WO2008083453A1

WO2008083453A1 - Process to produce biomass and proteins by microalgae

Info

Publication number: WO2008083453A1
Application number: PCT/BR2008/000004
Authority: WO
Inventors: Carlos Ricardo Soccol; Cristina Elisabete Knoerr; Jorge Alberto Vieira Costa
Original assignee: Ouro Fino Participacoes e Empreendimentos Ltda
Current assignee: Ouro Fino Participacoes e Empreendimentos Ltda
Priority date: 2007-01-08
Filing date: 2008-01-08
Publication date: 2008-07-17
Anticipated expiration: 2009-07-08
Also published as: PA8796301A1; MX2009007354A; AU2008204737A1; NI200900136A; CR10905A; ECSP099498A; NZ578233A; ZA200904706B

Abstract

The present invention refers to a process to produce biomass and proteins from microalgae, advantageously using as a source of development of said microalgae rejects from the alcohol industry, notably sugar cane husks and carbon dioxide originating from fermentation vats. The process according to the present invention comprises basic steps of preparation of cane husks, adaptation and preparation of inoculum with the microalga Spirulina platensis OF 25, cultivation of the microalga under controlled conditions and use of CO2, separation of algal biomass and optional recirculation of the water phase in the process until acceptable DQO and DBO levels by environmental regulations are reached.

Description

"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 microalgae rejects from the sugar and alcohol industries, notably sugar cane husks and carbon dioxide originating from fermentation vats.

The process of the present invention also contributes as a solution to reduce the emission of pollutant loads to water courses, soil desertification by 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 cane husks, as well as the emission of pollutant loads to the atmosphere, bearing in mind the reuse of carbon dioxide (CO₂) 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 sugar cane juice and molasses into ethanol. This is a biological process which may be represented by means of the stoichiometric equation of Gay Lussac, as reproduced below: C₁₂H₂₂O₁₁+ H₂O -» C₆H₁₂O₆ + C₆H₁₂O₆ (a)

C₆H₁₂O₆ ^■* 2CH₃CH₂OH + 2CO₂ + 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.

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 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. Cane husks are taken at the base of said 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 cane husks depends on various factors, such as the composition of raw material, characteristics and mode of operation of the distillation columns. Table 1 presents qualitative and quantitative characteristics of cane husks 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 Cane Husks (average of 64 samples from 28 plants in the State of Sao Paulo - Source: ELIA NETO, A. & NAKAHODO, T. (in Relatόrio da Copersucar, Project n⁰ 95000278, Piracicaba, 1995, 26 p).

Cane husks 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 cane husks were launched to surface springs, mainly 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 cane husks 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 cane husks into water courses causes bad odor and contributes to worsening various endemic parasitary diseases.

Estimates show that the Brazilian production of cane husks for the 2006/2007 crop has been of about 190 billion liters. Currently, the fate of cane husks has been their pulverization into soil, particularly in sugar cane plantations and/or their stocking in depuration lagoons. However, continued spraying of cane husks 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 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 to the handling of said cane husks since 1978, forbidding the discharge of cane husks to surface waters. One of said rules regulating the use of cane husks establishes that cane husks 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 sugar cane in the year at issue, i. e. cane husks equivalent to 185 kg/ha of K₂O. With such regulations in force, various sugar cane culture areas suffer restrictions, and the sector already develops projects aiming to transport cane husks to longer distances than used today.

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

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 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 condiςδes 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 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.

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 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:

- 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, 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 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).

Macron utrients 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 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 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, 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 and Agriculture Organization), a United Nations organism.

Specifically, microalga Spirulina is a filamentous cyanobacteria with 1 to 12 μm 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, 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) 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 SuI (DURANTE, A. J., REICHERT, C. C, DALCANTON₁ F., MORAIS, M. lsolamento e cultivo de uma cepa de Spirulina nativa da Lagoa 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 β- 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. 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% above any natural food (FALQUET, J. Th e 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 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 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 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 glucidiques elaborees par les Spirulines. Ann. Nutr. Aliment, 29, n⁰ 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 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 (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 β- carotene, represents about 80% of carothenoids as present in Spirulina. In 1 kg of dry biomass of Spirulina, β-carotene concentration is about 700 and 1700 mg. Biomass of Spirulina also contains tocopherols with antioxidizing power, at 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 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, the Applicant concluded that the use of cane husks originating from distillation of must in alcohol plants, as well as CO₂ 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.

