacillus lactis. The yeast and/or bacteria can be inoculated in the same algal growth medium at a pH of 6.8 to 7.5. The included microbe generate and provide inbuilt lactic acid and acetic acid that helps to lower pH. The process of the present invention comprises harvesting the two algae and microorganisms.

The harvesting of grown microbial biomass along with fermentative yeast and bacteria results in growth as a consortium biomass. This microbial biomass is procured to make a feed formulation which finds use as dairy animal feed under a feed based brand.

In an embodiment, two different classes and family of algae such as Cyanophyceae and Chlorophyceae with useful yeasts and probiotic bacteria grown according to the process of the present invention finds application in poultry and aquaculture industry as natural feed bio-ingredient/s.

Thus, the present invention provides a process to suitably and ably grow a co-culture of atleast two algae; the Spirulina and Chlorella with useful yeast and bacillus bacterial forms as add-ons for their use in the fast growing feed sector in a cost effective manner in given single growth unit under same time period for effective and efficient delivery of the given algal
biomass.

The process is easy to operate and accommodates growth of two algae and useful microbes simultaneously in one algae growth system. This makes the process economical and saves on cost on money invested in making a growth system, the manpower employment and in usage of electric power. The main reason for expenditure in such processes would be land and making of biomass culture unit, manpower and electrical utility employment as it needs to manage the growth of two algae of different classes namely the Cyanophyceae and Chlorophyceae, with microbes the yeast and the ever versatile and useful bacillus bacterial forms.

The harvested algae and the useful microbial biomass have direct application in dairy feeds and aqua feed lines. Thus, the present invention encompasses the use of the harvested algae with microbial biomass in diary feed and aqua feed lines.

The present invention also provides a system for growing algae of different class or strain by regulating pH of the growth medium and by altering the carbon source as described herein. The system would include a cemented or HDPE or a PVC or the polycarbonate grade pond liner housed in a round, or a raceway or rectangular shape algae growth unit fitted with a manual or mechanically driven paddle wheel or agitators to move algal cultures so as to allow enough sunlight availability to all algal cells to grow better and to remove Oxygen generated by the growing and active co-culture algal cells and the useful microbes.

Chlorella vulgaris ,Yeast and the Bacillus forms have been obtained from Talegaon Dhabade, Pune, Maharashtra, Lactobacillus forms have been obtained from Pune, Maharashtra and Spirulina platensis has been obtained from Sambhar Lake, Rajasthan.

WORKING EXAMPLES:

Example 1: Process of manufacture of co-culture of Spirulina and Chlorella biomass using an algal growth unit.

A fresh litre of algae Spirulina and Chlorella were kept in growth mediums under light and appropriate temperature and allowed to grow as a co-culture.

The growth medium was prepared by using good quality of fresh or brackish water with some salinity and adding low dosage of inorganic carbon source like sodium bicarbonate, low dosage of glycerol, medium dosage of nitrogen source such as NPK, NaNO3 ,urea and phosphorous source from Di or Mono Potassium phosphate or DAP wherein NPK is 15:15:15 followed by low dosages of magnesium and calcium salts with iron releasing compounds and micronutrients like zinc, cobalt, molybdenum, copper and manganese.
The growth media for the co-culture is as under in grams/litre dosage:-
Ingredient g/l
Sodium bicarbonate 1.7 – 2.4
To rise pH towards alkaline range
Sodium/Potassium Nitrate 0.50 – 0.75
Glycerol 0.075 – 0.10
Urea 0.05 – 0.075
K2HPO4 0.060 – 0.075
KH2PO4 0.05 – 0.60
MgSO4 0.1 – 0.18
CaCl2 0.03 – 0.04
FeSO4 0.01 – 0.015

About 0.05 gm/l of Spirulina platensis and Chlorella vulgaris was inoculated in a culture medium A, mixed, and oxygen was released out and the interaction with light was improved to speed up the growth.

