NZ578233A - Process to produce biomass and proteins by microalgae by providing a medium including vinasse and additional carbon dioxide - Google Patents

Abstract

Disclosed is a process for the production of biomass and proteins from microalgae, intended for human or animal feed or for other uses, wherein said process comprises the following steps: - providing a culture medium including vinasse and carbon dioxide for culturing said microalgae; - addition of the said microalgae to said culture medium; and - collecting a fraction of microalgae biomass, in which the vinasse is, optionally, diluted and the carbon dioxide is in a concentration of between 0.1 to 100%. Further disclosed is a process for the production of biomass and proteins from microalgae, characterized by comprising the following basic steps: (i) adequation of the vinasse by adding water and alkali until a pH value of about 6.0-11.0 is reached; (ii) delivery of pre-adjusted vinasse to an inoculation tank; (iii) addition of microalgae to the inoculation tank until a concentration of about 0.2 g/I of initial biomass is reached in the cultivation medium based on vinasse; (iv) delivery of inoculated vinasse to an algal biomass cultivation tank; (v) injection of air containing 0.1-100% highly pure carbon dioxide to the algal biomass cultivation tank; (vi) keeping algal biomass at an average temperature between 25 and 35°C under natural light intensity; (vii) delivery of algal biomass to a separation unit, where a fraction of algal biomass and a fraction of water supernatant will be generated; (viii) recycle of the fraction of water supernatant to the inoculation tank.

Description

RECEIVED at IPONZ on 18 January 2012 "PROCESS TO PRODUCE BIOMASS AND PROTEINS BY MICROALGAE" Field of the Invention The present invention refers to a process to produce biomass and proteins from microalgae, advantageously using as a culture medium of said 5 microalgae rejects from the sugar and alcohol industries, notably vinasse (a byproduct of the alcohol destilation) and carbon dioxide originating from fermentation vessels.

The process of the present invention also contributes as a solution to reduce the emission of pollutant loads to water courses, soil desertification by 10 the accumulation of minerals, since the present process offers a drastic reduction of DQO (Chemical Oxygen Demand) and DBO (Biochemical Oxygen Demand) values as present in vinasse, as well as the emission of pollutant loads to the atmosphere, bearing in mind the reuse of carbon dioxide (C02) from the fermenting process.

Background of the Invention In Brazil, ethanol is only produced by means of fermenting processes, wherein yeasts transform juice, molasses and/or a mixture of sugarcane juice and molasses into ethanol. This is a biological process which may be represented by means of the stoichiometric equation of Gay Lussac, as 20 reproduced below: C12H22O11+ H2O C6H12O6 + C6H12O6 (a) C6Hi206 -» 2ch3ch2oh + 2C02 + 23,5 kcal (b) Equation (b) shows that, for each consumed 180 grams of sugar, 92 grams of ethanol and 88 grams of carbon dioxide are produced. 25 At the end of fermentation, the obtained liquid is named wine.

Wine, or fermented juice, has an ethanol concentration, percentage by volume, which may be between 6° and 10° GL, besides other liquid, solid and gaseous components. Within wine, besides alcohol (ethanol), we can find water under RECEIVED at IPONZ on 18 January 2012 2 rates which may vary between 89% and 93%, minerals and other substances under lower concentrations. Alcohol as present in such wine is recovered at the top of distillation columns, where present volatile substances are separated by their different points of ebullition. Vinasses are taken at the base of said 5 columns and constitute a liquid residue, generated under average proportion of 12 to 15 liters for each liter of produced hydrated alcohol. Said liquid residue, rich in minerals, among other chemicals, represents the largest source of pollution in the alcohol (ethanol) industry as obtained by fermenting processes.

The composition of vinasse depends on various factors, such as 10 the composition of raw material, characteristics and mode of operation of the distillation columns. Table 1 presents qualitative and quantitative characteristics of vinasse originating from juice must, molasses must and mixed must as collected in plants in the state of Sao Paulo.

Table 1 - Physical-Chemical Characterization of Vinasse (average of 64 15 samples from 28 plants in the State of Sao Paulo - Source: ELIA NETO, A. & NAKAHODO, T. (in Relatorio da Copersucar, Project n° 95000278, Piracicaba, 1995, 26 p).

Parameter Unit Minimu m value Average value Maximu m value Standard /I of alcohol Process data Must brix °B 12,00 18,65 23,65 Alcohol content of wine °GL ,73 8,58 11,30 Sugar husk rate (l/l of alcohol) ,11 ,85 16,43 ,851 Flow of reference (m3/day) 530,00 1908,86 4128,00 RECEIVED at IPONZ on 18 January 2012 3 Characteristics of sugar husk: pH - 3,50 4,15 4,90 Temperature °C 65 89,16 110,5 DB05 mg/l O2 6680,00 16949,76 75330,0 0 175,13g DQO mg/l 02 9200,00 28450,00 97400,0 0 297,60g Total solids (ST) mg/l 10780,00 25154,71 38680,0 0 268,90g Total Solids in Suspension (SST) mg/l 260,00 3966,84 9500,00 45,71g Fixed Solids in Suspension (SSF) mg/l 40,00 294,38 1500,00 2,69g Volatile Solids in Suspension (SSV) mg/l 40,00 3632,16 9070,00 43,02g Total Dissolved Solids (SDT) mg/l 1509,00 18420,06 33680,0 0 223,19g Dissolved Volatile Solids (SDV) mg/l 588,00 6579,78 15000,0 0 77,98g Fixed Dissolved Solids (SDF) mg/l 921,00 11872,36 24020,0 0 145,21g Residues which may settle (RS) (1 hour) ml/l 0,20 2,29 ,00 24,81 mL Calcium mg/l CaO 71,00 515,25 1096,00 ,38g Chloride mg/l CI 480,00 1218,91 2300,00 12,91 g RECEIVED at IPONZ on 18 January 2012 4 Copper mg/l CuO 0,50 1,20 3,00 0,01g Iron mg/l Fe203 2,00 ,17 200,00 0,27 Total Phosphorous mg/l P204 18,00 60,41 188,00 0,65g Magnesium mg/l MgO 97,00 225,64 456,00 2,39g Manganese mg/l MnO 1,00 4,82 12,00 0,05g Nitrogen mg/l N 90,00 356,63 885,00 3,84g Nitrogen in Ammonia mg/l N 1,00 ,94 65,00 0,12g Total Potassium mg/l K20 814,00 ,34,89 3852,00 21,21 g Sodium mg/l Na 8,00 51,55 220,00 0,56g Sulphate mg/l SO4 790,00 1537,66 2800,00 16,17g Sulphite mg/l S04 ,00 ,90 153,00 0,37g Zinc mg/l ZnO 0,70 1,70 4,60 0,02g Ethanol - CG ml/l 0,10 0,88 119,00 9,1ml_ Glycerol ml/I 2,60 ,89 ,00 62,1mL Yeast (dry basis) mg/l 114,01 403,56 1500,15 44,1g Vinasse contain minerals, organic matter and water, being characterized as a highly aggressive residue to the environment, for having high DBO and DQO levels. Up to the end of the 1970s, when that practice was forbidden, growing volumes of vinasse were launched to surface springs, mainly 5 to water courses such as rivers, streams and small rivers, near sugar and alcohol plants. The effects caused by said practice have been known for a long time. The organic load as present in vinasse causes proliferation of microorganisms consuming oxygen as dissolved in water, destroying water flora and fauna and causing difficulties to the use of drinkable water supply sources.

