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Title:
PREPARATION OF ETHANOL BY SACCHAROMYCES CEREVISIAE IMMOBILIZED ON CHRYSOTILE IN AN AIRLIFT REACTOR
Document Type and Number:
WIPO Patent Application WO/2009/012553
Kind Code:
A1
Abstract:
It is described an ethanol obtainment process by Saccharomyces cerevisiae immobilized on chrysotile in an inner circulation pneumatic bioreactor from a culture medium with carbon source from 160 to 300 g/L, chrysotile from 0.5 to 5% w/v and anti-foaming, the reactor operating between 30°C and 35°C, while its content is homogenized with the aid of a gas injection with a flow from 0.3 to 0.9 vvm, with inoculation between 5 and 30 g/L in dry base of Saccharomyces cerevisiae cells, where cell immobilization from the culture medium on the chrysotile support occurs concomitantly to the ethanol production process. The fermentative period extends between 7 and 14 hours, after which the reactor is exhausted, keeping the fibers containing immobilized cells in the interior of the reactor. The wine without yeast, reaction product, is recovered with an ethanol final concentration higher than would be possible through processes with free cells. The process is made in batchwise or in repeated batchwise.

Inventors:
GALVÃO, Célia Maria Araújo (Rua treze de Maio, 1270 BL.01, apt.401,Centr, São Carlos -647 São Paulo, 13560, BR)
ALMEIDA, Paulo Ignácio Fonseca de (Rua Nestor de Campos nº287, São Carlos, -020 São Paulo, 13562, BR)
BADINO JUNIOR, Alberto Colli (Rua Célio Barbosa da Silva, 212São Carlos, -060 São Paulo, 13564, BR)
FILHO, Rubens Rela (Av. dos Limoeiros, S/nº casa L-16,Vila Residencial Sam, Minaçu 76.450-000 Goiás, BR)
Application Number:
BR2008/000219
Publication Date:
January 29, 2009
Filing Date:
July 25, 2008
Export Citation:
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Assignee:
FUNDAÇÃO UNIVERSIDADE FEDERAL DE SÃO CARLOS - UFSCAR (Rodovia Washington Luís, km 235São Carlos, -905 São Paulo, 13565, BR)
SAMA S.A - MINERAÇÕES ASSOCIADAS (Mina da Cana Brava, S/nºCx. Postal 0, Minaçu -000 Goiás, 76450, BR)
GALVÃO, Célia Maria Araújo (Rua treze de Maio, 1270 BL.01, apt.401,Centr, São Carlos -647 São Paulo, 13560, BR)
ALMEIDA, Paulo Ignácio Fonseca de (Rua Nestor de Campos nº287, São Carlos, -020 São Paulo, 13562, BR)
BADINO JUNIOR, Alberto Colli (Rua Célio Barbosa da Silva, 212São Carlos, -060 São Paulo, 13564, BR)
FILHO, Rubens Rela (Av. dos Limoeiros, S/nº casa L-16,Vila Residencial Sam, Minaçu 76.450-000 Goiás, BR)
International Classes:
C12P7/06
Other References:
VICENTE ET AL: "Flocculation bioreactors - engineering based problems" 28TH CONFERENCE SSCHE, TATRANSKÉ MATLIARE (SK), 21-25 MAY, 2001, XP002502863 Retrieved from the Internet: URL:repositorium.sdum.uminho.pt/bitstream/ 1822/3676/1/SSChe2001-L28%5B2%5D.pdf> [retrieved on 2008-11-06]
RUSSELL ET AL: "The influence of vessel height and top-section size on the hydrodynamic characteristics of airlift fermentors" BIOTECHNOLOGY AND BIOENGINEERING, vol. 43, 1994, pages 69-76, XP002503278
MERCHUK ET AL: "Liquid flow and mixing in concentric tube air-lift reactors" JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY, vol. 66, 1996, pages 174-182, XP000625193
WENDHAUSEN ET AL: "Continuous fermentation of sugar cane syrup using immobilized yeast cells" JOURNAL OF BIOSCIENCE AND BIOENGINEERING, vol. 91, 2001, pages 48-52, XP002502864 cited in the application
SÁNCHEZ ET AL: "Trends in biotechnological production of fuel ethanol from different feedstocks" BIORESOURCE TECHNOLOGY, vol. 99, 26 December 2007 (2007-12-26), pages 5270-5295, XP022647362
Attorney, Agent or Firm:
DI BLASI, Gabriel et al. (Rua do Ouvidor, 121 11º anda, Centro -030 Rio de Janeiro, 20040, BR)
Download PDF:
Claims:

CLAIMS

1. Ethanol obtainment process by Saccharomyces cerevisiae immobilized on chrysotile in an inner circulation pneumatic bioreactor, wherein it comprises the following steps: a) In a pneumatic bioreactor with a hollow inner cylinder for water recirculation, provide a culture medium with glucose or sucrose as carbon sources, with concentrations in a range of 160 to 300 g/L; b) Add to the said medium a chrysotile amount that corresponds to concentrations between 0.5 and 5% w/v and anti-foaming; c) Stabilize the culture medium in a temperature between 30°C and 35°C with the aid of water circulating through the hollow inner cylinder of the said bioreactor; d) Promote homogenization of the reaction medium, composed by culture medium and chrysotile fibers, through gas injection with an specific flow of 0.3; 0.6 or 0.9 vvm; e) Inoculate the said bioreactor with an initial Saccharomyces cerevisiae cell concentration in the fermentation broth between 5 and 30 g/L in dry basis, close the bioreactor and initiate the fermentative process; f) Keep the said fermentative process for a period from 7 to 14 hours, with the pH range between 4.3 and 5.5, where cell immobilization from the culture medium on a chrysotile support occurs concomitantly with the ethanol production process, recovering the dragged ethanol with the aid of a jacketed condenser located on the cap of the said inner circulation pneumatic bioreactor; g) Finalize the fermentative process when any sugar residue is no more detected in the culture broth; h) Exhaust the bioreactor keeping fibers containing

immobilized cells and recover the wine without yeast with an ethanol final concentration higher than would be possible to obtain through processes with free cells; and i) Analyze the ethanol content in the wine without yeast.