SUMMARY OF THE INVENTION Microalgae, when cultivated in appropriate means, may duplicate their biomass daily. This characteristic, added to simple cultivation skills, makes microalgae the main object of interest of the present invention.

The present invention has therefore the specific object to provide a process to produce biomass and proteins from microalgae, advantageously using cane husks and carbon dioxide originating from fermenters, as generated by the alcohol industry, as a medium or culture substrate.

More specifically, the present invention has the object to provide a process to produce microalga biomass from cane husks and carbon dioxide, generated as rejects by the alcohol industry, using sugar cane and its derivatives. Even more specifically, the present invention has the object to provide a microalga production process from cane husks and carbon dioxide, generated as rejects in the alcohol industry, by using sugar cane and its derivatives, being said microalgae selected from one or more genus (species) of the group comprising Spirulina (sp, platensis, maxima, major, subsalsa, geitleή, subtilissima, labyrinthiforms); Skeletonema sp; Chaetoceros sp; Scenedesmus sp (bijugatus, incrassatulus, ocultus, quadricauda, dimorphus); Anacystis sp (nidulans, cyanea, thermalis); Porphyήdium omentum; Crypthecodinium cognii; Euglena sp (gracilis); Crypthecodinium cohnii; Haematococcus pluvialis; Anabaena sp (variabilis, cylindrica, hassali, planctonica); Dunaliella sp (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); lsochrysis galbana (Parke); Tetraselmis sp. (tetrathele, suecia); Oscillatoria sp (limnetica, curviceps, splendida); Nostoc muscorum and Botrycoccus braunii,. However, the present invention may comprise other genus (species) besides the ones as reported herewith.

A specially contemplated object by the present invention is the use of microalga Spirulina platensis OF 25 in a process to produce biomass and proteins from cane husks and carbon dioxide, generated as rejects in the alcohol/ethanol industry. Therefore, in summary, the present invention has the objects to recycle and use cane husks as a medium of culture for the production of alga biomass rich in proteins and other products with commercial interest, notably Spirulina biomass, as well as to make use of the effect of CO₂ originating from fermentation vats in the growth of said microalga and to promote the reduction of DQO and DBO levels from cane husks as discharged in the fermenting process.

DESCRIPTION OF FIGURES The attached figures will serve to provide for better understanding 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 production process for hydrated alcohol, notably ethanol, from sugar cane derivatives.

Figure 2 shows a flow diagram showing the main steps of the cultivation process for Spirulina platensis OF 25 in cane husks 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.

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 present invention.

Figure 6 shows an arrangement of photobioreactors on the oven shelves with photoperiod during experiments to test cane husks under different air:CO₂ ratios as per the present invention.

Figure 7 is a graph showing the evolution of growth in terms of biomass of Spirulina platensis OF 25 as produced in a diluted cane husk culture (50%) under different levels of CO₂ and its comparison with Zarrouk medium. DETAILS OF THE INVENTION

Studies carried out by the Applicant have shown that cane husks contain practically all the mineral elements, as well as numerous organic 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 cane husks and carbon dioxide (CO₂) produced as residues in the process to ferment sugar cane juice, molasses or their mixtures to produce alcohol, notably hydrated and anhydrous ethanol.

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

TABLE 2 - COMPOSITION OF JARDEST CANE HUSKS

The process of the present invention comprises the production in large scale of algal biomass by using CO₂ as generated during alcohol fermentation, as well as the cane husks 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 sugar cane and/or another kind of carbohydrate. Cane husks 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 trucks up to the microalga production plant.