Furthermore, the discharge of vinasse into water courses causes bad odor and contributes to worsening various endemic parasitary diseases.

RECEIVED at IPONZ on 18 January 2012 Estimates show that the Brazilian production of vinasse for the 2006/2007 crop has been of about 190 billion liters. Currently, the fate of vinasse has been their pulverization into soil, particularly in sugarcane 5 plantations and/or their stocking in depuration lagoons. However, continued spraying of vinasse into soils, even at low dosages, may cause cation saturation, especially potassium, causing leaching of their components to underground waters. Potassium by itself is not a pollutant of waters and underground waters, but its presence in high concentrations in soil favors the 10 appearance of chemicals which, with neutral loads, are easily leached. The complex formed between (K)+ and (NO3)" provides a lot of environmental worries, since nitrate is an important pollutant for surface and underground waters.

In Brazil, governmental organisms attempt to impose restrictions 15 to the handling of said vinasse since 1978, forbidding the discharge of vinasse to surface waters. One of said rules regulating the use of vinasse establishes that vinasse may only be applied to soil when the total cation concentration (CTC) for that soil is below 5%. If that value has already been reached, the rule just allows the use of the potassium dosage equivalent to the consumption by 20 sugarcane in the year at issue, i. e. vinasse equivalent to 185 kg/ha of K2O. With such regulations in force, various sugarcane culture areas suffer restrictions, and the sector already develops projects aiming to transport vinasse to longer distances than used today.

One of the solutions being studied deals with the concentration of 25 vinasse as a form to reduce transport costs. The use of technical/scientific knowledge to better manage said vinasse, aiming at its more rational use with lesser environmental impact, is of vital importance.

RECEIVED at IPONZ on 18 January 2012 State of the Art Microalgae are organisms containing chlorophyll, which make photosynthesis, covering wide morphological, structural and metabolical variation, even including a few prokaryotic groups. A wide part of these 5 organisms is freely found in water, making part of phytoplancton, and is the base of the feeding chain in water ecosystems, being responsible for up to 50% carbon fixing and oxygen production on the planet (OLIVEIRA, A., Crescimento das diatomaceas bacillario phyceae Chaetocerus sp., Skeletonema costatum e Thalassiosira fluvia tilis em diferentes meios de cultura e em condigoes 10 controladas de temperatura e salinidade. Master grade monograph in Water Culture, Department of Water Culture, Federal University of Santa Catarina, Florianopolis, 1993).

Microalgae have been traditionally classified under various criteria, such as types of pigments, the chemical nature of reserve products and cell wall 15 constituents (TOMASSELI, L. The microbial cell, in RICHMOND, A. (Ed.), Handbook of Microalgal Culture: biotechnology and applied phycology. Oxford: Blackweel Science, p. 3-19, 2004). Microalgae form a heterogeneous group of organisms covering all photosynthesizer microorganisms, be them eukaryotic or prokaryotic. They are usually unicell and gram-negative. 20 The number of microalga species is very large, but still unknown. It is estimated that there may be between 200,000 and a few million species. Microalgae are unlimited sources of biomolecules of pharmaceutical and food interest, as well as other commercially interesting substances (PULZ, O., GROSS, W. Valuable products from biotechnology of microalgae. Applied 25 Microbiology and Biotechnology, 65 (6), p. 635-648, 2004).

According to RICHMOND, A. Handbook of Microalgal Mass Culture, CRC Press, U. S. A., 1986, microalgal production may be justified by numerous advantages, among them we may highlight: RECEIVED at IPONZ on 18 January 2012 - efficient biological process transforming sun energy into organic matter and many species grow more quickly than terrestrial plants, which enables higher biomass yielding; - its unicell nature assures biomass with one single biochemical composition, 5 which does not occur with terrestrial plants, presenting compounds located in specific parts, such as fruits, leaves, seeds or roots; - by controlling environmental culture conditions, such as light, temperature and nutrients, many species may be induced to synthesize and accumulate high concentrations of proteins, carbohydrates, lipids, etc. These compounds have high commercial value, mainly for being considered as of natural origin; - they may well grow in regions with extreme climate conditions. Cultures may develop under sea or estuary waters, which cannot be conventionally employed in the culture of plants with agricultural value, or with residual waters originating from various production processes, such as e. g. agriculture, cattle-raising, industry and domestic waste; - the life cycle of most microalgae is completed within a few hours, thus favoring selection of strains and genetic improvement of species.

Concerning nutrition, for optimum growth, microalgae need a number of nutrients. Among different genus and species, many variations 20 mainly related to the quantity of nutrients in the medium occur. Even so, these nutritional needs depend on different environmental conditions (ABALDEJ, C. A., FIDALGO, J. P., TORRES, E., HERRERO, C. Microalgas: Cultivo y Aplicaciones. La Coruna: Servicio de Publicaciones, p. 210, 1995. Microalgas: Cultivo y Aplicaciones. La Coruna: Servicio de Publicaciones, p. 210, 1995). 25 Macronutrients required by microalgae are carbon, nitrogen, oxygen, hydrogen and phosphorous, besides calcium, magnesium, sulfur and potassium. Concerning micronutrients, they usually need iron, manganese, copper, molybdenum and cobalt, while a few microalgae also require low vitamin RECEIVED at IPONZ on 18 January 2012 concentrations in the culture medium (GHILLARD, R. R. L. Culture of phytoplankton for feeding marine invertebrates. In: SMITH, W. L., CHANLEY, M. H. (Eds.), Culture of Marine Invertebrates Animals, Plenum Press, New York, p. 29-60, 1975).

The most important nutritional elements are carbon, nitrogen, phosphates and magnesium, potassium and calcium salts. Elements at lower concentrations such as manganese and cobalt are indispensable in various important metabolic activities. The most important carbon sources are carbohydrates. Nitrogen is found in proteic material and its degradation 10 products, being supplied through ammonia salts.

Spirulinas are classified as prokaryotic immobile beings with no spores. Their prokaryotic nature, their phycobilliproteic pigments and oxygen production by photosynthesis make them different from eukaryotic algae and photosynthetic bacteriae. Spirulinas live in liquid medium, rich in minerals, 15 mainly composed by sodium bicarbonate and carbonate, with pH between 8 and 11. Tropical and subtropical, hot and sunny regions are ideal for their cultivation. Furthermore, said microalgae are used as a source of food in human diet and animal feed, having high protein contents and containing all essential aminoacids under proportions following the recommendations by FAO (Food 20 and Agriculture Organization), a United Nations organism.

Specifically, microalga Spirulina is a filamentous cyanobacteria with 1 to 12 |im of diameter, spirally located, up to 1 mm long (TOMASELLI, I. Morphology, ultrastructure and taxonomy of Arthrospira (Spirulina). Physiology, cell-biology and biotechnology. London: Taylor & Francis, ISBN 0-484-0674-3, 25 1997). Natural occurrences of Spirulina are found in the lakes of Chad in Central Africa, Texcoco in Mexico, Nakaru and Elementeita in Kenya and Aranguadi in Ethiopia (VONSHAK, A. Spirulina platensis (Arthospira) RECEIVED at IPONZ on 18 January 2012 Physiology, cell-biology and biotechnology. London: Taylor & Francis, ISBN 0-484-0674-3, 1997). In Brazil, the occurrence of Spirulina at Mangueira Lagoon, Rio Grande do Sul (DURANTE, A. J., REICHERT, C. C„ DALCANTON, F„ MORAIS, M. Isolamento e cultivo de uma cepa de Spirulina nativa da Lagoa 5 Mangueira e influencia da Spirulina platensis no crescimento de uma cianobacteria toxigenica. Graduation Course conclusion work in Food Engineering, FURG, Rio Grande, 2003).

