Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/10—Protozoa; Culture media therefor
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
  • A01G33/00—Cultivation of seaweed or algae
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H17/00—Symbiotic or parasitic combinations including one or more new plants, e.g. mycorrhiza
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/12—Unicellular algae; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
  • C12P7/6436—Fatty acid esters
  • C12P7/6445—Glycerides
  • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management

Definitions

• Algae are important resources for many beneficial bio-products .
• Algae cells contain pigments and other intracellular matters useful for producing antioxidants, vitamins, aquaculture nutrients, bioplastics, dyes and colourants, feedstocks, pharmaceuticals, algae fuels and especially oils for energy and health care purposes.
• the biomass can give biofuel by various processes of pyrolysis (Chisti 2007, Giardi et al . 2010)
• Algal cultivation similar to culturing many other microorganisms, requires both macro and micronutrients that can be obtained from either organic or inorganic sources.
• Typical commercial algal growth methods rely on the use of exogenously added pure chemicals and micronutrients needed to sustain the algal cultures.
• algal cultivation occurs in proper photobioreactors which can be closed or open systems.
• closed photobioreactors for algal cultures are provided in U.S. Pat. Nos. 2,732,663; 4,473,970; 4,233,958; 4,868,123; and 6,827,036.
• open air photobioreactor systems used for cultivation of algae are disclosed, for example, in U.S. Pat. Nos. 3,650,068; 3,468,057; 3,955,318; and 4, 217, 728.
• the biomass produced generally ranges from 0.1 till a maximum of 2 g/1 in autotrophic conditions; higher yields can be obtained in cases of mixotrophic conditions and only under optimal (non commercially sustainable) growth conditions (pure salts, right levels of illumination, small volumes of culture) .
• algal scale up in big volumes is difficult due to several factors: i) difficulty in obtaining a correct mass movement, ii) selection of right levels of light inside the mass.
• algae For photosynthesis to work, light must reach the algae. If a layer of algae is more than a few centimetres thick, organisms on the surface shade those underneath, blocking the sunlight. An alternative is to spread horizontally the algae. However, algae would need to cover an area of about 9 million hectares to produce enough biodiesel to cover Europe's annual transport requirement of 370 billion litres.
• Imposing light inside the biomass by various lamp sources can be also dangerous: it is known that high levels of light, instead of promoting photosynthesis, can cause an inhibition called photoinhibition at the level of the activity of photosystem II apparatus (Barber and Andersson 1992; Mattoo et al . 1999) .
• Mutualistic symbioses are important ecological relationships that are generally defined as two or more species living together and providing benefit to each other.
• microalgal endosymbioses for example involving flatworms and various protists contribute globally to the primary productivity of aquatic ecosystems (Fujishima 2009).
• Such a cultivation process requires a minimal addition of exogenous nutrients but it is nevertheless affected by some drawbacks.
• This process for example, requires a cultivation medium suitable to induce at least one nitrogen stress response in the cultured algal cells to obtain a high lipid production. Nevertheless, as already emphasized stresses applied to the algal culture can reduce the content of overall biomass production.
• the present description concerns a process for producing an algal biomass comprising:
• a preferred embodiment of the production process described herein concerns use of a symbiosis deriving from a protozoan, preferably a ciliate and/or a flagellate protozoan, containing symbiotic algal organisms inside its cytoplasm.
• the process herein described allows to produce algal biomass by minimal addition of exogenous nutrients (like waste organic substances and technical salts) , thus maintaining production at low costs and environmentally friendly.
• a further embodiment of the present description concerns further inoculating in the cultivation medium algae and/or cyanobacteria as preys which, providing nutrients deriving from their metabolic products, contribute to increase the yield of biomass production.
• the production process disclosed in the present application is characterized by a continuous symbiotic co-cultivation system which provides several advantages in terms of high levels of biomass and lipid production.
• Such a production process enables algal growth with an enhanced bioproduct yield (e.g., on a per-algal cell basis) with an easy scale up process.
• the total biomass is increased also due to the high reproduction speed of the protozoan.
• both the alga and the protozoan have the ability to accumulate large amounts of lipids, which can be used as a feedstock for biofuel/bioliquid production without the need of generating any additional stress to the cultivating system.
• Another advantage of the production process herein disclosed is that it is broadly applicable to different algae photosymbionts and can be practised with a broad range of suitable symbiotic protozoa.