Medium A:
Ingredient g/l
Sodium bicarbonate
1.7 – 2.4
To rise pH towards alkaline range
Sodium/Potassium Nitrate 0.50 – 0.75

NPK 15:15:15 0.4 – 0.6

KH2PO4
0.05 – 0.60
MgSO4
0.1 – 0.15
CaCl2
0.03 – 0.04

Both the algae were allowed to grow autotrophically with a rise in pH near 8.8 which resulted in enhanced growth of Spirulina as opposed to Chlorella. Growth of Spirulina was noted by optical density (OD) and dry biomass gains on weight to volume basis. OD was in range of 0. 4 to 0.95 and dry weight gains per litre basis is 0.1 to 0.2 gram per litre of culture medium. NaCl (Sodium Chloride) and K2SO4 (Potassium sulphate) or NPK complex fertigation may be added intermittently to raise the salinity of the given medium and boost overall Spirulina platensis trichome’s growth. The shape of green alga Chlorella cells become oval to round with smaller cell size less than 3 to 4 microns when pH shifts from normalcy towards alkaline, and the blue green alga Spirulina filaments become more longer and spiral and luxuriant in growth and 50 micron or more in size.

Medium B was added to the culture to enhance the growth of Chlorella vulgaris. Glycerol was added adjust pH towards neutral range 6.8 – 7.5.

Medium B:
Ingredient g/l
Glycerol 0.075 – 0.10
Urea 0.05 – 0.075
MgSO4 0.015- 0.030
K2HPO4 0.060 – 0.075
FeSO4 0.01 – 0.015

Addition of glycerol as an organic carbon source lower the culture pH to enhance the growth of Chlorella vulgaris. The pH of the growth medium is then lowered to 6.8 to 7.5 to assist the faster growth of green alga in comparison to blue green alga Spirulina platensis. The algal growth was regulated using inorganic and organic carbon inputs such as N,P,K,Ca,Mg and Fe. The shape of green alga Chlorella cells become more round with bigger cell size more than 3 to 4 microns when pH shifts towards normalcy from alkaline, and the blue green alga Spirulina filaments become less longer and spiral and less luxuriant in growth and 50 micron or near to it in size.

The co-culture grew for a week’s time. The Spirulina alga which is bigger in size (about 50 microns or more) was harvested by a nylon cloth screen with a suitable mesh size. The alga Chlorella alga was easily harvested as mostly all of it got autoflocullated and collected at the bottom of the growth unit. The harvested biomass was collected and dried. The yield of Spirulina platensis was about 0.15 – 0.18 g/l and Chlorella vulgaris was about 0.2 – 0.25 g/l.

Example 2: Process of manufacture of co-culture of Spirulina and Chlorella biomass using an algal growth unit
A fresh litre of algae Spirulina and Chlorella were kept in growth mediums under light and appropriate temperature and allowed to grow as a co-culture. In this example, Chlorella was the first algae and Spirulina was the second algae. The pH conditions for co-culture were maintained accordingly.

The growth media for the co-culture is as under in grams/litre dosage:-

About 0.05 gm/l of Spirulina platensis and Chlorella vulgaris was inoculated in a culture medium B.mixed, and oxygen was released out and the interaction with light was improved to speed up the growth.

Medium B:
Ingredient g/l
Glycerol 0.075 – 0.10
Urea 0.05 – 0.075
K2HPO4 0.060 – 0.075
MgSO4 0.015- 0.030
FeSO4 0.01 – 0.015

Both the algae were allowed to grow autotrophically with a pH near 6.8-7.5 which resulted in enhanced growth of Chlorella as opposed to Spirulina. The algal growth was regulated by altering the pH using inorganic and organic carbon inputs such as N,P,K,Ca,Mg and Fe.

Medium A:
Ingredient g/l
Sodium bicarbonate
1.7 – 2.4
To rise pH towards alkaline range
Sodium/Potassium Nitrate 0.50 – 0.75

NPK 15:15:15 0.4 – 0.6

KH2PO4
0.05 – 0.60
MgSO4
0.1 – 0.15
CaCl2
0.03 – 0.04

Addition of inorganic carbon source raised the culture pH near 8.8 to enhance the growth of Spirulina platensis. The rise in pH of the growth medium was near 8.8 which resulted in enhanced growth of Spirulina as opposed to Chlorella. Growth of Spirulina was noted by optical density (OD) and dry biomass gains on weight to volume basis. OD was in range of 0. 4 to 0.95 and dry weight gains per litre basis is 0.1 to 0.2 gram per litre of culture medium. NaCl (Sodium Chloride) and K2SO4 (Potassium sulphate) or NPK complex fertigation may be added intermittently to raise the salinity of the given medium and boost overall Spirulina platensis trichome’s growth.