Spirulina highlights itself among other microalgae, mainly due to its protein content, vitamins like B12 and pigments like phycocianine and p-10 carotene. Said microalga is known as GRAS (Generally Recognized as Safe) by the U. S. FDA (Food and Drug Administration). Protein content in dry biomass varies between 64 and 74%. These proteins are considered as complete, since they have all essential aminoacids, summing up 47% of the total protein weight (COHEN, Z. The chemicals of Spirulina. In: VONSHAK, A. 15 Spirulina platensis (Arthrospira) Physiology, cell-biology and biotechnology. London: Taylor & Francis, ISBN 0-484-0674-3, 1997. Sulfur amino acids, methionine and cistine, are present in lower concentration and even so represent more than 80% of the ideal level as recommended by FAO. Spirulina biomass, in comparison with other foods in protein terms, is in average 65% 20 above any natural food (FALQUET, J. The nutritional aspects of Spirulina. Antena Technology, 1997. http://www.antenna.ch). NPU (Net Protein Utilization) is experimentally given by calculating the percentage of retained nitrogen when the researched protein source is the only limiting nutritional factor. NPU for Spirulina varies between 53 and 61% or 85 and 92% of NPU of casein as 25 original egg standard. PER (Protein Efficiency Ratio) is the ratio between mass gain of the animal being studied, usually rats, and the mass of ingested proteins. PER for Spirulina varies between 1.80 and 2.60, against PER of 2.50 RECEIVED at IPONZ on 18 January 2012 for egg casein (Falquet, 1997). Spirulina, as opposed to other microalgae, does not have a cellulose cell wall, but rather a relatively brittle murein envelope. The lack of cell wall is an advantage from the point of view of preserving the integrity of components such as vitamins and poliinsaturated fat acids, since it avoids 5 the use of cooking to make nutrients available (Falquet, 1997). Simple molecules such as glucose, fructose and sucrose are present in small quantities. From the nutritional point of view, the only carbohydrate occurring in interesting quantities is mesoinositol phosphate, an excellente source of organic phosphorous and inositol (QUILLET, M. Recherches sur les substances 10 glucidiques elaborees par les Spirulines. Ann. Nutr. Aliment., 29, n° 1, p. 553-561, 1975). Nucleic acids are usually a limiting factor for the consumption of proteins with microbial origin since, during their metabolism through the organism, uric acid is produced, and high rates may cause gout problems. It is advisable that the ingestion of nucleic acids does not overcome 4 g/day, in case 15 of an adult person. The concentration of nucleic acids in the biomass of yeasts is about 23%, while in Spirulina nucleic acids vary between 4.2 and 6% over the weight of dry biomass. Therefore, a higher daily ingestion of 80 g of Spirulina would be possible to reach the daily limit of nucleic acids. This quantity is about eight times higher than the dose of microalga as recommended for food supply 20 (FOX, R. D. Spirulina production & potential. France, Edisud, ISBN 2-84744-883-x, 1996). Spirulina produces high concentrations of vitamin B12, at about 11 mg/kg of dry biomass. Meats contain considerable concentrations of said vitamin, but it is practically absent in vegetables (CIFERRI, O. Spirulina the edible microrganism. Microbiol. Rev. 47, p. 551, 1983). Pro-vitamin A, or (3-25 carotene, represents about 80% of carothenoids as present in Spirulina. In 1 kg of dry biomass of Spirulina, p-carotene concentration is about 700 and 1700 mg. Biomass of Spirulina also contains tocopherols with antioxidizing power, at RECEIVED at IPONZ on 18 January 2012 11 about 50-190 mg/kg on dry basis, i. e. comparable levels to wheat germen. Spirulina also contains low quantities of niacin, folic acid, pantothenic acid and biotin (Cohen, 1997). Spirulina biomass is also rich in minerals such as calcium, iron, phosphorous, magnesium and potassium. In terms of calcium, iron and 5 phosphorous levels, contents are similar to milk. Spirulina contains higher iron contents than cereals (Falquet, 1997).

Generally speaking, algae need light, water, minerals and a certain quantity of carbon dioxide (CO2) to grow.

From this knowledge, and through long studies and experiments, 10 the Applicant concluded that the use of vinasse originating from distillation of must in alcohol plants, as well as C02 from the fermentative process, presents large potential to produce alga biomass from various genus and species, notably Spirulina, for application in human and animal feed, as well as for the production of other molecules of commercial interest.

In this specification unless the contrary is expressly stated, where a document, act or item of knowledge is referred to or discussed, this reference or discussion is not an admission that the document, act or item of knowledge or any combination thereof was at the priority date, publicly available, known to the public, part of common general knowledge; or known to be relevant to an 20 attempt to solve any problem with which this specification is concerned.

It is therefore an object of the present invention to provide an improved process for the production of biomass and proteins from microalgae entitled to human or animal feed as well as for other uses; and/or an improved process for the production of biomass and proteins from microalgae according 25 to certain method steps; or at least to provide the public with a useful choice.

Summary of the Invention Microalgae, when cultivated in appropriate medium, may duplicate RECEIVED at IPONZ on 18 January 2012 their biomass daily. This characteristic, added to simple cultivation techniques, makes microalgae an item of interest of the present invention.

The main aspect of the present invention is to provide a process to produce biomass and proteins from microalgae, advantageously using vinasse 5 and carbon dioxide originating from fermenters, as generated by the alcohol industry, as a medium or culture substrate.

Yet another aspect of the present invention is to provide a process to produce microalga biomass from vinasse and carbon dioxide, generated as rejects by the alcohol industry, using sugarcane and its derivatives. 10 Even more specifically, the present invention seeks to provide a microalga production process from vinasse and carbon dioxide, generated as rejects in the alcohol industry, by using sugarcane and its derivatives, being said microalgae selected from one or more genus (species) of the group comprising Spirulina (sp, platensis, maxima, major, subsalsa, geitleri, 15 subtilissima, labyrinthiformsy, Skeletonema sp; Chaetoceros sp; Scenedesmus sp (bijugatus, incrassatulus, ocultus, quadricauda, dimorphus)] Anacystis sp (nidulans, cyanea, thermalis); Porphyridium cruentum; Crypthecodinium cognii; Euglena sp (gracilis)] Crypthecodinium cohnii; Haematococcus pluvialis; Anabaena sp (variabilis, cylindrica, hassali, planctonica); Dunaliella sp 20 (salina,bardawil, tertioleta); Chlamydomonas sp (reinhardtii); Chlorella sp (vulgaris, kessleri, pyrenoidosa, mannophila, protothecoides, salina, homosphaera, stigmatophora, luteoviridis, regularis, ellipsoidea, variegata, sorokiniana, emersonii); Trichodesmium, Microcoleus; Ankistrodesmus sp (densus, braunii, falcatus, fusiformis, gracilis)-, Isochrysis galbana (Parke); 25 Tetraselmis sp. (tetrathele, suecia)-, Oscillatoria sp (limnetica, curviceps, splendid a)] Nostoc muscorum and Botrycoccus braunii,. However, RECEIVED at IPONZ on 18 January 2012 the present invention may comprise other genus (species) besides the ones as reported herewith.