In the microalga production plant, as shown by Figure 2, cane husks 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. Studies as made by the Applicant have shown that cane husks 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 cane husks 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 cane husks may be used to cultivate algae for the production of proteic 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, cane husks may be filtered and/or clarified by using activated charcoal, sand bed with different granulometries or flocculating agents, depending on the concentration of solids in suspension in the different types of cane husks. We have also noticed that pasteurization and/or sterilization of cane husks does not provide for significant difference in biomass production, and therefore a unit operation is 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, 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 cane husks, as disclosed by the present invention, may be made according to the flow diagram 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 make light penetration difficult, thus reducing the photosynthesis process. Tanks as used to produce algae from cane husks 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 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 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 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%. CO₂ as produced during alcohol fermentation in high quantities may be compressed and/or purified and stocked in pressurized reservoirs before being injected into alga production tanks. An example of CO₂ 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. 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.

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 rotating drum. Figure 3 shows a flow diagram of the process to produce proteins from microalgae from cane husks and carbon dioxide of the present invention, comprising the following basic steps:

The process to produce biomass and proteins from microalgae according to the present inventios comprises the following basic steps: (i) adequation of the cane husks by adding water and alkali until a pH value of about 6.0-11.0 is reached;

(ii) delivery of pre-adjusted cane husks 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 cane husks;

(iv) delivery of inoculated cane husks 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 ⁰C 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 O₂ 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 one single load of cane husks.

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 inorganic material as present in cane husks. 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 cane husks, microalga and other factors.

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 CO₂ will be dissolved into the culture medium. Recirculated water supernatants may also be mixed with pure cane husks 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% CO₂, which is packed in cylinders at 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 is of about 0.2 g/l biomass in the cultivation medium based on cane husks. Similarly, we prefer that light intensity in algal biomass tanks is of about 1,500

Iux 12h/12h day.

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

Furthermore, the biological treatment of cane husks 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 becomes more significant after three or more processing cycles from one single load of cane husks 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 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 cane husks 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 cane husks, 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 μm filter paper, subsequently washed with distilled water and dried for 24 hours in an oven at 100 ⁰C. Data is compiled at Table 3 below. TABLE 3 - ALGAL BIOMASS PRODUCED IN THE DIFFERENT RE-USE CYCLES OF CANE

HUSK SUPERNATANTS

We can notice on the above table that, throughout the cycles, micronutrients and macronutrients are consumed. W e 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, as shown by Table 4 below. DBO and DQO analysis have been taken according to Standard Methods for the Examination of Water and Wastewater, 20 ed., 1998, Hach Company and WTW. TABLE 4 - REDUCTION OF DQO AND DBO LEVELS ALONG RECYCLING CYCLES OF SUPERNATANTS FROM CANE HUSKS

The present invention will be additionally disclosed by means of the Examples that follow which, not limiting their scope, represent a preferred embodiment.

MlCROALGA SPIRULINA PLATENSlS OF 25 Spirulina platensis OF 25, selected from the Bank of Cultures of the company Ouro Fino Saύde 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 cane husks and carbon dioxide as generated by alcohol plants. Spirulina platensis OF 25 presents high growth under temperature ranges between 25 and 35 ⁰C under slightly alkaline pH. These physiological characteristics of Spirulina platensis OF 25 provide for large potential for their culture in cane husks, 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 Brazilian regions where sugar cane 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 micronutrϊents (Vonshak, 1997). Although Spirulina may grow photoautotrophically, the collection Of CO₂ from the air depends on the pH of the cultivation medium. The higher the pH of the medium, more easily CO₂ from the atmosphere migrates to inside it and is converted into CO3^2". 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 to assimilate carbon in the form of CO₃ ^2". Therefore, in cultivations of the microalga Spirulina, an external source of carbon is usually required in the form of HCO₃ ^", a species participating in the equilibrium:

2-

CO₂ ±> HCO₃ t; CO₃" This is the source of carbon most probably assimilated by 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 (ZARROUK, C. Contribution a I'etude d'une cyanophycee: Influence de divers facterurs physiques et chimiques sur Ia 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 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.

The mother line of Spirulina platensis OF 25 was cultivated in Zarrouk medium and preserved in a freezer at a temperature of -80 ⁰C.