The co-culture grew for a week’s time. The Spirulina alga which is bigger in size (about 50 microns or more) was harvested by a nylon cloth screen with a suitable mesh size. The alga Chlorella alga was easily harvested as mostly all of it got autoflocullated and collected at the bottom of the growth unit. The harvested biomass was collected and dried. The yield of Spirulina platensis was about 0.15 – 0.18 g/l and Chlorella vulgaris was about 0.2 – 0.25 g/l.

The biochemical content of Spirulina and Chlorella obtained by the co-culture process of the present invention is given below in Table 1:
Table 1
Sr.No. Biochemical Content Spirulina platensis Chlorella vulgaris
1 Moisture (%) 6 – 8 5 – 7
2 Ash (%) 7 – 9 6 – 8
3 Proteins (%) 62 – 65 54 – 56
4 Carbohydrates(%) 16 – 18 18 – 20
5 Lipids(%) 5 – 6 12 – 15
6 Chlorophyll (%) 1 – 1.2 1.8 – 2.5
7 Phycocyanin (%) 10 – 12 None
8 Carotenoids (%) 0. 25 – 0.40 0.20 – 0.28
9 Colour Blue Green Dark Green

Example 3: Process of manufacture of co-culture of Spirulina and Chlorella biomass with yeast using an algal growth unit

A fresh litre of algae Spirulina and Chlorella was kept in growth mediums under suitable light and appropriate temperature and allowed to grow as a co-culture with yeast. The yeast can be Saccharomyces cerevisiae.

The growth medium was prepared by using good quality of fresh or brackish water with some salinity based on NaCl and adding low dosage of inorganic carbon source like sodium bicarbonate, low dosage of glycerol, medium dosage of nitrogen source such as NPK, NaNO3, urea and phosphorous source from Di or Mono Potassium phosphate or DAP wherein NPK is 15:15:15 followed by low dosages of magnesium and calcium salts with iron releasing compounds and micronutrients like zinc, cobalt, molybdenum, copper and manganese.
The growth media for the co-culture was as under in grams/litre dosage:-
Ingredient g/l
Sodium bicarbonate 1.7 – 2.4
To rise pH towards alkaline range
Sodium/Potassium Nitrate 0.50 – 0.75
NPK 15:15:15 0.4 – 0.6
Glycerol 0.075 – 0.10
Urea 0.05 – 0.075
K2HPO4 0.060 – 0.075
KH2PO4 0.05 – 0.60
MgSO4 0.1 – 0.18
CaCl2 0.03 – 0.04
FeSO4 0.01 – 0.015
The pH of the medium was maintained closer to 8.2 and inoculated with fresh live culture of Spirulina platensis and Chlorella vulgaris species.

About 0.5 gm/l of inoculum of each algal culture is taken. The cultures of the different class of algae were mixed in Medium A, oxygen is released out and the interaction with light is improved to speed up the growth.

Medium A:
Ingredient g/l
Sodium bicarbonate
1.7 – 2.4
To rise pH towards alkaline range
Sodium/Potassium Nitrate 0.50 – 0.75

NPK 15:15:15 0.4 – 0.6

KH2PO4
0.05 – 0.60
MgSO4
0.1 – 0.15
CaCl2
0.03 – 0.04

Both the algae were allowed to grow autotrophically with a rise in pH near 8.8 which resulted in enhanced growth of Spirulina as opposed to Chlorella. Growth of Spirulina was noted by optical density (OD) and dry biomass gains on weight to volume basis on per litre basis. Medium B was added to the culture to enhance the growth of Chlorella. Glycerol was added adjust pH towards neutral range 6.8 – 7.5. About 0.05 gm/l of inoculum of yeast is added to the medium.

Medium B:
Ingredient g/l
Glycerol 0.075 – 0.10
Urea 0.05 – 0.075
K2HPO4 0.060 – 0.075
MgSO4 0.015- 0.030
FeSO4 0.01 – 0.015

The pH of the growth medium was then lowered to 6.8 to 7.5 to assist the faster growth of green alga Chlorella in comparison to blue green alga Spirulina. The algal growth was regulated using inorganic and organic carbon inputs such as N,P,K,Ca,Mg and Fe. The micronutrients speed up the growth of both the algae along with yeast.