2. Process according to claim 1, wherein it operates alternatively in repeated batchwise mode.

3. Process according to claim 1, wherein the culture medium is constituted of 3 g/L urea and 0.5 g/L MgSO 4 .7H 2 O. 4. Process according to claim 1, wherein the culture medium is alternatively constituted of 5.32 g/L urea, 6.8 g/L yeast extract, 1.4 g/L MgSO 4 .7H 2 O and 5.6 g/L KH 2 PO 4 .

5. Process according to claim 1, wherein the anti-foaming amount is 2, 4 or 6 drops as used in a 2 L, 5 L or 10 L bioreactor, respectively.

6. Process according to claim 1, wherein the gas used to homogenize the reactor content is CO 2 or N 2 .

7. Process according to claim 1, wherein the gas used to homogenize the reactor content is CO 2 . 8. Process according to claim 1, wherein it prepares chrysotile fibers by abundant wash with water, followed by immediate cell immobilization.

9. Process according to claim 1, wherein the chrysotile fibers are alternatively activated by sonication before immobilization.

10. Process, according to Claim 1, wherein the chrysotile fibers are alternatively used without the washing step and without activation before being immobilized.

Description:

PREPARATION OF ETHANOL BY SACCHAROMYCES CEREVISIAE IMMOBILI ZED ON CHRYSOTILE IN AN AIRLIFT REACTOR

FIELD OF THE INVENTION

The present invention belongs to the field of fermentation ethanol production processes in the presence of microorganisms, more specifically, to a fermentation ethanol production process in the presence of Saccharomyces cerevisiae immobilized on chrysotile in an inner circulation pneumatic bioreactor. BACKGROUND OF THE INVENTION

The alcohol industry has been developed in Europe since the middle of the 19 th century and only in the last quarter of the last century alcohol production began in Brazil. In 1931, the first anhydrous alcohol distillery was installed and the Federal Government established the obligatory addition of 5% ethanol to gas. In 1974, when the great international oil crisis broke out, ethanol Brazilian production initiated a new cycle aiming alternatives to the liquid fuel problem. With "Proalcool" launching in 1975, the alcohol nation production significantly experienced a boom, once the produced volume should, by now, attend a car fleet of nearly four millions automobiles, that was put in motion with pure alcohol, and also accomplish the obligation of mixture this fuel with all gas used in the country according to Amorim et al., "Produςao de Etanol", Biotecnologia Industrial Processos Fermentativos e Enzimaticos, Sao Paulo, vol. 3, pp. 1 - 43, 2001. Nowadays, the demand for this "green fuel" obtained from a renewable raw material is growing greatly by many

factors, namely:

- Increasing of anhydrous alcohol proportion in the alcohol-gas mixture, these days defined as 23%;

Increasing reality of "flex-fuel" or "bi- combustible" car, that in August 2006 has reached the mark of two million produced and marketed cars, although the forecast is that from 2007 on, 100% of cars sold in Brazil will be "flex-fuel" (Mendonςa de Barros e colaboradores, 2005; http: //www. ambientebrasil . com.br/noticias/index.php37action =ler&id=27368, in October 2006) .

- Beginning of biodiesel commercial production, that incorporates in its main obtainment process (transesterification) a chemical reaction of vegetable oils or animal fats with ethanol, in a major sense (http: //www. biodiesel . gov.br/) ; and

- Appearance of new big consumer markets as Japan and China.

There is estimation that in the 2006/2007 crop, the 17 billion ethanol liters initially forecasted will be overcome in function of new crops, crop anticipation and new cane processing plants installation (http: //www. cnpm. embrapa.br/conjuntura/0603 Sucroalcooleiro .pdf, in March/2006) . The ethanol production happens, in a global sense, by two main pathways; the synthetic and the fermentative. The synthetic pathway is characterized by ethanol obtainment from non-saturated hydrocarbons (ethene and ethine) , oil and coal gases, but with restricted importance to countries with big oil reserves and advanced petrochemical industries. The fermentative pathway is the model used in

Brazil for ethanol obtainment as a consequence of the great quantity of raw material, commonly sugar cane, that exists in the whole country and its geographical distribution allows its production in almost the whole territory and during the whole year, as the already cited reference Amorim et al. "Produgao de Etanol", Biotecnologia Industrial Processus Fermentativos e Enzimaticos, Sao Paulo, vol. 3, pp. 1 a 43, 2001.

Many ethanol production processes through the fermentative pathway are described in literature, but the most popular of them is still the batchwise process with cells reutilization (Melle-Boinot ) . By these days, a significant part of the ethanol producer industries has been substituting the batchwise process by multi-step continual cultivations with cell centrifugation, treatment and recycle for the first step, a process that shows advantages as high substrate conversion and productivity in ethanol. See the article by Wheals, A. et al., Trends in Biotechnology, London, vol. 17, pp. 482 - 487, 1999. A common step to both referred processes and that requires great attention is cell separation at the end of the process. This operation is commonly made by centrifugation. Because of it is an onerous operation, representing about 25% of the overall cost of a distillery, it should, if possible, be avoided, as described by Stroppa, C. T. et al., "Consumo de acύcar por bacterias contaminantes da Fermentaςao Alcoόlica Associados ao Uso de Antibiόticos", STAB - Aςύcar Alcool e Subprodutos, Piracicaba, vol. 16 (3), pp; 22 - 25, 1998. One of the most commonly cited forms in literature aiming to solve this cell separation problem consists on

the use of immobilized cells in different supports as, for example: porous glass and silica treated with glutaraldehyde, see the article by Navarro, J. M and Durand, G., "Modification of Yeast Metabolism by Immobilization into Porous Glass", European J. Appl. Microbiol., vol. 4, pp. 243 - 254, 1977; Rasching ceramic covered with gelatin and pulverized with a glutaraldehyde solution, as described in Marcipar, A. C. N. and Lebeult, B. L., "Immobilization of Yeasts on Ceramic Supports", Biotechnology Letters, Vol. 1, pp. 65 - 70, 1980; cellulose, see article by Szajani, B. et al., "Continuous Production of Ethanol Using Yeast Cells Immobilized in Preformed Cellulose Beads", Applied Microbiology and Biotechnology, vol. 46, pp. 122 - 125, 1996; agar and activated carbon, see Chaudhary, A. B. and Chinkholkar, S. B., "Cell Immobilization: A Critical Approach to Ethanol Production by Saccharomyces cerevisiae and Schizosaccharomyces pombe", Indian Journal of Microbiology, vol. 36, pp. 75 - 83, 1996; calcium alginate and carrageen k, see article by Ryu, S. and Lee, K. "Comparison of Immobilization Matrix for Ethanol Fermentation by Zymomonas mobilis and Saccharomyces cerevisiae" , Journal of Microbiology Biotechnology, vol. 6, pp. 438 - 440, 1997.