• the algal biomass obtained according to the instant description is useful for bioproduction .
• the obtained algal biomass can be employed for production of (but not limited to) bioliquids, biofuel, biodiesel, bioethanol, biogasoline, biocrude, biogas, and also pharmaceuticals, therapeutics, antioxidants, nutraceuticals , cosmetics, cosmeceuticals , food, feedstock, dyes, colorants and bioplastic.
• An embodiment of the present description concerns a process for producing an algal biomass comprising: i) inoculating in an aqueous cultivation medium at least a first algal species and at least a first protozoan species obtaining a symbiont co-culture, the at least first protozoan species being suitable to generate symbiosis with the at least first algal species ;
• the process object of present description provides commercially adequate algal biomass yield with a high lipid content which, unlike stressed algae grown alone in culture without any other symbiont organism, can be sustained on a continuous symbiotic basis in both open and closed systems.
• the instant description provides a process for a sustainable continuous cultivation of algae-bearing protozoa with minimal addition of exogenous nutrients.
• a significant proportion of the macronutrients, necessary for the symbiotic culture derives from the photosymbiont products continuously produced during symbiotic cultivation.
• the source of a significant proportion of carbon, CO 2 , magnesium, potassium, calcium, oxygen and other macronutrients for the living organisms of the symbiotic culture come, in fact, from metabolic cellular products deriving from the co-cultivation itself. More specifically, a portion of CO 2 and oxygen present in the cultivation medium is endogenously derived from the aerobic metabolism of the protozoan component.
• the algae can supply the host Paramecium with photosynthetic products, mainly maltose and sugars. Inside the host cell, the algae show a higher rate of photosynthetic oxygen production than in the isolated condition, thereby guaranteeing an oxygen supply for their host.
• the Paramecium can supply the algae with nitrogen components and CO 2 .
• Algae species suitable for the co-culture described in the present application include but are not limited to marine, brackish water and freshwater algae and include species that are derived from acidic or basic water.
• the algae species include micro and macro algal species like eukaryotic algae such as diatoms, green, red and brown algae and cyanobacteria.
• Different algal species may require for their metabolic activity different cultivation conditions (temperature in the range from 21 to 35 °C, cultivation medium containing different salt concentrations, pH ranging from 6 to 8) .
• the lipid content of the algal biomass obtained by means of the process herein described is significantly higher than that obtained through stressed algae grown alone in culture without any other symbiont organism.
• the increased content of lipids ranges from 57-90% of the dried weight biomass, for the specific case of Chlorella minutissima infected by a Paramecium sp. , compared to the range of 30-50% obtained with the algae alone .
• a particularly preferred embodiment of the present description concerns the cultivation of symbiotic systems comprising a Paramecium spp. (a ciliate protozoan) with Chlorella spp.
• the process for production of an algal biomass provided very good results in terms of algal biomass yield using as the first algal species Chlorella minutissima and two different protozoa species able to generate symbiosis with Chlorella minutissima, namely Tetrahymena pyriformis and Chilomonas Paramecium.
• the algal species and the protozoan species can be present in the cultivation medium in a ratio comprised between 500:0.1 and 50:5, preferably about 100:1.
• the inoculum in the cultivation medium of a second algal species and/or cyanobacteria as source of nutrients is provided.
• Such an addition increases the algal biomass yield in a long term cultivation.
• suitable algal species and/or cyanobacteria are not able to generate symbiosis with the protozoa species.
• the algal biomass yield can be increased by harvesting a portion of algal biomass, optionally followed by adding new cultivation medium or water, wherein said operations may be performed repeatedly during a long term period.
• the harvested portion of the algal biomass may comprise algal and Paramecium biomass, as well as part of the cultivation medium.
• table 1 a list of protozoa species which can infect algal species suitable to create a symbiotic system efficiently usable in the process object of the instant application is provided (first and second columns, respectively) .
• the third column provides a list of algae and cyanobacteria that can be used as exogenous nutrient of the symbiotic system.
• Nannochloropsis spp. Mesodinium rubrum Nannochloropsis occulata Paramecium sp.
• Neochloris oleoabundans Nitzschia laevis Neochloris oleoabundans Nitzschia laevis
• algal biomass means the biomass obtained by the symbiont culture of the algal species and the protozoan species. Therefore, the algal biomass contains both the algal cells and the protozoan cells, as well as part of the aqueous cultivation medium.