Example 4: Spirulina and Chlorella Co-culture with yeast and bacteria
A fresh litre of algae Spirulina and Chlorella was kept in growth mediums under light and appropriate temperature and allowed to grow as a co-culture along with yeast and bacteria. About 0.5 gm/l of inoculum of each algal culture is taken. The yeast can be Saccharomyces cerevisiae. The bacteria can be Bacillus subtilis and Lactobacillus plantarum.

The growth medium is prepared by using good quality of fresh or brackish water with some salinity and adding low dosage of inorganic carbon source like sodium bicarbonate, low dosage of glycerol, medium dosage of nitrogen source such as NPK, NaNO3, urea and phosphorous source from Di or Mono Potassium phosphate or DAP wherein NPK is 15:15:15 followed by low dosages of magnesium and calcium salts with iron releasing compounds and micronutrients like zinc, cobalt, molybdenum, copper and manganese.

The pH of the medium is maintained closer to 8.2 and inoculated with fresh live culture of both algae; Spirulina platensis and Chlorella vulgaris of the class Cyanophyceae and Chlorophyceae.

The growth media for the co-culture was as under in grams/litre dosage:-
Ingredient g/l
Sodium bicarbonate 1.7 – 2.4
To rise pH towards alkaline range
Sodium/Potassium Nitrate 0.50 – 0.75
NPK 15:15:15 0.4 – 0.6
Glycerol 0.075 – 0.10
Urea 0.05 – 0.075
K2HPO4 0.060 – 0.075
KH2PO4 0.05 – 0.60
MgSO4 0.1 – 0.18
CaCl2 0.03 – 0.04
FeSO4 0.01 – 0.015
The cultures of the different class of algae are mixed, oxygen is released out and the interaction with light is improved to speed up the growth.

Medium A:
Ingredient g/l
Sodium bicarbonate
1.7 – 2.4
To rise pH towards alkaline range
Sodium/Potassium Nitrate 0.50 – 0.75

NPK 15:15:15 0.4 – 0.6
KH2PO4 0.05 – 0.60
MgSO4 0.1 – 0.15
CaCl2 0.03 – 0.04

Both the algae were allowed to grow autotrophically with a rise in pH near 8.8 which resulted in enhanced growth of Spirulina as opposed to Chlorella. Growth of Spirulina was noted by optical density (OD) and dry biomass gains on weight to volume basis.

Medium B was added to the culture to enhance the growth of Chlorella. Glycerol was added adjust pH towards neutral range 6.8 – 7.5. About 0.05 gm/l of inoculum of yeast and bacteria were added to the medium.

Medium B:
Ingredient g/l
Glycerol 0.075 – 0.10
Urea 0.05 – 0.075
K2HPO4 0.060 – 0.075
MgSO4 0.015 – 0. 030
FeSO4 0.01 – 0.015

The pH of the growth medium is then lowered to 6.8 to 7.5 to assist the faster growth of green alga in comparison to Spirulina. The algal growth was regulated using inorganic and organic carbon inputs such as N,P,K,Ca,Mg and Fe. The micronutrients speed up the growth of both the algae along with yeast and useful bacillus bacteria.

The co-culture grew for a week’s time and then the Spirulina alga which is bigger in size was harvested by a nylon cloth screen with a suitable mesh size. The alga Chlorella got easily harvested as mostly all of it got autoflocullated and collected at the bottom of the growth unit. Or both the algae along with suitably grown yeast and useful bacillus bacteria can be harvested together with acidified chitin solution at 0.1% solution strength. The harvested biomass was collected and dried. Procured biomass consortium of both algae with microbes as wet bio-form was used as a feed additive in traditional feed or in CATTLACT brand.

Example 5: Spirulina and Chlorella Co-culture with yeast and bacteria
A fresh litre of algae Spirulina and Chlorella were kept in growth mediums under light and appropriate temperature and allowed to grow as a co-culture along with yeast and bacteria. About 0.5 gm/l of inoculum of each algal culture is taken. The yeast can be Saccharomyces cerevisiae. The bacteria can be Bacillus subtilis, Lactobacillus plantarum and Lactobacillus lactis.