Nevertheless, due to the elevated cost of traditional supports as the previously cited, the use of alternative supports as vegetable bush {Luffa cylindrica) , sugar cane stems, sugar cane bagasse, wood and chrysotile, among others, for cell immobilization has been proposed. See Brazilian Patent No. PI 9300474-5, entitled "Processo de Produgao de Etanol com Microrganismos Imobilizados em Colmos de Cana de agύcar, Sistema para a Produςao de

Etanol, e Processo para Imobilizaςao de Microrganismos em Colmos de Cana de agύcar"; and the articles by Debabrata D. A. S. et al., Journal of Fermentation and Bioengineering, vol. 75, pp. 132 - 137, 1993; Chaudhary, A. B and Chinkholkar, S. B., "Cell Immobilization: A Critical Approach to Ethanol Production by Saccharomyces cerevisiae and Schizosaccharomyces pombe", Indian Journal of Microbiology, vol. 36, pp. 75 - 83, 1996; Fregonesi, A. A. thesis, "Adesao de Celulas de Saccharomyces sp. em Materials Inorganicos para a Produςao de Etanol", Master dissertation, Instituto de Quimica, Unicamp, Campinas, 1998; Ogbonna, J. C. et al . , Bioresource Technology, vol. 76, pp 1 - 8, 2001 and Ogbonna, J. C, Liu, Y., Loofa {Luffa cylindrica) as a Carrier for Microbial Cell Immobilization", J. Ferm. Bioeng., vol. 78 (6), pp. 472 - 473, 1994 and Rigo, M. thesis, "Estudo de Fermentagao Alcoόlica por Celulas de Saccharomyces cerevisiae Imobilizadas em Crisotila", Master dissertation, Instituto de Quimica, Unicamp, Campinas, 2001. Nevertheless, we should mention that although many supports could be used for cell immobilization, many of them show serious problems for an industrial scale implementation, in accordance with Monte Alegre, R. et al. article, "Ethanol Fermentation of a Diluted Molasses Medium by Saccharomyces cerevisiae Immobilized on Chrysotile", Braz. Arch. Biol. Technol., vol. 46 (4), 2003.

Chrysotile is magnesium silicate with fibrous aspect and its chemical formula is 3MgO.2SiO 2 .2H 2 O, whose formation is related to metamorphic modifications occurred in volcanic ultra-basic rocks. Its structure is unique and highly organized, constituted by 16 - 18 brucite-silicate

double layers coaxially coiled that, in accordance with the assumed configuration, can originate long or short fibers, the last being more commonly called fibrils. Chrysotile has been used for a long time in fibril cement industry in large scale. More recently, it has been used as support for cells (see Brazilian Patent applications PI 8930849-5 and PI 9700635-1) in alcoholic fermentation processes, bio- reductions and sucrose hydrolysis (Fregonesi, A. A., "Adesao de Celulas da Saccharomyces sp. em Materials Inorganicos para a Produςao de Etanol", Master dissertation, Instituto de Quimica, Unicamp, Campinas, 1998; Wendhausen, R. et al., "Continuous Fermentation of Sugar Cane Syrup Using Immobilized Yeast Cells", Journal of Bioscience and Bioengineering, Vol. 91, pp. 48 - 52, 2001; Rigo, M., "Estudo de Fermentaςao Alcoόlica por Celulas de Saccharomyes cerevisiae Imobilizadas em Crisotila", Master Dissertation, Instituto de Quimica, Unicamp, Campinas, 2001) .

According to literature data, yeast adhere themselves preferably to chrysotile fibrils, originating a cell- support complex that does not exhibit any type of hindrance to development and/or cellular metabolism, in accordance with Cassiola, F. et al., "Interaction between Saccharmoyces cerevisiae and Chrysotile", European Cells and Materials, vol. 2, pp. 30 - 35, 2001.

Among the advantages of using chrysotile as support for ethanol production we can say:

- High yeast cell immobilization capacity through absorption; - High stability, which permits its use for prolonged periods of time and its reuse;

- High resistance to thermal treatments;

- Low cost; and

Stimulus of ethanol production by yeast, thus increasing the specific process productivity. The pointed advantages are possibly associated with an increase of physiological stability and cellular metabolism when the positive interaction between yeasts and the solid support, as suggested by Jokes, I. et al., "Characterization of Saccharomyces cerevisiae Immobilized onto Chrysotile for Ethanol Production", J. Chem. Technol. Biotechnol., vol. 73, pp. 54 - 58, 1998 and Monte Alegre, R., "Ethanol Fermentation of a Diluted Molasses Medium by Saccharomyces cerevisiae Immobilized on Chrysotile", Braz. Arch. Biol. Technol., vol. 46 (4), 2003. This interesting finding originated the deposit of Brazilian patent application PI 9700635-1 and, from this point on, studies related to immobilization technique, interaction between S. cerevisiae and chrysotile and ethanol production in submerged discontinued and continued cultivations has been published with promising results in terms of substrate conversion, ethanol production and productivity, see Joekes, I. et al., "Characterization of Saccharomyces cerevisiae Immobilized onto Chrysotile for Ethanol Production", J. Chem. Technol. Biotechnol., vol. 73, pp. 54 - 58, 1998; Fregonesi, A. A., "Adesao de Celulas de Saccharmoyces cerevisiae sp. em Materials Inorganicos para a Produςao de Etanol", Master Dissertation, Instituto de Quimica, Unicamp, Campinas, 1998; Rigo, M., "Estudo de Fermentaςao Alcoόlica por Celulas de Saccharomyces cerevisiae Imobilizadas em Crisotila", Master Dissertation, Instituto de Quimica, Unicamp, Campinas, 2001; Filloy, P.