Another aspect of the present invention is the use of microalga Spirulina platensis OF 25 in a process to produce biomass and proteins from 5 vinasse and carbon dioxide, generated as rejects in the alcohol/ethanol industry.

Therefore, in summary, the present invention seeks to recycle and use vinasse as a medium of culture for the production of alga biomass rich in proteins and other products with commercial interest, notably Spirulina biomass, 10 as well as to make use of the effect of CO2 originating from fermentation vessels in the growth of said microalga and to promote the reduction of DQO and DBO levels from vinasse as discharged in the fermenting process.

Description of Figures The attached figures will serve to provide for better understanding 15 of the objects and process of the present invention. Some of them refer to the cultivation of microalga Spirulina platensis OF 25, but it should be understood that the process is neither exclusive nor limited to the culture of said microalga, and may clearly be used for other genus and species.

Figure 1 shows a flow diagram showing the main steps of a typical 20 production process for hydrated alcohol, notably ethanol, from sugarcane derivatives.

Figure 2 shows a flow diagram showing the main steps of the cultivation process for Spirulina platensis OF 25 in vinasse and CO2 of the present invention.

Figure 3 shows a flow diagram of the production process for algal biomass of the present invention, using the microalga Spirulina platensis OF 25 and inoculation conditions of the first cycle.

RECEIVED at IPONZ on 18 January 2012 14 Figure 4 shows a scheme model of column photobioreactors as used in the experiments of the process of the invention.

Figure 5 shows an oven model with its corresponding dimensions with photoperiod as used in experiments with tube photobioreactors of the 5 present invention.

Figure 6 shows an arrangement of photobioreactors on the oven shelves with photoperiod during experiments to test vinasse under different air:C02 ratios as per the present invention.

Figure 7 is a graph showing the evolution of growth in terms of 10 biomass of Spirulina platensis OF 25 produced in a diluted cane husk culture (50%) under different levels of C02 and its comparison with Zarrouk medium.

Details of the Invention Studies carried out by the Applicant have shown that vinasse contain practically all the mineral elements, as well as numerous organic 15 compounds as requied for the growth of various genus and species of microalgae.

Therefore, the process to produce biomass and proteins from microalgae of the present invention advantageously uses vinasse and carbon dioxide (C02) produced as residues in the process to ferment sugarcane juice, 20 molasses or their mixtures to produce alcohol, notably hydrated and anhydrous ethanol.

Vinasse as used for the studies and experiments of the process of the present invention were supplied by the company Jardest S. A. Agucar e Alcool, Jardinopolis/SP, Brazil, which will now be called "Jardest Vinasse". 25 Table 2 presents the typical composition of "Jardest Vinasse".

RECEIVED at IPONZ on 18 January 2012 Table 2 - Composition of Jardest Vinasse Component Unit Jardest Vinasse Nitrogen % 0,60 Phosphorous ppm P205 450,00 Potassium % k2o 0,42 Calcium Ppm 357,00 Magnesium Ppm 615,00 Sulfur Ppm 145,00 Iron Ppm 330,00 Manganese Ppm ,00 Copper Ppm ,00 Zinc Ppm 18,00 Boron Ppm 37,00 Sodium Ppm 38,00 Cobalt % ANS Molybdenium % ANS Aluminum Ppm ANS Chlorine % ANS Nickel % ANS Organic Carbon % ANS Organic Matter % ,00 pH - 3,80 Density g/rnl 0,98 C/N Ratio - 4/1 Electric Conduction pS/s ANS DQO mg/l 02 .150,00 DBO mg/l 02 19.740,00 RECEIVED at IPONZ on 18 January 2012 16 The process of the present invention comprises the production in large scale of algal biomass by using C02 as generated during alcohol fermentation, as well as the vinasse originating from the distillation step in alcohol plants.

Figure 1 presents a simplified flow diagram of the most important unitary operations of an ethanol production plant from the fermentation of sugars as derived from sugarcane and/or another kind of carbohydrate. Vinasse as generated by the distillation of fermented must is conducted by pumping through tubes and/or by the use of gravity and/or gutters, or by using cistern 10 trucks up to the microalga production plant.

In the microalga production plant, as shown by Figure 2, vinasse may be directly transferred to culture tanks or stored in appropriate recipients, preferably sealed to avoid external contamination, and may suffer pre-treatment with physical, chemical and/or biological preserving purposes. 15 Studies as made by the Applicant have shown that vinasse include, besides water, considerable concentrations of minerals, especially potassium, phosphorous, sulfur, cobalt, molybdenium, manganese and zinc, among others. We also noticed that organic compounds such as residual sugars, biomass and yeast fragments, soluble proteins, etc. as present in 20 vinasse make it an excellent substrate for the culture of various groups (species) of algae.

Yieldings in terms of biomass as obtained are compatible with classical means as disclosed by the international literature. We also concluded that vinasse may be used to cultivate algae for the production of proteic 25 biomass as disclosed by alcohol distillaries, and if required may be diluted in water, adding or not other chemicals, with the purpose to adjust their pH and/or to complement given macronutrients and/or micronutrients. In some given cases, vinasse may be filtered and/or clarified by using activated charcoal, sand RECEIVED at IPONZ on 18 January 2012 17 bed with different granulometries or flocculating agents, depending on the concentration of solids in suspension in the different types of vinasse. We have also noticed that pasteurization and/or sterilization of vinasse does not provide for significant difference in biomass production, and therefore a unit operation is 5 not required, although it may be used when necessary.

Inoculation for biomass production is spread from cultures in laboratory scale, passing through reactors in growing volume scale up to forming sufficient algal biomass to start the culture in production tanks. Tanks to propagate inoculates may have different shapes and/or sizes, open or closed, 10 aerated or not, shaken or not, continual, semicontinual or discontinual, fed or not; horizontal or vertical, raceway type, in plates or tubes, oval, circular, rectangular, square, etc.

Microalga production in cultivation medium based on vinasse, as disclosed by the present invention, may be made according to the flow diagram 15 as shown by Figure 2. Cultures in open air comprise the use of natural or artificial tanks, with volume which may vary between a few dozen liters to several million liters. These tanks occupy large areas and may even reach, in average, 10,000 m2 in the case of one single tank. It is not advisable to use deep tanks, they generally should not overcome 0.5 m water column so not to 20 make light penetration difficult, thus reducing the photosynthesis process. Tanks as used to produce algae from vinasse may be horizontal or vertical, raceway, in plates or tubes, oval, circular, rectangular, square, etc, continual, semicontinual or discontinual, fed or not, shaken or not. When used, the most common shaking system uses blades, which are mechanically shaken and 25 distributed in regular spaces throughout the surface of the tank, or then located at the ends or in the center of the tank.

Simultaneously with shaking with blades, we may inject air, compressed or not. Such air may be filtered or not. Reactors may be closed by RECEIVED at IPONZ on 18 January 2012 18 using a removable cover, but for obvious reasons, such cover shall be constructed with transparent or translucid material to natural or artificial light.