TABLE 5 - COMPONENTS OF THE ZARROUK MEDIUM FOR THE CULTIVATION OF SPIRULINA PLA TENSIS OF 25

TABLE 5A - COMPOSITION SOLUTION A5

TABLE 5B- COMPOSITION SOLUTION B6

EXAMPLE 1 : ADAPTATION OF MICROALGAE TO CANE HUSKS Previous adaptation studies for Spirulina platensis OF 25 under growing mixtures (5, 25, 50, 75, 100%) of cane husks to the Zarrouk culture medium were evaluated. This same procedure may be applied if justifiable for other genus and/or species of microalgae when cultivated in cane husks, 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 containing 5% cane husks + 95% Zarrouk medium was used to inoculate the medium containing 25% cane husks and then subsequently until the final culture in pure cane husks 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 same objects. Cultures were incubated for a 14-day period, during which the following preferential standards were kept as constant: temperature 30 ⁰C (± 2 ⁰C), 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 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 μm pores, followed by washing with distilled water and dried for 24 hours in an oven at 100 ⁰C. The results as contained in Tables 6 and 7 below represent the average of three determinations for each one of the studied conditions. TABLE 6 - ADAPTATION OF SPIRULINA PLATENSIS OF 25 UNDER DIFFERENT CANE

HUSK CONCENTRATIONS

This process to previously adapt microalgae to cane husks allows obtaining more expressive results in terms of daily final yielding of biomass in comparison with a process in which microalgae do not pass through this previous adaptation (Table 7). TABLE 7 - EFFECT OF ADAPTATION OF SPIRULINA PLATENSIS OF 25 IN CANE HUSKS

DILUTED OVER THE PRODUCTION OF ALGAL BIOMASS

EXAMPLE 2: PREPARATION OF INOCULUM

This process was made in 500 ml Erlenmeyer flasks containing 90 ml of pure cane husks 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 + cane husks (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 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 CANE HUSKS AND DILUTED IN WATER

Spirulina platensis OF 25 was cultivated in pure cane husks and 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 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 cane husks and non sterilized diluted cane husks and inoculated with an active culture of Spirulina platensis OF 25 as previously adapted in cane husks, following a method as disclosed by Example 2, 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 cane husks, as shown by 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 ⁰C (± 2 ⁰C), 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 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 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 schematically represented by Figure 6.

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

TABLE 8 - CULTIVATION OF SPIRULINA PLATENSIS OF 25 UNDER GROWING CANE

HUSK CONCENTRATIONS

The algal biomass was vacuum filtered through 0.45 μm Millipore filter paper and subsequently washed with distilled water and dried for 24 hours in an oven at 100 ⁰C. 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 cane husks containing respectively 75% and 50% water, but 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 cane husks constitute an excellent substrate for the cultivation of Spirulina platensis OF 25. EXAMPLE 4: INFLUENCE OF CO? FOR THE CULTIVATION OF SPIRULINA PLATENSIS OF 25 IN 50% DILUTED CANE HUSKS

Experiments in bench scale have been made in tubular photobioreactors. Cultivations were kept at 30 ⁰C with a twelve-hour photoperiod and 1500 lux (μE/(m²*s)) illumination.

Experiments were conducted in 14 (fourteen) day batches in 52 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 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 CO₂ 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/CO₂ mixture of the outlet of gas mixer was conducted 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 cane husks since, under conditions of Example 3, considerable algal biomass yielding in volume was obtained, as well as cane husks.

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 CO₂ exerts a positive effect in the production of biomass from Spirulina platensis OF 25 cultivated in medium based on diluted cane husks (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% CO₂), while in the culture receiving just air, final concentration of biomass was of 2.98 g/l. When Spirulina platensis OF 25 was cultivated in Zarrouk medium with the addition of 15% CO₂, the concentration of biomass was 5.094 g/l. We highlight, however, that it is an extremely expensive cultivation medium in comparison with cane husks, which is an undesirable 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 not tested in these Examples. The same occurs with the algal biomass production medium, i. e. obtained results were for the sample of "Jardest Cane Husks" and clearly higher and/or lower results to those presented in the Examples may be reached, since new samples of cane husks originating from different alcohol plants located in different regions, different varieties of sugar cane, different reactor models and cultivation scales, for laboratorial production, brench, pilot or industrial, are used.