H. et al., "Activation of Brazilian Chrysotile Surface Using Gas Flow in Batch Reactors", Paper presented at Congress Proceedings of the International Conference on Advanced Materials Processing Technology (AMPT 01), vol. 3, Universidad Carlos III de Madrid, Leganis/Spain, 2001; Wendhausen, R., "Continuous Fermentation of Sugar Cane Syrup Using Immobilized Yeast Cells", Journal of Bioscience and Bioengineering, vol. 91, pp. 48 - 52, 2001 and Monte Alegre, R., "Ethanol Fermentation of a Diluted Molasses Medium by Saccharomyces cerevisiae Immobilized on

Chrysotile", Braz. Arch. Biol. Technol., vol. 46 (4), 2003.

Nevertheless, citations about ethanol production by S. cerevisiae immobilized on chrysotile in natura in an airlift bioreactor, object of the present invention, were not found.

The patent literature points out many documents related with the field of the invention.

The Brazilian patent application PI 0204931-7, entitled "Processo de Obtenςao de Microrganismos Aspergillus terreus, Rhizopus oryzae, Pseudomonas oleovorans e Serratia rubidaea imobilizados em crisotila para aplicaςao em processos biocataliticos e biotecnolόgicos" refers to the microorganism and organism immobilization process on chrysotile asbestos fibers, whose purpose is the obtainment of the immobilized microorganism or organism for biocatalyzed organic reactions. Fungus (Aspergillus terreus and Rhizopus oryzae) and bacteria {Pseudomonas oleovorans and Serratia rubidaea) are immobilized on chrysotile asbestos to be used as biocatalysts aiming transformation, through enzymatic reactions, of xenobiotic compounds (structurally distinct

substrates of the natural cell substrates) , intentionally inserted in the microorganism and/or organism environment, in products in which there is a great commercial interest. The microorganisms immobilized on chrysotile can be used in continuous, semi-continuous and batchwise processes, sequentially reused, as also can be stored for long periods, without modification of their biotransforming capacities. It should be noted that although the object of the cited application is the chrysotile use as an immobilization support, there is neither the description nor the suggestion to immobilize Saccharomyces cerevisiae yeast aiming ethanol production, and hence, does not show anteriority to the present application.

Publication CN 1403579, "Alcohol preparing airlift fermentation and separation coupling technological process and special equipment", refers to an alcohol production and separation process from a system construed by an airlift bioreactor, where occurs the properly said fermentative process, coupled to the product separation unit, which is composed by a condenser and two adsorption/desorption towers connected in parallel. This system, referred as special equipment, works under a 4000 to 8000 Pa vacuum with the aid of a pump; the two towers are used to adsorb and desorb the gaseous mixture pumped and the condenser is used to collect the desorbed alcohol. As can be seen, although the presented system also uses an airlift bioreactor, the global system does not show any similarity with the object of the present invention, that does not operate under vacuum, does not use adsorbent material for ethanol recuperation and yet use cells supported on chrysotile.

The publication MXPA05006992, "Proceso para Ia producciόn de etanol utilizando cristales estables de levedura en un reactor en lotes convencional modificado", shows the use of stabilized yeast crystals as an alternative to the need of addition of fresh microorganism cultures in each reactor operating in batchwise for ethanol production. Conventionally, the yeast crystals that grow after the beginning of the fermentative process tend to float on the broth surface during fermentation, reducing the contact of microorganisms with the culture medium, and hence, increasing significantly the culture period and lowering the fermentation speed. In this context, the innovation in which is based the described process refers to the use of stable yeast crystals in a batchwise reactor modified exclusively for this purpose, resulting in fermentation time reduction and in the increased speed of ethanol production. By the previously described, we can conclude that the process presented does not have any similarity with the object of the present invention. The Brazilian Patent Application PI 0306523-5, entitled "Processo de Produςao de Etanol com microrganismos imobilizados em sabugos de milho e processo para imobilizacao de microrganismos em sabugos de milho", refers to the use of corncob as an alternative support for microorganism immobilization that produces alcohol and presentation of a system where the centrifugation step is not observed. The system that operates in continuous mode is constituted by three closed serial fermenters and by a specific column to wash gases produced with water. There are screens situated in the body of the fermenters that keep the semi-fluidized bed, preventing the corncobs

containing immobilized microorganisms to freely circulate through the reactor and, occasionally, obstruct the entries/exits of the reactor. The described process greatly differs from the object of the present invention, where chrysotile fibers containing adhered microorganisms describe an ascendant movement through the interior of the inner tube and, by reaching its top, return to the basis in a descendent flow. As is possible to observe, although both systems are destined to ethanol production with immobilized cells in alternative supports, the cited application does not constitute anteriority to the present invention.

The Brazilian patent PI 9300474-5, entitled "Processo de produgao de etanol com microrganismos imobilizados em colmos de cana-de-acύcar, sistema para produgao de etanol e processo para imobilizagao de microrganismos em colmos de cana-de-agύcar", refers to a continuous ethanol production by immobilized microorganisms in sugar cane stems. The system is constituted by two serial reactors. The must is continuously feed by the inferior side of the first fermenter, has an ascendant flow, exits through the superior part, feeds the second reactor also by the superior part and is withdrawn, completely fermented, by its lower part. In this system, the support containing the adhered microorganism is hold in the reactor through a sustentation structure that hinders the free circulation of cell-support complex through the body of the reactor. The system here described has a narrow relation with the previously mentioned (PI 0306523-5) , although it does not present similarity with the present invention. The Brazilian patent application PI 9700635-1, entitled "Processo de produgao de etanol em alto rendimento

em regimes continuo e batelada" provides an ethanol production process using Saccharomyces cerevisiae cells obtained from fresh bakery ferment, adsorbed in treated and activated chrysotile. The first step in the development of said process constituted of chrysotile treatment and activation for a posterior S. cerevisiae cell immobilization. Initial batchwise cultures in a rotary incubator, with 15 mL of culture medium reaction volume containing glucose 10 to 50% (w/v) had duration from 90 to 122 hours and used free and immobilized cells with different cell mass/chrysotile mass ratio, for comparison purposes. Such assays showed that the final yield of the immobilized system overcame the free system up to 20%, although the process development has been accompanied by CO 2 evolution through Erlenmeyer weighing, which does not distinguish between CO 2 from the said fermentative process to the CO 2 from cell breathing. The first continuous culture, developed in a PBR type reactor ("packed-bed reactor") jacketed and with 1 L net volume, operated with different feed flows, at 30°C and with duration of 30 days.