Carbon dioxide (CO2) as produced in alcohol plants during fermentation will be recovered on the top of fermenters by means of a collector 5 coupled to it, from which such gases are delivered through appropriate tubes to the alga production tanks, where it is distributed through bubblers or diffusers into the cane husk-based cultivation medium. The quantity of CO2 as released to the medium should be enough for its concentration to reach a value of about 0.1 to 100%. C02 as produced during alcohol fermentation in high quantities 10 may be compressed and/or purified and stocked in pressurized reservoirs before being injected into alga production tanks. An example of C02 purification may be through the passage of gases originating from fermenters through three filled in washing towers. The first tower contains a diluted alcohol solution acting as a preliminary purifier and removes most of the alcohol as carried by the gas. 15 The two depurators that follow, in which the washing liquid is unaired water, remove most water-soluble impurities. The washing liquid returns to fermenters or distillation unit by pumping to recover the residual alcohol carried in it and the depurated gas is subsequently treated to supply an odorless gas which may be stocked by compression in tanks to be later used for microalga culture. 20 The separation or harvest of algal biomass as produced may be made continuously, semicontinuously or discontinuously, manually or mechanically, flocculated or not, by using centrifuges, filters, press filters, screens, decanters or vortexes. Biomass may be extruded or not, dried naturally or in fixed or mobile bed driers, or by atomization (spray drier) or 25 rotating drum.

Figure 3 shows a flow diagram of the process to produce proteins from microalgae from vinasse and carbon dioxide of the present invention, comprising the following basic steps: RECEIVED at IPONZ on 18 January 2012 19 The process to produce biomass and proteins from microalgae according to the present inventios comprises the following basic steps: (i) adequation of the vinasse by adding water and alkali until a pH value of about 6.0-11.0 is reached; (ii) delivery of pre-adjusted vinasse to an inoculation tank; (iii) addition of microalgae to the inoculation tank until a concentration of about 0.2 g/l of initial biomass is reached in the cultivation medium based on vinasse; (iv) delivery of inoculated vinasse to an algal biomass cultivation tank; (v) injection of air containing 0.1-100% highly pure carbon dioxide to the algal 10 biomass cultivation tank; (vi) keeping algal biomass at an average temperature between 25 and 35 °C under natural light intensity; (vii) delivery of algal biomass to a separation unit, where a fraction of algal biomass and a fraction of water supernatant will be generated; (viii) recycle of the fraction of water supernatant to the inoculation tank.

Optionally, the process includes a step (ix) of repetition of steps (iii) to (viii) until a DQO value of about 17 mg/l 02 and lower DBO than about 5 mg/l O2 in the fraction of water supernatant as produced in step (vii) is reached. Also, the process may optionally include three or more processing cycles for 20 one single load of vinasse.

As disclosed above, in the process of the present invention, the first supernatant as generated by step (vii) is inoculated to serve as a substrate for a second cycle of production of algal biomass, thus allowing to establish an optimized cultivation procedure for the microalga, fully using all the organic and 25 inorganic material as present in vinasse. The time of algal cultivation in the tank is usually about 14 days, but longer or shorter periods may be used, depending on processing conditions, origin and quality of vinasse, microalga and other factors.

RECEIVED at IPONZ on 18 January 2012 After cultivation and first filtering (first cycle), the supernatant usually presents pH of about 8.5 to 9.0, with no need for correction, since it is within an ideal range for microalga cultivation. The higher the pH, more easily C02 will be dissolved into the culture medium. Recirculated water supernatants 5 may also be mixed with pure vinasse in different stages, as a form to enrich them with organic and mineral compounds before the inoculation with active microalga biomass to conduct a new cycle or load.

Advantageously, the flow of feeding air from the cultivation tank of algal biomass is enriched with about 5-15% C02, which is packed in cylinders at 10 58.3 kgf/cm2 pressure and contains high degree of purity of more than 99.8%. Carbon dioxide percentage preferred in the present invention is about 15%.

Still more advantageously, at the completion of each cycle and after the removal of algal biomass, water supernatants are inoculated again with active microalga biomass so that the initial concentration in algal biomass tanks 15 is of about 0.2 g/l biomass in the cultivation medium based on vinasse. Similarly, we prefer that light intensity in algal biomass tanks is of about 1,500 lux 12h/12h day.

Besides not passing through sterilization processes, water supernatant recycle guarantees the economic and environmental success of the 20 process of the present invention, since it provides for the full use of organic and inorganic elements as present in vinasse to produce algal biomass.

Furthermore, the biological treatment of vinasse by using microalgae is obtained with a consequent reduction of the high DQO and DBO rates as present in such rejects from alcohol plants. Such biological treatment 25 becomes more significant after three or more processing cycles from one single load of vinasse as per the process of the present invention.

The process of the present invention also contributes to the current environmental laws, since it is an ecologically correct and sustainable RECEIVED at IPONZ on 18 January 2012 21 technology and has as its final product algal biomass rich in proteins, besides promoting the release of oxigen to the environment. Therefore, the process of the present invention is extremely important in terms of reduction of the environmental impact as generated by alcohol plants.

Laboratory studies have shown that, at each cultivation cycle, the final quantity of biomass as obtained decreases. This occurs as a function of the fact that micronutrients and macronutrients as present in the recycled supernatant become exhausted while the algal biomass is produced in the different cycles. Therefore, to keep subsequent cycles under equivalent yielding rates in terms of global biomass, it is required to supplement the supernatant with a fraction of fresh vinasse to compensate the quantity of micronutrients and macronutrients as lost in previous cycles. However, in cases in which the main object is to reduce the pollutant load of vinasse, said substitutions may be avoided.

To evaluate the growth of algae under different experimental conditions as studied, (duplicate) samples have been collected each two days for analysis by dry weight. The biomass was vacuum filtered through 0.45 p,m filter paper, subsequently washed with distilled water and dried for 24 hours in an oven at 100 °C. Data is compiled at Table 3 below.

Table 3 - Algal biomass produced in the different re-use cycles of cane husk supernatants Time of cultivation (days) Supernatant Condition x (g/l) Air + 15% C02 0 Beginning 0,211 14 1st Cycle 4,470 28 2nd Cycle 3,043 42 3rd Cycle 2,154 RECEIVED at IPONZ on 18 January 2012 22 We can notice on the above table that, throughout the cycles, micronutrients and macronutrients are consumed. We have also noticed a reduction in DQO and DBO values after the first and the second cultivation cycle, arriving at rates very close to zero after the conclusion of the third cycle, 5 as shown by Table 4 below. DBO and DQO analysis have been taken according to Standard Methods for the Examination of Water and Wastewater, ed., 1998, Hach Company and WTW.

Table 4 - Reduction of DQO and DBO levels along recycling cycles of supernatants from vinasse Condition Time (days) DQO (mg/l 02) DBO (mg/l 02) Beginning 0 14790 10034 1 st Cycle 14 2935 1115 2nd Cycle 28 308 83 3rd Cycle 42 17 < 5 The present invention will be additionally disclosed by means of the Examples that follow which, not limiting their scope, represent a preferred embodiment.