• exogenous nutrients means nutrient compounds that are added by the operator during the cultivation phase of the symbiont co-culture.
• Exogenous nutrients useful in the process herein described comprise, i.a. technical salts, glycerol, molasses, hormones (preferably vegetal natural hormones like for example cytochines or indoleaceutic acid) , aminoacids (for example glutammic acid, asparagine, alanine, lysine) , vitamins (for example A, B, C, PP, K vitamins) , microelements (like boron, iron, manganese, molybdenum, zinc, copper, cobalt) , humic substances like humic and fulvic acids, agro-industrial waste materials and plant detritus.
• hormones preferably vegetal natural hormones like for example cytochines or indoleaceutic acid
• aminoacids for example glutammic acid, asparagine, alanine, lysine
• Symbioses in general, are defined as two or more species living together in beneficial coexistence. This type of mutualistic interaction plays an important role in maintaining populations living under precarious environmental conditions.
• algal-bearing protozoa are ubiquitous and abundant components in oceanic and freshwater systems of different trophic interactions.
• the mixotrophic nutrition mode of algal-bearing ciliates is considered to be an adaptation allowing exploitation of oligotrophic environments.
• phototrophic endosymbionts are ingested by the host, but are able to escape digestion and to utilize the waste products of the metabolism of their host.
• Mixotrophic organisms combine the advantages of a heterotrophic nutrition mode with autotrophic energy gain, through algal symbionts.
• a stable symbiosis is the mutualistic relationship between the ciliate Paramecium bursaria (Hymenostomatia) and unicellular green alga Chlorella (Trebouxiophyceae) .
• This symbiosis represents a permanent association with hereditary symbionts, where each algal cell is enclosed in an individual perialgal vacuole derived from the host digestive vacuole to protect from lysosomal fusion.
• the exclusive mutualistic relationship of P. bursaria with ⁇ zoochlorellae' in natural conditions has long been considered as a fact, but aposymbiotic P. bursaria natural populations have been recently reported (Fujishima, 2009; Summerer, 2008).
• Chlorella species like for example, C. vulgaris, P. kessleri , C. variabilis , C. sorokiniana and C. minutissima
• C. vulgaris a species of Chlorella
• P. kessleri a species of Chlorella
• C. variabilis a species of Chlorella
• C. sorokiniana a species of Chlorella
• C. minutissima Chlorella species
• Certain strains of M. rubrum may have a stable association with their cryptophyte organelles, while others need to acquire a cryptophyte nucleus through feeding .
• ciliates form symbiotic relationship preferably with algae of Chlorella genus.
• Paramecium cells may harbour several hundreds of symbiotic Chlorella cells in their cytoplasm.
• Each symbiotic alga is enclosed in a special membrane called Perialgal Vacuole (PV) , derived from the host Digestive Vacuole (DV) membrane of the protozoan.
• PV Perialgal Vacuole
• DV Digestive Vacuole
• the Perialgal Vacuole is able to protect algal cells preventing the fusion to the host lysosoms.
• the alga appears in the cytoplasm by budding of the digestive vacuole membrane of the protozoan.
• the vacuole enclosing a single green alga differentiates into the perialgal vacuole from the digestive vacuole.
• the alga localizes beneath the host cell cortex. At about 24 h after mixing, the alga increases by cell division and establishes endosymbiosis .
• Alga-bearing Paramecium cells can divide better than the alga-free cells. Alga-bearing Paramecium cells show a higher survival rate than the alga-free cells under various stressful conditions, like for example administration to the culture of 0.5 mM nickel chloride (N1CI 2 ) or 150 mM hydrogen peroxide or exposing the culture to high temperatures (40°C) .
• N1CI 2 nickel chloride
• H2 high temperatures
• the host paramecia can receive protection against UV damage by their symbiotic algae which contain protecting substances which confers their capability to thrive in sunlit UV-exposed waters.
• the symbiotic algae can be distributed to the daughter cells.
• Alga-free Paramecium cells can be produced easily from alga-bearing cells using one of the following methods: rapid cell division; cultivation under the constant dark condition, X-ray irradiation, treatment with 3- (3, 4-dichlorophenyl) -1, 1-dimethylurea (DCMU - a blocker of electron flow in photosystem II), treatment with the herbicide paraquat or treatment with cycloheximide (an eukaryotic protein synthesis inhibitor) .