The complex cell/support and compact glass spheres were used as fillings and sucrose was used as culture medium

(20% w/v) with other nutrients. As a result, the food flow was determined (2,25 mL/min) that conducted the major productivity (16 g/L.h) . The following continuous culture, also with duration of 30 days, was developed in optimized conditions and, as a mean, kept its productivity in 16 g/L.h with good operational stability. Then, a new assay in continuous mode was accomplished using as a filler the cell/support complex and rough sand. In such culture, the feed flow was changed (1,30 to 4,12 mL/min) and the

greatest obtained productivity was similar to that of the previous assays (16 g/L.h) to a feed flow of 2,62 mL/min. Besides these, other assays were also accomplished varying the process temperature, where it was possible verify that 35 0 C was the optimum fermentation temperature. As can be seen, the batchwise cultures showed in this invention used an extremely tiny reaction volume (15 mL) , used previous support activation and immobilization processes and were excessively long, what difficult the industrial implementation of the proposed technique. Differently from the patent application PI 9700635-1, in the present invention, batchwise cultures duration varied from 7 to 14 hours, at maximum, a reactor with a fixed bed was not used and any process for previous cell immobilization to the chrysotile fibers was not used.

The U.S. Patent 4.952.503, entitled "Process for the production of ethanol", refers to a process for ethanol production through continuous fermentation of a substrate containing carbohydrate, in which a fermentation broth stream is continuously removed from the fermenter and divided by centrifugation in a stream rich in yeast, which is re-circulated to the reactor, and a stream essentially- free from cells and rich in ethanol. This last stream is submitted to a distillation process, generating an ethanol enriched fraction and other poor in this species, which is re-circulated to the reactor entry stream.

The U.S. Patent 4.769.324, entitled "Ethanol Production", relates to ethanol production through molasses fermentation in the presence of Saccharomyces cerevisiae and Saccharomyces castellii. Saccharomyces cerevisiae converts hexoses from molasses directly in ethanol and

Saccharomyces castellii produces amylase enzyme in a medium containing molasses. The produced amylase acts on starch and on sugars with higher chains present in the non- fermentable portion of the medium, turning them into hexoses, which are converted into ethanol by S. cerevisiae. Thus, higher ethanol yields are obtained from a given molasses quantity when compared to other processes. It should be said, however, that Saccharomyces castellii that produces amylase is a mutant that needs a laborious treatment for its perfect cell adaption to the medium containing molasses.

Thus, there is still a technical need for a fermentative process for ethanol production using Saccharomyces cerevisiae cells immobilized on in natura chrysotile fibers in inner circulation pneumatic bioreactors with 2, 5 and 10 L of net capacity, operated in batchwise and repeated batchwise mode, such process being described and claimed in the present application. SUMMARY OF THE INVENTION In a broad sense, the process of the invention for ethanol obtainment through fermentation in the presence of Saccharomyces cerevisiae immobilized on chrysotile in an inner circulation pneumatic bioreactor comprise the following steps: a) In an airlift or pneumatic bioreactor, provide a culture medium with glucose or sucrose as carbon sources, with concentrations from 160 to 300 g/L; b) Add to the said medium a chrysotile amount that corresponds to concentrations between 0,5 and 5% w/v and anti-foaming; c) Stabilize the culture medium in a temperature

between 30°C and 35°C with the aid of water circulating through the hollow inner cylinder of the said bioreactor; d) Promote homogenization of the reaction medium, here composed by culture medium and chrysotile fibers, through the injection of a gas as CO 2 with an specific flow of 0,3; 0,6 or 0,9 vvm; e) Inoculate the said bioreactor with an initial concentration of Saccharomyces cerevisiae cells in the fermentation broth between 5 and 30 g/L in dry basis, close the bioreactor and initiate the fermentative process; f) Keep the said fermentative process for a period from 7 to 14 hours, with a pH range between 4,3 and 5,5, where cell immobilization of the culture medium on the chrysotile support occurs concomitantly to the ethanol production process, recuperating the dragged ethanol with the aid of a jacketed condenser located on the cap of the said airlift bioreactor; g) Finalize the fermentative process when any sugar residue is no more detected in the culture broth; h) Exhaust the bioreactor keeping fibers containing immobilized cells and recover the wine without yeast with an ethanol final concentration higher than would be possible to obtain through processes with free cells; and i) Analyze the ethanol proportion in the wine without yeast.

Thus, the present invention provides a process for alcohol obtainment with immobilized S. cerevisiae on chrysotile in an inner circulation pneumatic bioreactor.

The present invention also provides a process for ethanol obtainment with immobilized S. cerevisiae on chrysotile in an inner circulation pneumatic bioreactor

that operates in a batchwise or repeated batchwise mode.

The present invention also provides an ethanol obtainment process with immobilized S. cerevisiae on chrysotile in a pneumatic bioreactor where the cells present in the culture broth are physically entrapped on chrysotile fibers and the suspension circulation inside the reactor is impelled by a gas stream as CO 2 . BRIEF DESCRIPTION OF THE DRAWINGS

The attached Figure 1 is a graphic that illustrates the comparison between the percentage increase in ethanol yields obtained in cultures using different in natura (-•-) and activated (-o-) chrysotile concentrations related to the free cell system (0% w/v) . DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a fermentative process for ethanol production using Saccharomyces cerevisiae cells immobilized on in natura chrysotile fibers in inner circulation pneumatic bioreactors with 2, 5 and 10 L of net capacity operated in batchwise and repeated batchwise mode.