Microalga Spirulina platensis OF 25 Spirulina platensis OF 25, selected from the Bank of Cultures of the 15 company Ouro Fino Saude Animal Ltda., was the considered microalga for specific studies of the process of the present invention, although other microalgae, alone or in mixtures, may be equally employed for the production of biomass and proteins from vinasse and carbon dioxide as generated by alcohol plants.

Spirulina platensis OF 25 presents high growth under temperature 20 ranges between 25 and 35 °C under slightly alkaline pH. These physiological characteristics of Spirulina platensis OF 25 provide for large potential for their RECEIVED at IPONZ on 18 January 2012 culture in vinasse, since that residue, when discharged by alcohol distilleries, presents high organic and mineral loads.

Furthermore, temperature ranges considered as optimum for the cultivation of Spirulina platensis OF 25 are close to average temperatures of the 5 Brazilian regions where sugarcane is cultivated, exactly where alcohol plants are installed. Therefore, the requirement to heat algal production tanks, also called photobioreactors, is practically eliminated.

Spirulina, as well as other microalgae, requires, besides a source of carbon, a source of nitrogen, phosphorous and other micronutrients (Vonshak, 10 1997). Although Spirulina may grow photoautotrophically, the collection of C02 from the air depends on the pH of the cultivation medium. The higher the pH of the medium, more easily C02 from the atmosphere migrates to inside it and is converted into CO32". At pH above 11, however, Spirulina does not grow, probably due to the effect of large alkalinity over metabolic processes or also to the microalga's inability 15 to assimilate carbon in the form of CO32". Therefore, in cultivations of the microalga Spirulina, an external source of carbon is usually required in the form of HC03", a species participating in the equilibrium: C02 ±5 HCO3 ±5 C032" This is the source of carbon most probably assimilated by 20 Spirulina (BINAGHI, L„ BORGHI, A. D„ LODI, A., COVERTI, A., BORGHI, M. D. Batch and feed-batch uptake of carbon dioxide by Spirulina platensis. Process Biochemistry, 38, p. 1341-1346, 2006).

In the production of microalgae, the highest financial terms are firstly handwork and subsequently costs with cultivation medium. The ZARROUK medium 25 (ZARROUK, C. Contribution a I'etude d'une cyanophycee: Influence de divers facterurs physiques et chimiques sur la croissance et photosynthese de Spirulina maxima Geitler. PhD Thesis, University of Paris, 1966) is traditionally used for the cultivation of Spirulina. Therefore, the possibilities to reduce costs of the Zarrouk RECEIVED at IPONZ on 18 January 2012 24 medium for the cultivation of Spirulina are significantly desirable.

Tables 5, 5a and 5b relate concentrations of all chemical elements present in the Zarrouk medium as used in handling and piercing Spirulina platensis OF 25 during the whole experimental process of the present invention. 5 The mother line of Spirulina platensis OF 25 was cultivated in Zarrouk medium and preserved in a freezer at a temperature of -80 °C.

Table 5 - Components of the Zarrouk Medium for the Cultivation of Spirulina platensis OF 25 Component Concentration g/l NaHC03 16.8 K2HP04 0.5 NaN03 2.5 K2SO4 1.0 NaCI 1.0 MgS04.7H20 0.2 CaCI2 0.04 FeS04.7H20 0.01 EDTA 0.08 Solution A5 1 ml Solution B6 1 ml Table 5a - Composition Solution A5 Component Concentration (g/i) H3BO3 2,86 MnCI2 4H20 1,81 RECEIVED at IPONZ on 18 January 2012 ZnS04 7H20 0,222 CuS04 5H20 0,079 NaMo04 0,015 Table 5b- Composition Solution B6 Component Concentration (g/l) NH4VO3 22.96 K2Cr2(S04)2.24H20 96.00 NiS04.7H20 47.85 Na2W04.2H20 61.10 Ti0S04.H2S04.8H20 0,015 C0(N03)2. 6H20 43.98 Example 1: Adaptation of microalgae to vinasse Previous adaptation studies for Spirulina platensis OF 25 under growing mixtures (5, 25, 50, 75, 100%) of vinasse to the Zarrouk culture 5 medium were evaluated. This same procedure may be applied if justifiable for other genus and/or species of microalgae when cultivated in vinasse, including by employing other media than Zarrouk, but more specific for each algal group. That adaptation was made in 250 ml Erlenmeyer flasks containing 50 ml of medium or in another similar cultivation system. Considering that the medium 10 containing 5% vinasse + 95% Zarrouk medium was used to inoculate the medium containing 25% vinasse and then subsequently until the final culture in pure vinasse of a previously adapted culture. Flasks were delivered to a "Shaker" type incubator trademark TECNAL, model TE-421, containing controlled photoperiod, temperature and shaking or in other systems having the 15 same objects. Cultures were incubated for a 14-day period, during which the RECEIVED at IPONZ on 18 January 2012 26 following preferential standards were kept as constant: temperature 30 °C (± 2 °C), 110 rpm shaking, 1500 Lux light irradiation intensity for 12 hour periods alternated with 12 hours in darkness. The light intensity inside the incubator was daily evaluated, in this case using a digital light meter Minip MLM 101. To follow 5 the growth of algae, samples were taken each two days for analysis by dry weight. The algal biomass as formed after 14 days of culture was vacuum filtered through filter paper Milipore with 0.45 jim pores, followed by washing with distilled water and dried for 24 hours in an oven at 100 °C. The results as contained in Tables 6 and 7 below represent the average of three 10 determinations for each one of the studied conditions.

Table 6 - Adaptation of Spirulina platensis OF 25 under different cane husk concentrations ZARROUK Vinasse Starting Final algal A Algal medium (%) algal biomass (g/l) biomass (%) biomass (g/l) (g/i) 100 0 0,197 2,951 2,754 95 0,211 2.780 2.569 75 0,199 2,162 1.963 50 50 0,195 1,875 1,680 75 0,205 1,377 1,172 95 0,199 0,845 0,646 0 100 0,203 0,543 0,340 This process to previously adapt microalgae to vinasse allows obtaining more expressive results in terms of daily final yielding of biomass in 15 comparison with a process in which microalgae do not pass through this previous adaptation (Table 7).

RECEIVED at IPONZ on 18 January 2012 27 Table 7 - Effect of adaptation of Spirulina platensis OF 25 in vinasse diluted over the production of algal biomass Condition Biomass (g/l) Production of biomass from Spirulina platensis OF 25 cultivated in vinasse (50%) + distilled water (50%) with previously adapted inoculum. 1,464 Production of biomass from Spirulina platensis OF 25 cultivated in vinasse (50%) + distilled water (50%) with not previously adapted inoculum. 0,633 Example 2: Preparation of Inoculum This process was made in 500 ml Erlenmeyer flasks containing 90 ml of 5 pure vinasse or mixed with water. Non-sterilized flasks were inoculated with 10 ml of an active culture of Spirulina platensis OF 25 adapted in a medium containing Zarrouk + vinasse (1:1), so that the initial algal biomass concentration remains under values of at least 0.15 g/l. Flasks were delivered to a "Shaker" type incubator and cultivated under the same conditions as 10 established by Example 1. The algal biomass as obtained was employed to inoculate tubular photobioreactors such as those shown by Figure 4.

Example 3: Cultivation of Spirulina platensis OF 25 under concentrations of pure vinasse and diluted in water Spirulina platensis OF 25 was cultivated in pure vinasse and 15 diluted in water as the sole culture medium, just having its initial pH adjusted to 8.0 with 3N NaOH. Table 8 below presents the main tested dilutions.