• symbiotic algae can be isolated from alga-bearing Paramecium cells by sonication, homogenization or treatment with some detergents.
• Paramecium species need simple cultivation conditions to get mass culture.
• symbiosis between an algal species and a protozoan species is very effective in terms of production of algal biomass.
• the presence of the protozoan in the co- cultures provides for adequate and sustained algal growth and preserves the capability of high rate lipid production without needing application of any stress conditions to the culture.
• Tetrahymena pyriformis is a teardrop-shaped, unicellular, ciliated freshwater protozoan about 50 ym long. Tetrahymena species are very common in aquatic habitats and are non-pathogenic, have a short generation time of about 2 h and can grow to high cell density in inexpensive media. Tetrahymena pyriformis structure is characteristic since it exhibits striking nuclear dimorphism: two types of cell nuclei, a large somatic macronucleus and a small germline micronucleus, exist in a single cell at the same time and carry out different functions with distinct cytological and biochemical properties. In addition, Tetrahymena possesses hundreds of cilia and microtubules in its cytoskeleton .
• Tetrahymena genus includes several species and the most common are: T. pyriformis, T. thegewischi , T. hyperangulari , T. malaccensis, T. pigmentosa , T. thermophila and T. vorax. Recently, the whole macronuclear genome has been sequenced for Tetrahymena thermophila .
• Chilomonas Paramecium (19-30ym) occurs in 8 species, recently ascribed to Cryptomonad flagellatae. It is a colourless organism which contains a leucoplast. Chilomonas Paramecium shows a peculiar swaying swimming behaviour, caused by a stereospecific asymmetry in cell shape with clearly definable dorso- ventral/right-left sides, and are easy to recognize due to a unique set of characters. It can be grown in relatively simple solutes even only with inorganic salts, it divides at a fairly uniform rate and it lives and thrives in a temperature range extending over twenty degrees.
• Chilomonas Paramecium when grown in a favourable environment, contains a large quantity of stored food material in the form of starch granules and neutral fats.
• Mast and Pace (1933) demonstrated that it produces starch, fats and proteins in a wholly inorganic medium, with one part of CO 2 added to 5 parts of air at atmospheric pressure.
• Starch and fat are by ⁇ products of metabolism which can be utilized as food materials under adverse conditions of nutrition.
• KOB medium contains: 2g/l sodium acetate trihydrate; 2g/l yeast extract; O.lg/1 magnesium chloride anhydrous; O.Olg/1 ferrous sulphate heptahydrate ; 0.02g/l calcium chloride dihydrate.
• - Chalkley' s medium with grass seeds is obtained by dissolving 5ml of a stock solution (containing 20g/l NaCl; 0.8g/l KC1; 1.56g/l CaCl2 2H20) in 1L of distilled water and adding 0.7g/l of grass seeds and boiled for 15-20 min compensating water lost.
• a stock solution containing 20g/l NaCl; 0.8g/l KC1; 1.56g/l CaCl2 2H20
• TAP medium contains the following components that are added in the order: 10 ml/1 2M tris-acetate (pH 7); 10 ml/1 2M phosphate buffer (pH 7); 10 ml/1 Nutrient stock; 1 ml/1 Trace elements solution (10X dilution) .
• the different components are prepared as stock solutions as follows:
• Tris-acetate stock (100X) is obtained by dissolving 242 g Tris base in 600 mL water while titrating to pH 7 with glacial acetic acid and then brought to 1 L.
• Phosphate buffer (100X) is obtained by dissolving in water 10.8 g K2HPO4 , 5.6 g KH2 PO4 and then bringing to 1 L.
• Nutrient stock (100X) is obtained by dissolving 40 g NH4C1, 10 g MgS04 7H20 e 5 g CaCl2 2H20 in water and then bringing to 1 L.
• Glycerol used in this procedure is not pure, but derived from an industrial purification of vegetables from biodiesel plants. Before use, glycerol is purified with active carbon and after it is autoclaved at 1 Bar, 121 °C for 20 minutes.
• Vigor ultra is a commercial growth promoter (Hydrofert) used generally for plant growth and comprises: Aspartic acid 0.157g/100g; Glutamic acid 2.96g/100g; Alanine 0.252g/100g; Glycine 0.2g/100g; Leucine 0.112g/100g; Proline 0.139g/100g; 3- indoleaceutic acid 6.68 mg/kg.