In the described process of the present invention chrysotile is used as a support for Saccharomyces cerevisiae cell immobilization, having evidenced significant advantages in terms of production, productivity and ethanol yield (up to 15% in comparison with a similar system without chrysotile) .

Under this context, the process of the invention proposes the ethanol production by immobilized yeasts on chrysotile in an airlift pneumatic or inner circulation "ascending gas flux" bioreactor, equipment of the object of the patent application PI 0404703-6 of the applicant and

here entirely incorporated as reference, operating in a discontinuous mode.

This bioreactor is constituted by two concentric cylinders that generates an annular space by which the fermentation medium circulates that, in turn, is impelled by the injected gas by an sprinkler installed on the lower portion of the inner cylinder. The inner cylinder has a height lower than the external cylinder and is completely immersed in the reaction volume, actuating as a heat exchanger with the reaction medium. The apparatus has a superior cap, with orifices for pH, temperature, dissolved oxygen sensor allocation, among others, besides one condenser which retains the dragged ethanol by CO 2 generated during culture. The configuration of this bioreactor promotes an efficient fermentation medium circulation containing immobilized cells through CO 2 injection, so that along the whole experiment the immobilized cells remain in contact with the nutrient medium. The bioreactor used in the present process shows to be robust, of low construction cost, of simple operation and with efficient heat and mass transfer devices.

In the development of the present process two culture mediums were evaluated with glucose or sucrose as carbon sources, with concentrations in the range from 160 to 300 g/L.

Besides the carbon source, the "culture medium 1" shows the following composition: urea (3 g/L) and MgSO 4 .7H 2 O (0,5 g/L) . The "culture medium 2" shows a composition that simulates the diluted sugar cane molasses used as raw

material in the ethanol industrial production. Besides the carbon source, the "culture medium 2" presents the following composition: urea (5,32 g/L) , yeast extract (6,8 g/L) as a nitrogen source, MgSO 4 .7H 2 O (1,4 g/L) as magnesium source and KH 2 PO 4 (5,6 g/L) as phosphorous and potassium source.

After the preparation of the said mediums, a chrysotile amount is added onto them corresponding to the desired concentration (from 0,5 to 5% m/v) and anti- foaming. The anti-foaming amount added to the suspension depends on the reactor volume to be used (2, 4 or 6 drops for 2, 5 or 10 L bioreactors, respectively) . The useful anti-foaming for the practice of the invention include the ones with silicon and without silicon (http: //www. silaex.com.br) .

The cultures are made in a temperature range from 30°C to 35°C, controlled by water recirculation from a thermostat controlled bath. This water circulates through the bioreactor hollow inner cylinder, which acts as a heat exchanger.

The reaction medium mixture is preferentially accomplished through CO 2 or N 2 injection without being limited by these gases, preferably CO 2 from a pressurized CO 2 cylinder and the specific flow (0,3; 0,6 or 0,9 vvm, where "vvm" means gas volume per broth volume per minute or specific gasification flow) controlled by a mass flux meter.

Afterwards, the bioreactor is inoculated with Saccharomyces cerevisiae cells, in a pressed bakery ferment pattern, to perform at the beginning of the process a cellular concentration in the fermentation broth between 10

and 30 g/L in dry basis.

After inoculation, the bioreactor is properly closed, beginning the fermentative process. It should be pointed that, oppositely to the processes contained in the state of the art, the present process dispenses any method for the previous cell immobilization on chrysotile fibers.

Cell immobilization on the chrysotile support occurs concomitantly with the ethanol production process and is not characterized by the establishment of any type of chemical linkage between cell and support, but only by the physical cell entrapment to the solid support, which occurs in a completely random manner as the suspension circulates inside the reactor, impelled by the CO 2 stream. From the point of view of a possible implementation in industrial scale, this procedure presents great advantages over others related in literature, because it does not require the previous yeast cell immobilization step, what demands time and, consequently, augment the whole process costs.

During the fermentative process, ethanol is dragged by CO2, which is also produced during the culture. Thus, to recover the dragged ethanol a jacketed condenser is used located on the airlift bioreactor cap, kept at low temperature by cold water circulation from a second thermostat controlled bath. The cultures have 7 to 14 hour duration, depending on the initial concentration of offered substrate.

By the end of the processes, when any sugar residue in the culture broths is no more detected, the suspension is filtered to separate fibers containing immobilized cells and to obtain wine without yeast, that is then submitted to a distillation process to evaluate the ethanol proportion.

Alternatively, in a repeated batchwise process, the wine is separated and immobilized cells are kept in the bioreactor and participate on a new batchwise reaction.

In the three studied scales (2, 5 and 10 L) different experimental conditions were tested, varying the initial concentration of energy and carbon source (160 to 300 g/L) , the chrysotile concentration (0,5 to 5% w/v) and the specific CO 2 feed flow (0,3 to 0,9 vvm) .

In all evaluated conditions and in different scales, a superiority of the system with immobilized cells in comparison to the free cell traditional system was observed, with substrate yields in ethanol for the immobilized system up to 15% higher than those obtained with a free cell system, in the same experimental conditions.

The present process will now be described with the aid of the Examples and comparative Tables that should not be considered as limitative of the invention.

In the development of the process of the invention, cultures in a rotary incubator (shaker) were accomplished and in inner circulation pneumatic bioreactors (airlift) with 2 liters, 5 liters and 10 liters of net volume. Example 1

Preliminary assays are developed in a rotary incubator (shaker) aiming to reach the optimized culture conditions, regarding the initial substrate concentration (Cs 0 ) , initial cell concentration (Cx 0 ) and chrysotile concentration (C C τ) • In such assays cellular concentrations from 5 to 30 g/L; fermentation medium volumes from 15 to 100 mL; agitation frequencies from 0 to 350 rpm; chrysotile concentrations from 0 to 6,67% (w/v) (in natura and

activated by supersonic sonication at 25 Hz for 30 minutes for fiber opening); pH of the fermentation medium from 4,5 to 5,3; temperature of 30°C and two different culture mediums are tested, here called "culture medium 1" and "culture medium 2".