In these experiments, 52 cm high tubular glass photobioreactors RECEIVED at IPONZ on 18 January 2012 with 8 cm diameter were used, with total volume of 2 I, just like shown by Figure 4. Photobioreactors were filled in with 1.8 I of vinasse and non sterilized diluted vinasse and inoculated with an active culture of Spirulina platensis OF 25 as previously adapted in vinasse, following a method as disclosed by Example 2, 5 until the concentration of algal biomass at the start of culture reaches about 0.2 g/l. Shaking and aeration of photobioreactors were provided through an atmospheric air flow of 1 v/v/m (volume of air per volume of medium), passed through small glass canes with porous stones at their ends to increase the diffusion of gases into the liquid culture medium based on vinasse, as shown by 10 Figure 4.

The experiments were conducted in a climatized 3.5 m x 2.5 m x 2.5 m room with controlled temperature within the range of 30 °C (± 2 °C), by using a split air conditioner, trademark Consul Ambiense (12,000 BTU/h). In that room, two ovens with photoperiods for weather control Full Gauge 15 trademark digital cyclomatic model PROGS I with direct 220 V AC supply containing twelve 20 Watts daylight fluorescent lamps, with two electronic reactors and an auxiliary four-point supplier per shelf, automatic and manual functioning system and a steel structure covered by white Formica to improve lighting, as shown by Figure 5. The illumination of photobioreactors was 1,500 20 Lux as supplied by daylight type fluorescent lamps for a twelve-hour period, alternating with twelve hours of darkness. Culture time was fourteen days for all experiments.

Each oven has three shelves with photoperiod, each one comprising six photobioreactors. The arrangement of said photobioreactors is 25 schematically represented by Figure 6.

Volume of cultures was kept constant by daily reposition of distilled water to compensate losses by evaporation.

RECEIVED at IPONZ on 18 January 2012 29 Table 8 - Cultivation of Spirulina platensis OF 25 under growing cane husk concentrations Vinasse (%) Distilled H2O (%) Initial algal biomass (g/l) Final algal biomass (g/l) A Algal biomass (g/i) 75 0,205 3,374 3,169 50 50 0,195 3,162 2,967 75 0,199 1,435 1,236 100 0 0,197 0,974 0,777 The algal biomass was vacuum filtered through 0.45 |j,m Millipore filter paper and subsequently washed with distilled water and dried for 24 hours 5 in an oven at 100 °C. Results as presented by Table 8 represent the average of two photobioreactors for one single experimental condition in the cultivation of Spirulina platensis OF 25 under different cane husk concentrations. The best result in terms of algal biomass as formed after fourteen days of cultivation was reached with diluted vinasse containing respectively 75% and 50% water, but 10 we may also observe that, in other studied conditions, an expressive production of algal biomass also occurred.

Results as obtained have shown that pure or diluted vinasse constitute an excellent substrate for the cultivation of Spirulina platensis OF 25.

Example 4: Influence of CO? for the cultivation of Spirulina platensis OF 15 25 in 50% diluted vinasse Experiments in bench scale have been made in tubular photobioreactors. Cultivations were kept at 30 °C with a twelve-hour photoperiod and 1500 lux (|iE/(m2*s)) illumination.

Experiments were conducted in 14 (fourteen) day batches in 52 20 cm long glass tubular photobioreactors with 8 cm diameter and total volume of two liters (1.8 liters of working volume) as represented by Figure 4. All RECEIVED at IPONZ on 18 January 2012 cultivations were kept under constant shaking with a flow of filtered atmospheric air of 1.0 v/v/m. The addition of a supplementary source of carbon was made by adding C02 to air through a mixer under the concentrations of 5%, 10% and 15% (v/v) as schematically represented by Figure 6.

Cultivations were started with bioactive algal cell concentration of about 0.20 g/l. Cultures were kept throughout the time of culture (fourteen days) with no pH correction or adjustment. Culture volumes were kept constant by means of daily reposition of water as lost by evaporation.

The air/C02 mixture of the outlet of gas mixer was conducted 10 through 8 mm silicone hoses up to tubular photobioreactors, as per the schematic model of Figures 4 and 6. In that study, tubular photobioreactors were filled in just with 50% diluted vinasse since, under conditions of Example 3, considerable algal biomass yielding in volume was obtained, as well as vinasse.

Inoculation and incubation conditions of photobioreactors were identical to Example 3. All experiments have been conducted in three copies and results express the average of said determinations.

From the above disclosed Examples and the graph as shown by Figure 7, it is possible to realize that the addition of C02 exerts a positive effect 20 in the production of biomass from Spirulina platensis OF 25 cultivated in medium based on diluted vinasse (50%) in comparison with the culture receiving just atmospheric air. The higher concentration of algal biomass as obtained was 4.47 g/l after 14 days of culture in a tubular photobioreactor with the mixture (air + 15% C02), while in the culture receiving just air, final 25 concentration of biomass was of 2.98 g/l. When Spirulina platensis OF 25 was cultivated in Zarrouk medium with the addition of 15% C02, the concentration of biomass was 5.094 g/l. We highlight, however, that it is an extremely expensive cultivation medium in comparison with vinasse, which is an undesirable RECEIVED at IPONZ on 18 January 2012 31 industrial residue of which hundreds of billions of liters are produced in Brazil.

We also highlight that the experts in the art will recognize that higher and/or lower values may be obtained when Spirulina platensis OF 25 and/or other genus and/or species of microalgae are cultivated at CO2 levels 5 not tested in these Examples. The same occurs with the algal biomass production medium, i. e. obtained results were for the sample of "Jardest Vinasse" and clearly higher and/or lower results to those presented in the Examples may be reached, since new samples of vinasse originating from different alcohol plants located in different regions, different varieties of 10 sugarcane, different reactor models and cultivation scales, for laboratorial production, brench, pilot or industrial, are used.

RECEIVED at IPONZ on 18 January 2012 32

Claims (19)

Claims

1. PROCESS FOR THE PRODUCTION OF BIOMASS AND 5 PROTEINS FROM MICROALGAE, intended for human or animal feed or for other uses, wherein said process comprises the following steps: - providing a culture medium including vinasse and carbon dioxide for culturing said microalgae; - addition of the said microalgae to said culture medium; and 10 - collecting a fraction of microalgae biomass, in which the vinasse is, optionally, diluted and the carbon dioxide is in a concentration of between 0.1 to 100%.

2. PROCESS FOR THE PRODUCTION OF BIOMASS AND PROTEINS FROM MICROALGAE, according to claim 1, characterized by the 15 fact that vinasse and carbon dioxide from fermentation vats are generated as rejects from the hydrated and anhydrous alcohol industry.

3. PROCESS FOR THE PRODUCTION OF BIOMASS AND PROTEINS FROM MICROALGAE, according to claim 2, characterized by the fact that the hydrated and anhydrous alcohol industry uses sugarcane and its 20 derivatives as a source of raw material.