• the cultivation process occurred in a bioreactor known to the expert in the field, such as a cylindrical open plastic container containing surface water as aqueous cultivation medium.
• the bioreactor is provided with suitable detection systems for measuring at least pH and temperature in the cultivation medium. Chlorella minutissima from UTEX collection with accession number Utex#2341 was used as algal species.
• Chlorella minutissima was cultured using tap water sterilized by a UV lamp, and containing glycerol and vigor ultra as nutrients.
• Chilomonas differs considerably in its metabolism from the ciliate Tetrahymena. The latter does not live in extremely high concentrations of CO 2 in which Chilomonas flourishes even when the carbon dioxide pressure reaches 400 mmHg.
• Tetrahymena pyriformis and Chilomonas Paramecium can grow together and this association is often positive.
• Microstomatous forms of Chilomonas are observed in mixture with Tetrahymena.
• the percentage of microstomatous forms of Chilomonas is larger at the beginning of population growth.
• Tetrahymena and Chilomonas mutually advantage in a sort of symbiotic association.
• disappearance of Chilomonas and cannibalism has been observed in old mixed cultures.
• Chlorella minutissima is stored at 26 °C in petri dishes on KOB medium. C. minutissima is then inoculated in TAP medium for a week at constant agitation, with temperature at 26°C and exposed to white light of 20
• Protozoa culture containing Tetrahymena pyriformis and Chilomonas Paramecium (in the following Ps culture), is stored at 28°C in two alternative different media: TAP or Chalkley' s medium. To keep the culture alive, it is advisable to refresh every week the culture medium with fresh medium.
• the cultured protozoa are used to infect Chlorella minutissima grown in 150 ml TAP medium in the ratio (protozoa/alga) 1:10. Then, after 5 days culturing, the 150 ml mixture culture is used as inoculum to obtain a culture of 1.5L in the same TAP medium.
• This new culture is kept in constant stirring at 28°C and exposed to a white light of 20-50 micromoles/m 2 s, for other five days.
• this culture is used as inoculum for a new culture with a volume of 15L. This volume is obtained adding to the culture 2.5 g/L glycerol and 100 ⁇ /L vigor ultra in tap water treated with UV and colloidal Ag (1:250) .
• the culture of algae plus protozoa is aerated and exposed to white environmental light at 28 °C. Every day, culture is added of 2.5 g/1 glycerol; 100 ⁇ / ⁇ vigorultra .
• This culture can live up to 6 months or more. Every day 2.5 g/1 glycerol and 100 ⁇ / ⁇ vigor ultra are added to the culture.
• the culture is refreshed harvesting the volume by 20 or 50 or 70%.
• the harvesting volume is taken at the bottom of the culture and can be used to fill other 200L containers to get an industrial module of hundred containers.
• the paramecia In case purchased Chilomonas and Tetrahymena paramecia are employed, the paramecia have to be conditioned to reach a good symbiosis with the algal species and consequently a good algal biomass yield.
• Protozoa are cultured singularly in Chalkley' s medium with grass seeds or TAP medium.
• Chilomonas and Tetrahymena are mixed together (ratio 1:1) or are used singularly to infect Chlorella minutissima in TAP medium (ratio protozoa (n) /alga 1:10) to obtain a mixed culture with all two/three microorganisms.
• mixture culture can be grown for a long time; for adaptation at least 2 months are required, refreshing it every week (half volume) by Chalkley' s medium with grass seeds or TAP medium.
• the quantity of algae in endosymbiosis increases with time of co-culturing and also the size of the bearing-algae protozoa and of algae increase as seen in (Nakajima T., Sano A. and Matsuoka H., 2009) .
• This culture can be further adapted to the nutrients glycerol and vigorultra in quantities of one fourth of the optimum concentration, increasing gradually with each refresh with an amount of 20% until arriving at the optimum 100% (of 2.5 g/L glycerol and 100 ⁇ /L vigor ultra) .
• the two paramecia can coexist in the same culture or one type, mainly Tetrahymena , can be preponderant ;
• cyst Non motile, with algae inside: in this state, called cyst, the Paramecium suspends animation and cell metabolic activities are slowed down.