Claims

1. PROCESS FOR THE PRODUCTION OF BIOMASS AND PROTEINS FROM MICROALGAE, entitled to human or animal feed, as well as for other uses, characterized by having as the culture medium of said microalgae cane husks and carbon dioxide.

2. PROCESS FOR THE PRODUCTION OF BIOMASS AND PROTEINS FROM MICROALGAE, according to claim 1, characterized by the fact that cane husks 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 sugar cane and its 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 selected from one or more of genus (species) of the group comprising Spirulina (sp, platensis, maxima, major, subsalsa, geitleri, subtilissima, labyrinthiforms); Skeletonema sp; Chaetoceros sp; Scenedesmus sp φijugatus, incrassatulus, ocultus, quadricauda, dimorphus); Anacystis sp (nidulans, cyanea, thermalis); Porphyridium omentum; Crypthecodinium cognii; Euglena sp (gracilis); Crypthecodinium cohnii; Haematococcus pluvialis; Anabaena sp (variabilis, cylindrica, hassali, planctonica); Dunaliella sp (salina,bardawil, tertioleta); Chlamydomonas sp (reinhardtii); Chlorella sp (vulgaris, kessleri, pyrenoidosa, mannophila, protothecoides, salina, homosphaera, stigmatophora, luteoviridis, regularis, ellipsoidea, variegata, sorokiniana, emerson /V); Trichodesmium, Microcoleus; Ankistro desmus sp (densus, braunii, falcatus, fusiformis, gracilis); lsochrysis galbana (Parke); Tetraselmis sp. (tetrathele, suecia); Oscillatoria sp (limnetica, curviceps, splendida); Nostoc muscorum and Botrycoccus braunii.

5. PROCESS FOR THE PRODUCTION OF BIOMASS AND

PROTEINS FROM M I C ROALGAE, according to claim 4, characterized by the fact that microalga is Spirulina platensis OF 25.

6. PROCESS FOR THE PRODUCTION OF BIOMASS AND

PROTEINS FROM MICROALGAE, characterized by comprising the following basic steps:

(i) adequation of the cane husks by adding water and alkali until a pH value of about 6.0-11.0 is reached; (ii) delivery of pre-adjusted cane husks to an inoculation tank;

(iv) delivery of inoculated cane husks to an algal biomass cultivation tank; (v) injection of air containing 0.1-100% highly pure carbon dioxide to the algal biomass cultivation tank;

(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 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%.

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

PROTEINS FROM MICROALGAE, according to claim 6, characterized by the fact that cane husks and carbon dioxide originate from the industry of hydrated and/or anhydrous alcohol using sugar cane and its derivatives as a source of raw material.

11. PROCESS FOR THE PRODUCTION OF BIOMASS AND

PROTEINS FROM MICROALGAE, according to claim 6, characterized by the fact that microalga is Spirulina platensis OF 25.

12. PROCESS FOR THE PRODUCTION OF BIOMASS AND PROTEINS FROM MICROALGAE, according to claim 6, characterized by the 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 fact that, after each cycle is completed and after the removal of the algal biomass, supematants 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 cane husks.

14. PROCESS FOR THE PRODUCTION OF BIOMASS AND PROTEINS FROM MICROALGAE, according to claim 6, characterized by the fact that cane husks 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 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 claim one of claims 6 to 15, characterized by comprising the following basic steps:

(i) adequation of the cane husks by adding water and alkali until a pH value of about 6.0-11.0 is reached;

(ii) delivery of pre-adjusted cane husks 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 cane husks;

(iv) delivery of inoculated cane husks 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 ⁰C under natural light intensity;

(viii) recycle of the fraction of water supernatant to the inoculation tank; and (ix) repetition of steps (iii) to (viii) until a DQO value of about 17 mg/l O2 and lower DBO 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 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.