The growth medium was prepared by using good quality of fresh or brackish water with some salinity and adding low dosage of inorganic carbon source like sodium bicarbonate,low dosage of glycerol, medium dosage of nitrogen source such as NPK, NaNO3 ,urea and phosphorous source from Di or Mono Potassium phosphate or DAP wherein NPK is 15:15:15 followed by low dosages of magnesium and calcium salts with iron releasing compounds and micronutrients like the zinc, cobalt, molybdenum, copper and manganese.

The pH of the medium was maintained closer to 8.2 and inoculated with fresh live culture of algae Spirulina and Chlorella of the class Cyanophyceae and Chlorophyceae.

The growth media for the co-culture was as under in grams/litre dosage:-
Ingredient g/l
Sodium bicarbonate 1.7 – 2.4
To rise pH towards alkaline range
Sodium/Potassium Nitrate 0.50 – 0.75
NPK 15:15:15 0.4 – 0.6
Glycerol 0.075 – 0.10
Urea 0.05 – 0.075
K2HPO4 0.060 – 0.075
KH2PO4 0.05 – 0.60
MgSO4 0.1 – 0.18
CaCl2 0.03 – 0.04
FeSO4 0.01 – 0.015
The cultures of the different class of algae are mixed, oxygen is released out and the interaction with light is improved to speed up the growth.

Medium A:
Ingredient g/l
Sodium bicarbonate
1.7 – 2.4
To rise pH towards alkaline range
Sodium/Potassium Nitrate 0.50 – 0.75

NPK 15:15:15 0.4 – 0.6
KH2PO4
0.05 – 0.60
MgSO4
0.1 – 0.15
CaCl2 0.03 – 0.04

Both the algae were allowed to grow autotrophically with a rise in pH near 8.8 which resulted in enhanced growth of Spirulina as opposed to Chlorella. Growth of Spirulina was noted by optical density (OD) and dry biomass gains on weight to volume basis.

Medium B was added to the culture to enhance the growth of Chlorella. Glycerol was added adjust pH towards neutral range 6.8 – 7.5. About 0.05 gm/l of inoculum of yeast and bacteria were added to the medium.

Medium B:
Ingredient g/l
Glycerol 0.075 – 0.10
Urea 0.05 – 0.075
K2HPO4 0.060 – 0.075
MgSO4 0.015 – 0. 030
FeSO4 0.01 – 0.015

The pH of the growth medium was then lowered to 6.8 to 7.5 to assist the faster growth of green alga Chlorella in comparison to Spirulina. The algal growth was regulated using inorganic and organic carbon inputs such as N,P,K,Ca,Mg and Fe. The micronutrients speed up the growth of both the algae along with yeast and useful bacillus bacteria.

The co-culture grew for a week’s time and then the Spirulina alga which is bigger in size was harvested by a nylon cloth screen with a suitable mesh size. The alga Chlorella was easily harvested as mostly all of it got autoflocullated and collected at the bottom of the growth unit. Or both the algae along with suitably grown yeast and useful bacillus bacteria can be harvested together with acidified chitin solution at 0.1% solution strength. The harvested biomass was collected and dried. Procured biomass consortium of both algae with microbes as wet bio-form was used as a feed additive in traditional feed.

The yield obtained by the process of the present invention is given below in Table 2:
Table 2
Spirulina
platensis Chlorella vulgaris Saccharomyces
cerevisiae Lactobacillus
plantarum Bacillus
Subtilis Lactobacillus
lactis
Biomass Algae Algae Yeast Bacteria Bacteria Bacteria
Product Colour Blue green Dark green Yellow
Green Light
Yellow Yellow
Red Yellow
Red
Cell Shape Spiral to straight Oval to Round Ellipsoid to round Straight rods round ends Rod
Oval Rods with round ends
Cell Size 50 plus microns 3–5 microns 5 to 6 microns 1 width 6 micron long 0.25 to 1 micron 0.5 width 1.5 micron long
Yield 0.15 – 0.18 g/l 0.2 – 0.25 g/l 10000-20000 CFU/litre 5000-12000
CFU/litre 1500 – 3000 CFU/litre 3000 – 6000
CFU/litre
Time 1 week 1 week 1 week 1 week 1 week 1 week
pH 8.5 – 9.2 6.8 -7.5 6.8 – 8.2 6.8 – 7.0 6.8 6.8

The foregoing description of the invention has been set merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to person skilled in the art, the invention should be construed to include everything within the scope of the disclosure.