• Unfavourable environmental conditions such as lack of nutrients or oxygen, extreme temperatures, lack of moisture, presence of toxic chemicals, which are not conducive for the growth of the microbe - trigger the formation of a cyst;
• the macro culture shows green, yellow and brown biomass with a state similar to glue, mostly precipitated and in part floated;
• culture colour pass through different colours: green, yellow and brown; for a high biomass production it is useful/advisable to maintain the co-culture in the above green or yellow stage since brown stage corresponds to partially digested alga, by means of repetitive dilutions of the culture medium. These repetitive dilutions cause an increased division of the alga-bearing Paramecium and the re- establishment of the green colour of the co- culture followed again by the various coloured phases .
• Chlorella algae result highly protected in this endosymbiotic process. Chlorella alone culture, using waste material as nutrient, does not survive over 7 and occasionally 30 days while, when infection with protozoa occurs, it can survive up to several months. Moreover, when the culture is not infected with the identified paramecia, it becomes prey of several other types of predators that do not establish endosymbiosis (e.g. rotifers) . Generally, culture of Chlorella alone rapidly decrease with a consequent high viscosity due to the release of microtubules in association with glycerol.
• Weight of algal biomass was determined by harvesting 20% of the culture at the base of the bioreactor. 5 samples of 100 mL of algal biomass were taken, under strong aeration for homogeni zat ion, and subsequently dried at 90°C for at least 10 days till a weight constancy and production of a fine powder. Therefore, as already stated, the algal biomass contains both the algal cells and the protozoan cells, as well as part of the aqueous cultivation medium.
• Weight of algal biomass produced by the above described co-culture symbiotic system was compared to the weight of algal biomass obtained by a culture of algae without protozoa.
• Example 1 Evaluation of algal biomass production by the co-culture symbiotic system (C. minutissima plus Tetrahymena pyriformis and Chilomonas Paramecium) vs . alga alone
• the relative standard deviation, calculated on n 6 sampling of one typical pattern of growth, was less than 10%.
• the symbiotic culture was obtained as described in Materials and Methods section, in two months culturing. The only difference is that further inocula of algae- bearing Paramecium in TAP medium were performed to the culture medium at different time intervals.
• algal cells/paramecium at the beginning were maintained in a ratio of about 100:1 and addition of algae-bearing Paramecium (in the same ratio of 100:1 for the algal cell/paramecium symbiont) was repeated every 3 days.
• the algae-bearing Paramecium were freshly prepared and added in the second stage of infection .
• the relative standard deviation, calculated on n 6 sampling of one typical pattern of growth, was less than 10%.
• the alga-bearing Paramecium was obtained as described in Materials and Methods section, in two months culturing.
• the culture was grown in a vessel of 15L for 6 months adding quantity of glycerol 2.5 g/1 and 100 ⁇ / ⁇ of vigor ultra per day; a process of harvesting 20-50-70% of the algal biomass and water re- addition to the initial volume was regularly applied on alternate days or on consecutive days.
• Table 5 shows that higher biomass yields were obtained one day after harvesting the algal biomass and cultivation medium re-addition to initial volume, that was accompanied by the typical cycle of colour steps: green-faintly yellow-brown. The process could be repeated without any production interruption.
• Results reported in table 5 show that during a 20 days production, harvesting alternatively 20-50% of the biomass, and re-addition of water in the co-culture on alternative days lead to high yield of algal biomass till about 10 g/1.
• culture realized according to the process herein described can be maintained for months.
• seasonal differences in the yields of accumulated biomass are observed from a minimum of 1-2 g/1 and occasionally till a maximum of 20-30 g/1.
• Example 5 Evaluation of inoculum (a) of algae/ cyanobacteria in the culture medium during the production process on overall algal biomass yield
• Symbiotic culture was obtained as described as described in Materials and Methods section, in two months culturing. The only differences are that glycerol was reduced to 1 g/L and Chlamydomonas reinhardtii cells were added to the co-culture as additional exogenous nutrient.
• This example illustrates that the addition of a second alga species (in TAP or Chalkley' s medium) to the co-culture is useful to reduce content of organic nutrient (in the specific case glycerol) .