The culture medium 1 is similar to that described by Wendhausen, R., "Continuous Fermentation of Sugar Cane Syrup Using Immobilized Yeast Cells", Journal of Bioscience and Bioengineering, vol. 91, pp. 48 - 52, 2001 and is constituted only by glucose (200 or 300 g/L) , urea (3 g/L) and MgSO 4 .7H 2 O (0.5 g/L).

The culture medium 2, by its turn, simulates the sugar cane molasses composition and shows the following formulation: glucose or sucrose (200 or 300 g/L), urea (5.32 g/L), MgSO 4 .7H 2 O (1.4 g/L), yeast extract (6.8 g/L) and KH 2 PO 4 (5.6 g/L) .

In the cultures made in the presence of chrysotile, the fibers are firstly washed with abundant tap water to eliminate powder and impurities from the extraction process. After an exhaustive wash, the fibers are used for immediate cell immobilization or are submitted to the previous activation process previously mentioned.

A fact observed in all the cultures accomplished in a rotary incubator is the chrysotile flotation, that is, the CO 2 generated by alcoholic fermentation push the immobilized yeast containing fibers to the broth surface, thus reducing the contact with microorganisms immobilized in it with the fermentation medium. The various strategies used in an attempt to grant a great homogeneity to the reaction suspension are deposed of success such that, in general, the free system yield is always superior to the

immobilized system. 123 cultures are made at all in an Erlenmeyer in a rotary incubator, from the combination of different variables cited above.

The total reducing sugar concentration (ART) is evaluated through dinitrosalicylic acid method (DNS), see Chaplin, M. F., "Monosaccharides", in Chaplin, M. F., Kennedy, J. F. (eds) : "Carbohydrate Analysis: A Practical Approach", IRL Press (Oxford, England), pp. 1 - 36, 1986, after acid hydrolysis with 0.9 N HCl at 60°C for 40 minutes. Ethanol concentration is measured by the oxidation method by potassium dichromate after sample distillation.

Ethanol yield (%) is calculated by a relation between ethanol amount produced and the ART amount consumed, divided by a stoichiometric factor (0.511 g e thanoi/gARτ) and multiplied by 100.

The main results obtained in the cultures accomplished in a rotary incubator does not refer to the obtainment of ethanol yield of the immobilized system more expressive than that observed for the free system, but to the definition of the medium type and the yeast origin to be used in later assays, that is, culture medium 2 and ferment purchased from Fleischmann Royal. Example 2

The cultures in a 2 L airlift bioreactor aim to evaluate homogeneity of a medium containing Saccharomyces cerevisiae cells immobilized on chrysotile fibers in natura, as a function of CO 2 injected through the basis of this type of reactor. There is an expectation of a better suspension circulation and, hence, an uninterrupted contact between immobilized cells and the culture medium, which represents the main problem detected in Erlenmeyer

cultures .

Such cultures are accomplished in the absence and in the presence of chrysotile, for comparison purposes, using the culture medium 2 with the following composition: sucrose, 200 g/L; urea, 5.32 g/L; MgSO 4 .7H 2 O, 1.4 g/L; yeast extract, 6.8 g/L and KH 2 PO 4 , 5.6 g/L. Other parameters are also kept fixed, that is: Cx 0 = 10 g/L, φ C o 2 = 0.3 vvm, 9 hours of culture time, 30 0 C temperature and pH = 5,3. The concentration (from 0 to 4% w/v) and the chrysotile type (in natura or activated) are manipulated, in order to maximize the ethanol yield. 19 cultures in a 2 L airlift bioreactor are made as a whole (3 with free cells, 12 with in natura chrysotile immobilized cells and 4 with activated chrysotile immobilized cells) . The concentration of total reducing sugars (ART) is evaluated by the dinitrosalicylic acid method (DNS), see Chaplin, M. F., "Monosaccharides", in Chaplin, M. F., Kennedy, J. F. (eds.): "Carbohydrate Analysis: A Practical Approach", IRL Press (Oxford, England), pp. 1 - 36, 1986, after the acid hydrolysis with 0.9 N HCl at 60°C for 40 minutes. Ethanol concentration is obtained using the oxidation method using potassium dichromate after sample distillation. Ethanol yield (in %) is calculated by the relation between ethanol amount produced and the ART amount consumed, divided by an stoichiometrical factor (0.511 g e thanoi/gARτ) and multiplied by 100.

For comparative purposes, Table 1 below shows the values of the percent increase of ethanol yields of the immobilized systems related to the free systems in the same culture conditions.

In all assays made with immobilized cells, ethanol

yields obtained are superior to the free cell systems in the same culture conditions. Nevertheless, the most expressive result (increase around 11% on ethanol yield) is obtained when in natura chrysotile 3% (w/v) is used (result in eminence on Table 1 below) . Table 1

* Assays made in triplicate

For comparative purposes, Figure 1 shows the percentage increases in ethanol yield for systems with activated and in natura chrysotile, in relation with the free cell system, obtained with the values from Table 1. Although the obtained results with activated chrysotile are superior to that obtained with free cells, these are inferior to that observed with in natura chrysotile. For this reason, systems using activated chrysotile are definitively discarded, in natura chrysotile being used in the continuity of research that lead the Applicant to the present results. Example 3 The relevance of the results obtained in a 2 L airlift bioreactor, from an ethanol yield point of view, motivated the study of new initial substrate concentrations, CO 2 flows and chrysotile concentrations {in natura), as well as

an scale increase study, from 2 L to 5 L airlift bioreactor. For this, a conjunction of experimental conditions is defined obeying a factorial plan (2 3 with sextuplicate of the central point) . For each assay with immobilized microorganism a correspondent one with free cells is made for comparative purposes, 19 cultures in total.

