WO2009067771A1

WO2009067771A1 - Production of ethanol from algae

Info

Publication number: WO2009067771A1
Application number: PCT/BR2008/000357
Authority: WO
Inventors: Nei Pereira Junior; Elis Cristina ARAÚJO ELEUTHERIO; Marcos Dias Pereira; Dárlio Inácio ALVES TEIXEIRA; Maria Isabel Madeira Liberto; Allen Norton Hagler; Julio DE MENDONÇA LOUZADA; Gustavo DE MENDONÇA VALLADARES
Original assignee: Universidade Federal do Rio de Janeiro UFRJ
Current assignee: Universidade Federal do Rio de Janeiro UFRJ
Priority date: 2007-11-26
Filing date: 2008-11-26
Publication date: 2009-06-04
Anticipated expiration: 2010-05-26
Also published as: BRPI0704200A2; BRPI0819746A8; BRPI0819746A2

Abstract

The present patent application refers to a process of producing alcohol from the fermentation of hydrolyzed sugars of gelling and/or thickening polysaccharides obtained from algae cultivated in natural or artificial aquatic environments, eutrophized or not eutrophized. The main steps of this process are: collection and cleaning of algae; fragmentation; drying; hydrolysis of the total sugars produced by the algae; fermentation of the sugars resulting from hydrolysis of the algae polysaccharides, with the use of yeast capable of promoting fermentation of galactosides; and alcohol distillation.

Description

PRODUCTION OF ETHANOL FROM ALGAE

Background of the Invention

Having economic and ecological interest, algae are cultivated with the aid of buoys, in areas where the wind and the current are not strong. Among the most common species are Kappaphycus, Gelidium, Gigardina, Gracilaria, Encheuma, Hypmea and Pterocladia. During growth, the algae selectively assimilate many of the minerals contained in water. They photosynthesize and synthesize the carbohydrate polymer that constitutes the structural framework of the alga body, which may be agar-agar, carrageen or others polymers.

The most known substances extracted from macro-algae are of three types: alginates, extracted from chestnut algae; agar-agar and carrageens, extracted from varies species of red algae. The agar-agar is a mucilage (vegetable gelatin) constituted of agarose and agaropectin polymers.

The alginates, agar-agar and carrageens are used, in the form of polymers, as thickening or gelling agents in the industrial manufacture of food products, cosmetics and pharmaceuticals, by virtue of the texture it gives them. The industrially-discarded algae residue constitutes an excellent raw material for transformation into ethanol according to the methodology that constitutes the object of this international patent registration.

For many centuries on the menu of oriental peoples, it is commonplace to use the agar-agar and carrageen polymer as a gelling and thickening product, respectively. The recipe for agar-agar was subsequently incorporated, serendipitously, to microbiology to solidify culture means designed for the growth of microbes. In the scope of applications known today, agar-agar and carrageen are used in various industrial and biotechnological sectors where these polymers are necessary (Renn, D. W., 1990. Seaweeds and biotechnology — inseparable companions. Hydrobiologia, 204-205 (1): 7-13; Marinho-Soriano, E. (2001) Agar polysaccharides from Gracilaria species

(Rhodophyta, Gracilariaceae) Journal of Biotechnology Volume 89, Issue 1 ,26

July, Pages 81-84).

In terms of production of fuels, patent application PI7701845 describes a process of obtaining ethanol from amide produced by green amilogenic microalgae. The production of alcohol using amide as a raw material is a process that was already known at the time.

Patent US7135308 protects a process for the production of ethanol from algae cultures of the Zygnemataceae, Cladophoraceae and Oedogoniales species, or a combination thereof, which after harvesting and drying are submitted to an anaerobic aqueous environment for fermenting in the presence of yeast typically used in the beer-making industry.

Patent application CA2005426 describes a process of producing ethanol in vivo, carried out by genetically-modified photosynthesizing bacteria with genes isolated from Saccharomyces cerevisiae or Zymomonas mobilis.