4. PROCESS FOR THE PRODUCTION OF BIOMASS AND PROTEINS FROM MICROALGAE, according to claim 1, characterized by the fact that said microalgae are one or more seclected from the group comprising: Spirulina sp, including Spirulina platensis, Spirulina maxima, Spirulina major, 25 Spirulina subsalsa, Spirulina geitleri, Spirulina subtilissima, Spirulina labyrinthiforms; Skeletonema sp; Chaetoceros sp; Scenedesmus sp, including Scenedesmus bijugatus, Scenedesmus incrassatulus, Scenedesmus ocultus, Scenedesmus quadricauda, Scenedesmus dimorphus; Anacystis sp, including RECEIVED at IPONZ on 18 January 2012 33 Anacystis nidulans, Anacystis cyanea, Anacystis thermalis; Porphyridium cruentum; Crypthecodinium cognii; Euglena sp, including Eugiena gracilis; Crypthecodinium cohnir, Haematococcus pluvialis; Anabaena sp, including Anabaena variabilis, Anabaena cylindrica, Anabaena hassali, Anabaena 5 planctonica; Dunaliella sp, including Dunaliella salina, Dunaliella bardawil, Dunaliella tertioleta; Chlamydomonas sp, including Chlamydomonas reinhardtii; Chlorella sp, including Chlorella vulgaris, Chlorella kessleri, Chlorella pyrenoidosa, Chlorella mannophila, Chlorella protothecoides, Chlorella salina, Chlorella homosphaera, Chlorella stigmatophora, Chlorella luteoviridis, 10 Chlorella regularis, Chlorella ellipsoidea, Chlorella variegata, Chlorella sorokiniana, Chlorella emersonir, Trichodesmium, Microcoleus; Ankistrodesmus sp, including Ankistrodesmus densus, Ankistrodesmus braunii, Ankistrodesmus falcatus, Ankistrodesmus fusiformis, Ankistrodesmus gracilis; Isochrysis galbana; Tetraselmis sp, including Tetraselmis tetrathele, Tetraselmis suecia\ 15 Oscillatoria sp, including Oscillatoria limnetica, Oscillatoria curviceps, Oscillatoria splendida)', Nostoc muscorum and Botrycoccus braunii.

5. PROCESS FOR THE PRODUCTION OF BIOMASS AND PROTEINS FROM MICROALGAE, according to claim 4, characterized by the fact that said microalgae is Spirulina platensis. 20

6. PROCESS FOR THE PRODUCTION OF BIOMASS AND PROTEINS FROM MICROALGAE, characterized by comprising the following basic steps: (i) adequation of the vinasse by adding water and alkali until a pH value of about 6.0-11.0 is reached; 25 (ii) delivery of pre-adjusted vinasse to an inoculation tank; (iii) addition of microalgae to the inoculation tank until a concentration of about 0.2 g/l of initial biomass is reached in the cultivation medium based on vinasse; (iv) delivery of inoculated vinasse to an algal biomass cultivation tank; RECEIVED at IPONZ on 18 January 2012 (v) injection of air containing 0.1-100% highly pure carbon dioxide to the algal biomass cultivation tank; (vi) keeping algal biomass at an average temperature between 25 and 35 °C under natural light intensity; 5 (vii) delivery of algal biomass to a separation unit, where a fraction of algal biomass and a fraction of water supernatant will be generated; (viii) recycle of the fraction of water supernatant to the inoculation tank.

7. PROCESS FOR THE PRODUCTION OF BIOMASS AND PROTEINS FROM MICROALGAE, according to claim 6, characterized by the 10 fact that, in step (v), the percentage of carbon dioxide in the air flow varies between 5 and 15%.

8. PROCESS FOR THE PRODUCTION OF BIOMASS AND PROTEINS FROM MICROALGAE, according to claim 5, characterized by the fact that the percentage of carbon dioxide in the flow of air is about 15%. 15 9. PROCESS FOR THE PRODUCTION OF BIOMASS AND

PROTEINS FROM MICROALGAE, according to claim 6, characterized by the fact that the algal biomass is kept for a period of about 14 days in the cultivation tank.

10. PROCESS FOR THE PRODUCTION OF BIOMASS AND 20 PROTEINS FROM MICROALGAE, according to claim 6, characterized by the fact that vinasse and carbon dioxide originate from the industry of hydrated and/or anhydrous alcohol using sugarcane and its derivatives as a source of raw material.

11. PROCESS FOR THE PRODUCTION OF BIOMASS AND 25 PROTEINS FROM MICROALGAE, according to claim 6, characterized by the fact that said microalgae is Spirulina platensis.

12. PROCESS FOR THE PRODUCTION OF BIOMASS AND PROTEINS FROM MICROALGAE, according to claim 6, characterized by the RECEIVED at IPONZ on 18 January 2012 fact that the addition of water at step (i) is made in proportion of about 50% (v/v).

13. PROCESS FOR THE PRODUCTION OF BIOMASS AND PROTEINS FROM MICROALGAE, according to claim 6, characterized by the 5 fact that, after each cycle is completed and after the removal of the algal biomass, supernatants are again inoculated with active biomass from microalga so that the initial concentration in the tanks of algal biomass remains around 0.2 g/l in the cultivation medium based on vinasse.

14. PROCESS FOR THE PRODUCTION OF BIOMASS AND 10 PROTEINS FROM MICROALGAE, according to claim 6, characterized by the fact that vinasse are kept under pH of about 7.0-11.0 and diluted with a quantity of water about 5 to 95% (v/v) during each cycle.

15. PROCESS FOR THE PRODUCTION OF BIOMASS AND PROTEINS FROM MICROALGAE, according to claim 6, characterized by the 15 fact that the intensity of light in the production tanks of algal biomass is about 1,500 lux 12/12 h day.

16. PROCESS FOR THE PRODUCTION OF BIOMASS AND PROTEINS FROM MICROALGAE, according to any one of claims 6 to 15, characterized by comprising the following basic steps: 20 (i) adequation of the vinasse by adding water and alkali until a pH value of about 6.0-11.0 is reached; (ii) delivery of pre-adjusted vinasse to an inoculation tank; (iii) addition of microalgae to the inoculation tank until a concentration of about 0.2 g/l of initial biomass is reached in the cultivation medium based on vinasse; 25 (iv) delivery of inoculated vinasse to an algal biomass cultivation tank; (v) injection of air containing 0.1-100% highly pure carbon dioxide to the algal biomass cultivation tank; (vi) keeping algal biomass at an average temperature between 25 and 35 °C RECEIVED at IPONZ on 18 January 2012 under natural light intensity; (vii) delivery of algal biomass to a separation unit, where a fraction of algal biomass and a fraction of water supernatant will be generated; (viii) recycle of the fraction of water supernatant to the inoculation tank; and 5 (ix) repetition of steps (iii) to (viii) until a Chemical Oxygen Demand (COD) value of about 17 mg/l O2 and lower Biochemical Oxygen Demand (BOD) than about 5 mg/l O2 in the fraction of water supernatant as produced in step (vii) is reached.

17. PROCESS FOR THE PRODUCTION OF BIOMASS AND 10 PROTEINS FROM MICROALGAE, according to claim 16, characterized by the fact that it comprises three or more processing cycles of one single cane husk load.

18. A process for the production of biomass and proteins from microalgae as claimed in claim 1, substantially as herein described with 15 reference to the examples and/or the figures of the accompanying drawings.

19. A process for the production of biomass and proteins from microalgae as claimed in claim 6, substantially as herein described with reference to the examples and/or the figures of the accompanying drawings.