On Table 2 the manipulated variables are presented, as well as its lower and higher limits for the execution of the factorial plan in a bioreactor of 5 L of net capacity. Table 2

Cs 0 ( g/L) φco2 (vvm) CcT ( % w/v)

160 ( - ) 0 . 3 ( - ) 1 . 0 ( - )

200 * ( 0 ) 0 . 6* ( 0 ) 2 . 5 * ( 0 )

240 ( + ) 0 . 9 ( + ) 4 . 0 ( + )

^Condition defined as central point: assays in sextuplicate .

Initially, cultures in a 5 L airlift bioreactor with in natura chrysotile previously washed with water are made. During the washing step, there is loosening of a great fibrils quantity from the higher fibers. In view of the fact that, in accordance with literature, Saccharomyces cerevisiae cells adhere preferably to fibrils, a choice was made to no more proceed with the fibers washing operation before the use to avoid significant fibrils losses. The obtained results in test cultures after this alteration in the operation protocol show an increment in the ethanol final yield in comparison to assays made with washed chrysotile .

Table 3 shows the percentage increase values of

ethanol yields (%) of immobilized systems related to free systems and in accordance with experimental conditions defined in the factorial plan 2 3 . Each line from Table 3 informs the percentage increase of the immobilized systems related to the free system at the same experimental conditions (C s0 and φ C o2) • In the "free cells" column the percentage increases related to the minor value obtained in this system are informed, in this case, in the following condition: Cs 0 = 200 g/L and φ C o 2 = 0,6 vvm. Globally evaluating the obtained results using a 5 L airlift bioreactor (Table 3) can be observed that in all the cultures accomplished with immobilized microorganism on chrysotile fibers, superior ethanol yields are obtained in relation to that obtained with free cells, similar to that observed in a 2 L airlift. The best result (with ethanol yield around 7% superior to the free system) is obtained when Cso = 160 g/L, φ C o2 = 0.9 vvm and C C τ = 4% (w/v) is used (emphasis at Table 3) . Table 3

Culture Chrysotile <concentration (% w/v) condition 0 (free 1 2.5 4 cells)

Cs 0 = 200 0.00 - 1.09* - g/L vvm

Cs 0 = 240 2.96 1.17 - 4.22 g/L vvm

Cs 0 = 24 0 5.25 1.16 - 2.71 g/L vvm

* Assays made in sextuplicate Example 4

This example refers to the culture made using an airlift bioreactor with 10 L of net volume. In this assays is possible to evaluate data reproducibility with scale increase (from 5 to 10 L), using the same factorial plan used on a 5 L bioreactor and identical experimental conditions .

Table 4 shows below the values of the percentage increases of ethanol yields (%) of the immobilized systems related to the free systems obtained in cultures in a 10 L bioreactor. Each line from Table 4 informs the percentage increases of immobilizes systems related to the free system in the same experimental conditions (Cso and φco2) • In the "free cells" column the percentage increases related to the

minor value obtained in this system are informed, in the case, in the following condition: Cs 0 = 160 g/L and φ C o 2 = 0 , 9 vvm.

The obtained results confirm the reproducibility of the experiments previously made in a 5 L reactor, as the best result is obtained at the same experimental conditions (Cs 0 = 160 g/L, φco2 = 0,9 vvm and C C τ = 4% w/v) and in accordance with the experimental conditions defined in the factorial plan 2 3 . Nevertheless, in this case, the immobilized system overcomes the free system, in terms of final ethanoϊ yield, in approximately 15% (emphasis on Table 4) . Table 4

* Assays made in triplicate.

After the cultures referred to experimental plan were made, the obtained data are statistically treated, obtaining a model that describes the percentage increase in ethanol yield as a function of the evaluated independent variables (Cso, φco2 and C C τ) • The optimized culture conditions that theoretically conduct to the best ethanol yield are found (Cso = 240 g/L, φ C o2 = 0.88 vvm and C C τ = 2,74% w/v) . Thus, two new cultures are made, one with immobilized cells and other with free cells for comparison effect, the obtained results proving the immobilized system superiority in comparison with the free one. Example 5 Once determined the optimized culture conditions

(Example 4), afterwards cultures in repeated batches are made. Nevertheless, in these cultures an initial 200 g/L substrate concentration is used. The explanation for this experimental change is that, in despite of being possible to exhaust all substrate when Cs 0 = 240 g/L is used, the system productivity is considerably reduced, that is, the

culture total time is far superior from the 9 hours that has been required on the other cultures. Thus, the initial culture of the repeated batches is made with Cso = 200 g/L, φco2 = 0,88 vvm, C = 2.74% (w/v) and Cx 0 = 10 g/L. The total consumption of substrate happens in 9 hours, as expected, obtaining a 4,32% percentage increase in ethanol yield in comparison with the free system and 11.46 g/L.h productivity.

Once the first batch is finished, the fermented wine is removed from the bioreactor through gravity and the immobilized cells containing fibers are kept in its interior. After its depletion, a new amount of culture medium is feed into the reactor, beginning the second batch. This second culture has duration of 14 hours for the complete depletion of the initial offered substrate, having reached a 4.70% percentage increase of ethanol yield in comparison with the free system, with 7.13 g/L.h productivity. Once finished the second batch, the same depletion and recharge procedure is used, to the third consecutive assay. This culture has 21 hour duration until the all substrate be consumed. There is a 4.74% percentage increase in ethanol yield in comparison with the free system and a 5.61 g/L.h productivity. With these results, we can note that the immobilization system here used is efficient, but as a consequence of the difficulty in determining the final concentration of adhered cells onto the support after each culture, we cannot guarantee that the initial cell concentration in all assays had been exactly the same. To bypass the cell wash problem during the bioreactor depletion, the cells removed could be separated by centrifugation and return to the reactor,

diminishing the culture time and increasing the process productivity.

Based on the five Examples presented above, we can assume that the process of the invention for ethanol production with Saccharomyces cerevisiae immobilized on chrysotile generates ethanol yields superior to that obtained in the traditional process, that does not use chrysotile, and that said superiority of the immobilized system is constant with scale enlargement in terms of ethanol yields.

Moreover, we can verify that different operational methods of trie airlift bioreactor always conduct to yields superior to the immobilized systems in comparison with the free ones, and this demonstrates a great versatility/viability of the process of the invention.