• CM C. minutissima
• CS C. sorokiniana H1986
• Algae were cultivated in TAP medium at 26°C, under red light and in continuous shaking. After three days, we added at each inoculum one Paramecium culture, in a ratio alga/paramecium of 1:100. After two days, we have transferred each culture in containers of 18L and we started scaling up procedure, doubling volume every two days adding UV sterilized tap water, 2.5 g/1 glycerol and 100 ⁇ 1/1 vigor ultra. After reaching the final volume to invigorate cultures, every day the harvesting of half volume was carried out, replaced by an equal volume of water. This experiment was carried out for 20 days and the results are reported in table 7.
• the relative standard deviation, calculated on n 4 sampling of one typical pattern of growth, was less than 16%.
• algae were cultivated in TAP medium at 26°C, under white light and in continuous shaking. After two days, we added at each inoculum one Paramecium culture, in a ratio of 1:100. After two days, we have transferred each culture in bottles of 1L and started scaling up procedure, doubling volume every two days, till 800 mL, adding UV sterilized water, 2.5g/l glycerol and 100 ⁇ 1/1 vigor ultra. After reaching the final volume of 800 mL, to invigorate the cultures, every two days the harvesting of half volume was carried out replaced by an equal volume of fresh nutrients.
• the relative standard deviation, calculated on n 4 sampling of one typical pattern of growth, was less than 16%.
• Algal biomass has been produced according to Example 4.
• the particular composition of the biomass was found to comprise cells, lipids, starch, microtubules, and soluble sugars such as glycogen, explaining that it appears as a "glue".
• the biomass was mostly present as precipitate in big aggregations, in part floating and in part in solution in a ratio depending on the age of the culture.
• the content of lipids is preponderant and increases with the age of the culture (table 10) .
• the age of the culture was calculated as days of culture starting from the first infection day, after mixing the two microorganisms, and when symbiosis was established, co-culture and algal culture were compared for the content of total lipids.
• Table 10 provides the average lipid content per dried biomass in percent determined by the gravimetric method of Logan 2008.
• Table 10 shows that the lipid content greatly increased in the co-cultivation system compared to the culture of the alga grown alone from 47 till 90% of lipid per dried biomass after infection of algae with Paramecium for a long time indicating that lipid content increases with the age of the culture after the infection .
• the relative standard deviation, calculated on n 6 sampling of one typical pattern of growth, was less than 10%.
• Method GC Injection split (50:1) of FAME on GC Agilent 6850 column 60m ⁇ 0.25mm ⁇ 0.25ym of (50%-cyanopropyl) -methylpolysiloxane DB23, Agilent) with flame ionization as revealing system. Temperature of the injector: 230 °C. Conditions: initial temperature 195 °C for 26 min, increased by 10 °C/min till 205 °C, maintained for 13 min, followed by a second increment of 30 °C/min till 240 °C, maintained for 10 min. Constant pressure (29 psi) with hydrogen gas carrier.
• the first sample is richer on PUFA (about 35%) than the CM sample (about 26%) .
• the sample PS+CM has a slightly major production of arachidonic acid omega-6 (about 29% against 20% for CM) and similar production of DHA omega-3 (about 6%) .
• CM+caudatum lipid distribution is more similar to CM+Ps production than CM+bursaria. Table 11 .
• the fatty acid profile of microalgae is an important characteristic as it ultimately affects the quality of biodiesel production.
• the length of carbon chain of saturated and unsaturated fatty acids affects biodiesel properties such as cetane number, oxidative stability and cold-flow properties.
• SFA saturated fatty acids
• MUFA monounsaturated fatty acids
• oils dominated by these fatty acids are prone to solidify at low temperature. While oils rich in polyunsaturated fatty acids (PUFAs) have very good cold-flow properties, they are, on the other hand, more susceptible to oxidation.
• results in tables 11 and 12 show that the majority of fatty acids presents in isolated cultures were C16:0 (about 23-25%), 16:1 (about 10-12%) and omega-3 and 6 which comprised 35-40% of the total FAME. SFA and MUFA were predominant at >60% of the total lipid content which is favourable for high cetane number.
• PUFAs in range of 35-45% exceeded the requirements in the International biodiesel Standard for Vehicles (EN14214) .
• PUFA are high-value fatty acids for nutrition, food additives and aquaculture nutrients. In order to comply with biodiesel standard on the PUFA ratio, these components can be hydrogenated or extracted before the rest of oil is converted into biodiesel. That makes the oils derived biodiesel less susceptible to oxidation in storage and takes full advantage of algae-paramecium symbiotic culture.

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