Patent document EP645456 describes a process and a system for the production of ethanol from saccharogenic microalgae. Patent JP52082785 describes a process of producing alcohol from the fermenting of amide accumulated in algae of the Chlamydomonas and Scenedesmus species cultivated in a liquid environment containing a source of carbon, a source of nitrogen and inorganic elements. Patent JP7031485 also claims a process of producing alcohol from saccharogenic algae using the step of adding a solution containing 80% of an organic solvent capable of denaturing the inner cellular membrane of the algae. Patent application US2008155890 discloses a system of cultivating algae of the Synechococcus and Chlorella species in addition to cyanobacteria, for the production of biodiesel. Patent application CN 1699516 describes a process for preparing biodiesel using oil extracted from microalgae.

Among the scientific documents, Horn and collaborators of The Norwegian University of Science and Technology NTNU published a series of articles, the most relevant dating from 1997, 2000 and 2001 , which demonstrate the possibility of D-galactosides being fermented by micro-organisms, particularly by Pichia angophorae, Zymobacter palmae, Pacchysolen tannophilus and Kluyveromyces marxianus.

As can be seen, today there is nothing in literature relating to the process of producing ethanol from algae storing gelling polysaccharides, in particular L- galactosides such as agar-agar and carrageenan.

Summary of the Invention

The present invention refers to a process of producing alcohol from fermenting of hydrolyzed sugars of gelling and/or thickening polysaccharides obtained from algae cultivated in natural or artificial aquatic environments, eutrophized or not. The main steps of this process are: collection, cleaning and treatment of algae to remove toxic elements and/or microbial inhibitors; fragmentation; drying; hydrolysis of the total sugars produced by the algae; fermentation of the sugars resulting from hydrolysis of the algae polysaccharides, with the use of yeast capable of promoting fermentation of galactosides; and alcohol distillation.

Drawings Figure 1 : Organization chart containing the steps of the process of producing alcohol from algae.

Detailed Description of the Invention

The process of producing alcohol from biomass of galactan-producing marine, red and/or dark macroalgae of this invention, occurs by the hydrolysis of gelling and/or thickening polysaccharides which are obtained from algae cultivated in natural or artificial aquatic environments; eutrophized or not.

For the purposes of this invention, the following definitions are hereby established: Carbohydrate polymers are understood to be those containing galactosides and/or glycosides, produced naturally and stored by the cells of the macroalgae.

Gelling and thickening polysaccharides are those formed by galactosides in their D and L forms, whereas the content of the glycoside polymers is represented by the algae cellulose.

Artificial environments are understood to be man-made environments for aquiculture, principally for pisciculture, carciniculture, malacoculture, herpetoculture, and other economically feasible cultures. The algae that can be used in this process are macroscopic, preferably those belonging to the Gelidium, Gigardina, Gracilaria, Encheuma and Pterocladia species.

The first step of this process comprises the collection of algae which are then cleaned and fragmented for submission to a partial or total drying process. This step is essential to control the carbohydrate content (Brix Degree) to be used in the fermentation phase.

After the cleaning step and treatment to remove the algae film, where microbial inhibitors are accumulated, fragmentation of the alga body can be performed by any mechanical procedure known in the state of the art. Preferably, fragmentation should be carried out by cutting into very small pieces.

Partial drying of the algae can be by processes known in the state of the art, such as: filter pressing, open-air drying; gas drying; solar collector; incident artificial light; chemical drying; and others. In the drying step, the algae may be completely or partially dried, and algae having a maximum humidity content of

45% in relation to their initial weight can be used.

For storage, the humidity content resulting in the algae, after the drying procedure, will be a maximum of 25%, and even more preferably, the maximum humidity should be 10% on average. The third step of this process comprises the initial hydrolysis of the carbohydrate polymers. The hydrolysis of said carbohydrate polymers can be carried out both by traditional physio-chemical processes and by enzymatic processes or by both processes in conjunction.

The physio-chemical hydrolysis used by this process is acid hydrolysis, and should be carried out in an aqueous environment containing an acid, at a temperature of between 60 and 120⁰C under pressure of between 1 and 3atm. Preferably, hydrolysis occurs at a pressure of between 1 and 2atm.

The physio-chemical process to be used in acid hydrolysis comprises the use of a solution containing on average the equivalent of: a. 20% fermentable substrate previously defined in the correspondent in dried algae; b. 5% acid in a concentration destined for each polymer type; c. 75 % water.