,CLAIMS:
1) A process for co-culturing algae of different class comprising the steps of:
a) inoculating two algae in a medium at a first pH, growing first algae and maintaining second algae in said medium at the first pH; and
b) regulating the first pH of the medium to a second pH by adding an organic or inorganic carbon source and mixotrophically growing the second algae and maintaining the first algae in said medium at the second pH; and
c) harvesting the first and second algae together or individually;
wherein the pH of the medium is regulated such that both the algae sustain and grow simultaneously in the said medium.
2) The process as claimed in claim 2, wherein the two algae are selected from the group comprising Cyanophyceae and Chlorophyceae.
3) The process as claimed in claim 2, wherein the Cyanophyceae algae is selected from Spirulina species and Chlorophyceae algae is selected from Chlorella species.
4) The process as claimed in claim 3, wherein Spirulina species is selected from Spirulina platensis and Chlorella species selected from Chlorella vulgaris.
5) The process as claimed in claim 1 to 4, wherein the first algae is Spirulina species or Chlorella species.
6) The process as claimed in claims 1 to 5, wherein the second algae is Chlorella species or Spirulina species.
7) The process as claimed in claims 1 to 6, wherein the process comprises growing Spirulina in pH 8.2. to 9.2.
8) The process as claimed in claims 1 to 6, wherein the process comprises growing Chlorella in pH 6.8 to 7.5.
9) The process as claimed in claims 1 to 8, wherein the process comprises:
(i) inoculating Spirulina and Chlorella in a medium at a pH 8.2 to 9.2, growing Spirulina algae and maintaining Chlorella in said medium at pH 8.2 to 9.2;
(ii) regulating the pH of the medium to pH 6.8 to 7.5 by adding an organic or inorganic carbon source and mixotrophically growing the Chlorella and maintaining the Spirulina in said medium at pH 6.8 to 7.5; and
wherein the pH of the medium is regulated an alkaline pH and a neutral pH such that Spirulina and Chlorella sustain and grow simultaneously in the medium.
10) The process as claimed in claims 1 to 8, wherein the process comprises:
(i) inoculating Spirulina and Chlorella in a medium at a pH 6.8 to 7.5, growing Chlorella algae and maintaining Spirulina in said medium at pH 6.8 to 7.5;
(ii) regulating the pH of the medium to pH 8.2 to 9.2 by adding an organic or inorganic carbon source and mixotrophically growing the Spirulina and maintaining the Chlorella in said medium at pH 8.2 to 9.2; and
wherein the pH of the medium is regulated between a neutral pH and an alkaline pH such that Spirulina and Chlorella sustain and grow simultaneously in the medium.
11) The process as claimed in claim 1 to 10, further comprises co-culturing a microorganism with said two algae wherein the microorganism is inoculated in the said medium containing two algae at a regulated pH for growing and sustaining said microorganism with said algae.
12) The process as claimed in claim 11, wherein the microorganism is selected from yeast and bacteria.
13) The process as claimed in claim 11 to 12, wherein the process comprises inoculating yeast in the medium containing Spirulina and Chlorella at a pH of 6.8 – 7.5.
14) The process as claimed in claims 11 to 13, wherein the process comprises inoculating bacteria in the medium containing Spirulina and Chlorella at a pH of 6.8 – 7.5.
15) The process as claimed in claims 11 to 14, wherein the yeast is selected from Saccharomyces cerevisiae, bacteria is selected from Lactobacillus plantarum, Bacillus subtilis, Lactobacillus lactis.
16) The process as claimed in claims 11 to 15 , wherein the process comprises harvesting the two algae and microorganisms together or individually.
17) The process as claimed in claim 1 to 16 , wherein the organic carbon source is glycerol.
18) The process as claimed in claim 1 to 16 , wherein the inorganic carbon source is selected from sodium bicarbonate or urea.