The acid used in this step can be hydrochloric acid or others that do not alter the chemical structure of the resulting monomers. The application of heat by microwaves, under pressure, may also be used. The efficiency of the heating by microwave allows a reduction of the reaction times of days and hours to minutes and seconds. This is the main but not the sole advantage of this mode of operation. It is evermore common to apply hydrolysis reactions assisted' by microwaves to produce monosaccharides from cellulose, amide and other natural polymers.

In enzymatic hydrolysis, the acid is substituted for an enzymatic preparation, containing an agarolitic and/or carragenolitic enzyme duly buffered or a mixture of agarolitic and/or carragenolitic enzymes duly buffered; the temperature and pressure conditions are identical to those used in physio-chemical hydrolysis. Depending on the alga origin of the polymers, mixed hydrolysis will be used, involving physio-chemical and enzymatic processes.

The fourth step in the process of producing alcohol according to this invention comprises the separation of total sugars obtained after the hydrolysis described in the prior step. Total fermentable sugars are understood to be those carbohydrates, in the form of monomers, dimers or trimers, resulting from the hydrolysis of galactan polymers of the macroalgae, fermentable by yeast of previously defined species.

The succus of fermentable sugars can be separated from the partially hydrolyzed mixture by filtering. The solutions of the sugars of galactosidic and/or glucosidic nature, to be deposited in the fermentation vat, receives the name 'liquid must' and should be prepared by appraising the Brix degree. Evaluation of the Brix degree will be considered satisfactory when the solution presents, preferably, between 13° and 17° Brix. This manner assures the best yield of the fermentative potential exercised by the yeast used in the process. Depending on the product of hydrolysis, the concentration of fermentable carbohydrates should be diluted so as to comply with the aforementioned Brix degree range.

The fifth step of the process for obtaining ethanol from the biomass of macroalgae, which corresponds to the fermentation stage, elements necessary to maintain the yeast should be added to the substrate in the vats.

The yeast to be added to the vat, to initiate the fermentation process, should be previously cultivated to propagate in culture environments containing growth factors, in addition to the macro and micro nutrients required for the development of cellular biomass. The sixth step begins by allocating said must to suitable equipment for fermenting such as, for example, open or sealed vats. This step will occur by adding the must to said vats that may or may not contain yeast capable of fermenting the galactosidic and glucosidic monomers. Preferably, the yeasts used are those capable of fermenting galactosides and glycosides, having high alcoholic tolerance and resistance to the microbial inhibitors present in the algae extracts. The yeasts that can be used for fermenting the algae products are those related to the following species or anamorphs thereof: Candida sp; Clavispora sp; Debaryomyces sp; Geotrichum sp; Kloeckera sp; Kluyveromyces sp; Metschnikowia sp; Pachysolen sp; Pichia sp; Kazachstania spp; Rhodotorula sp; Saccharomyces sp; Torulaspora sp; sp; Zygosaccharomyces sp. The yeasts used in the process of producing alcohol according to this invention are of the thermo-tolerant kind, capable of carrying out the fermentative process at high temperatures. Therefore, the fermentation step occurs by adding a fermentatively efficient quantity of yeast to said must. Preferably, fermentation occurs anaerobically, at a temperature between 25 and 45°C; pressure between 0.5 and 3atms and slightly acidic pH (3.5 to 5.5).

A fermentatively efficient quantity of yeast is understood to be such content that is capable of promoting fermentation of the must at an ideal velocity, that enables the yeast to develop at a low stress level, generating a higher ethanol yield and a lower production of secondary products of the fermentative process such as, for example, glycerol, acetic acid and succinic acid.

In the cultures, the yeast cells should remain mostly isolated, in view of the risks of aggregating together with others forming clusters. Additionally, antibiotics and bactericides should be added to the culture, capable of keeping the culture free of contaminations by exogenic bacteria during the fermentation process, so as to optimize the efficiency of the process described herein.

The efficiency of the fermentation can be evaluated by standard procedures used in the traditional sugar/alcohol industry, verifying the maximum conversion of galactosides, at which point the fermentation process should be interrupted. The efficiency of the fermentation can preferably be evaluated by using the following formula:

concentration of alcohol produced x 100

Theoretical volume of alcohol that could be produced Interrupting the fermentation can be carried out by separating the yeast from the unrefined alcohol. In this invention, unrefined alcohol is understood to be the product resulting from the fermented must that contains alcohol and impurities deriving from the fermentation process.

Separating the yeast can be achieved by methods known in the state of the art, such as centrifugation and induced flocculation. The yeast separated from the unrefined alcohol can be treated with antibiotics and diluted, to be used in a new fermentation, which may be subsequent or not.

The unrefined alcohol obtained can then be distilled to obtain more concentrated ethanol, in the form of hydrated alcohol, which can then be used in the production of anhydrate ethyl alcohol. The whole process should be carried out in an environment free of potential contaminants, principally those bacteria capable of interacting with the yeast causing undesirable flocculation, which will reduce the efficiency of the conversion of sugars into alcohol, thus impairing the productivity of the process. Decontamination of the yeast culture can be obtained by the final concentration of ethanol itself or by adding bactericides and antibiotics to said culture.

Claims

ClaimsPRODUCTION OF ETHANOL FROM ALGAE

1. Process of producing alcohol from algae characterized by being obtained by the hydrolysis of gelling polysaccharides obtained from algae cultivated in natural or artificial aquatic environments; eutrophized or not eutrophized; being obtained from the steps of collecting and cleaning of the algae; drying; fragmentation; hydrolysis of the total sugars produced by the algae; fermentation of the gelling polysaccharides due to the use of yeast capable of promoting the fermentation of galactosides; and distillation of alcohol.

2. Process according to claim 1 , characterized by using algae of the Gelidium, Gigardina, Gracilaria, Encheuma and Pterocladia species.

3. Process according to claim 1 , characterized by obtaining after the drying step algae with a maximum humidity content of 50%.

4. Process according to claim 3, characterized by obtaining algae with a maximum humidity content of 25%.

5. Process according to claim 1 , characterized by fragmenting the algae by using shredding and/or chopping procedures.

6. Process according to claim 1 , characterized by hydrolyzing the carbohydrate polymers by physio-chemical and/or enzymatic processes.

7. Process according to claim 1 , characterized wherein the physio- chemical process is acid hydrolysis, in a solution containing on average the equivalent of: 20% dried algae;

5% acid; 75% water.

8. Process according to claim 1 , characterized by using an agarolitic and/or carragenanolitic enzyme; or a mixture of agarolitic and/or carregenanolitic enzymes in the enzymatic hydrolysis.

9. Process according to claim 1 , characterized by separating the total sugars obtained in the prior step by filtration.

10. Process according to claim 1 , characterized by forming a fermentative liquid must free of solid particles and free of contaminations; containing between 13° to 17° Brix of fermentable carbohydrates.

11. Process according to claim 1 , characterized by adding the must to a vat that may or may not contain yeast capable of fermenting L-galactosides and glycosides.

12. Process according to claim 11 , characterized by using a fermentatively efficient quantity of the following yeast types: Candida sp; Clavispora sp;

Debaryomyces sp; Geotrichum sp; lssatchenkia sp; Kloeckera sp; Kluyveromyces sp; Metschnikowia sp; Pachysolen sp; Pichia sp; Kazachstania spp; Rhodotorula sp; Saccharomyces sp; Torulaspora sp; Williopsis sp; Zygosaccharomyces sp.

13. Process according to claim 12, characterized by preferably using the following yeast types: Candida sp, Kloeckera sp; Kluyveromyces sp; Pichia sp; Rhodotorula sp; Saccharomyces sp; Kazachstania spp; and Torulaspora sp.

14. Process according to claim 1 , characterized by being carried out at a temperature between 20 and 70⁰C, in acidic pH, at a pressure between 1 and 3atms.

15. Process according to claim 14, characterized by anaerobic fermentation; at a temperature between 30 and 55°C; pressure between 1 and 2atms and slightly acid pH.

16. Process according to claim 1 , characterized by separating the yeast from the unrefined alcohol.

17. Process according to claim 1 , characterized by distilling the unrefined alcohol to obtain pure ethylic alcohol.