WO2012085689A1

WO2012085689A1 - Improved method for obtaining fermentable sugars based on microalgae and macroalgae

Info

Publication number: WO2012085689A1
Application number: PCT/IB2011/052398
Authority: WO
Inventors: Laura Liliana GARZON FUENTES; Kafarov VIATCHESLAV; Andrés Fernando BARAJAS SOLANO; Manuel Laureano NUÑEZ ISAZA
Original assignee: Ecopetrol S.A.
Priority date: 2010-12-23
Filing date: 2011-05-31
Publication date: 2012-06-28
Also published as: CO6470093A1

Abstract

Improved method for obtaining fermentable sugars based on microalgae and macroalgae. The method comprises treating the algal biomass with a mixture of methanol and sulphuric acid at a temperature of between 100 and 200°C and at a pressure of between 101.14 kPa and 303.42 kPa. This treatment breaks down the cell wall, thereby facilitating extraction of carbohydrates and increasing the fermentable-sugar hydrolysis yield.

Description

IMPROVED PROCESS OF OBTAINING FERMENTABLE SUGARS FROM MICROALGAS AND MACROALGAS

TECHNOLOGICAL SECTOR

The present invention is in the field of obtaining fermentable sugars from the biomass of microalgae and macroalgae grown in natural or artificial media. The obtaining of fermentable sugars is carried out through physical-chemical processes capable of extracting the total carbohydrate content present in algal biomass.

The improved process of this invention weakens the cell wall, thus facilitating the extraction of carbohydrates and increasing the hydrolysis performance of fermentable sugars.

STATE OF ART

The constant change in the prices of fossil fuels, the uncertainty of the depletion of oil reserves in oil wells and the release of large amounts of pollutant gases that these fuels emit into the atmosphere, have led to the development of new technologies that promote the degradation and exploitation of plant biomass, without compromising food production for the production of biofuels.

In this search, the study of microalgae as a potential raw material to develop new energy sources has begun, which is advantageous since these microorganisms do not require large areas for cultivation, and reduce greenhouse gas concentrations, as They capture carbon dioxide as a nutrient for their growth.

Microalgae are photosynthetic, non-vascular microorganisms that contain chlorophyll, have simple reproductive structures and float freely, but with limited mobility. Depending on the way in which their nutrients are obtained, algae can be autotrophic, auxotrophic and heterotrophic.

The microalgae are rich in carbohydrates, which are mainly found in the cell wall, do not contain lignin, which facilitates the extraction of reducing sugars, avoiding later stages of hydrolysis, requiring only one stage to release the carbohydrates used in the stage of fermentation.

The chemical composition of microalgae varies depending on the species, nutrients, temperature, carbon source, intensity and color of light, among others. It has been found that in an average chemical composition they contain 30% protein, 25% carbohydrates, 21% lipids and the rest of unclassified compounds.

The extraction of starches contained in the algae is carried out by saccharification or hydrolysis with sulfuric acid in a concentration of 5% to 25% with respect to the starch to be treated.

There are different process reports whose raw material is algae. Among them is patent application GB1493480A, describes a process for producing ethanol from starch obtained by the cultivation of a unicellular algae with a culture medium containing a source of carbon and assimilable nitrogen and inorganic salts. After the culture, the cells are broken and subsequently the starch that is saccharified is extracted. The saccharified starch is then fermented with an ethanol producing microorganism. Preferred algae species are Chlorella vulgaris, Chlamydomonas sp., And Scenedesmus basilensis grown between 25 and 42 ° C between pH 5 to 9. The culture medium contains glucose, maltose or acetic acid from an industrial residue. The starch can be saccharified with sulfuric acid or through an enzymatic process. The saccharification process of starch is carried out with temperature and dilute sulfuric acid. Specifically in the Pretreatment with aqueous sulfuric acid (with a ratio of 2: 1 to 5: 1 water: sulfuric acid) is added in an amount of 5-25% sulfuric acid with respect to the amount of starch to be treated. The acidified medium is heated in a water bath at 100 ° C for 30 minutes. The acid solution is then diluted 2 to 5 times and the saccharification operation is completed by heating at 120 ° C under a pressure of 2 kg / cm2 for 30 minutes. The liquor obtained is then neutralized to a pH of 5 with a suspension of fine particles of calcium hydroxide in water.

Despite this process of using physical-chemical treatments capable of hydrolyzing carbohydrates, an amount of an organic solvent such as methanol or ethanol is not used in the hydrolysis process to promote the separation of hydrolyzed carbohydrates in a solid phase at a later stage than Hydrolysis process, there is no solids separation process and the process does not include the possibility of mixing the treated stream of oligosaccharides from the algae with a stream of sugar cane molasses.

Another patent application related to algae is WO 2009/067771 which is directed to a process for obtaining alcohol from the fermentation of hydrolyzed sugars from gelling agents and / or polysaccharides from algae grown in natural or artificial aquatic environments, eutrophied or not. The main steps of the process disclosed there are: collection, cleaning and treatment of the algae for the removal of toxic elements and / or inhibitory microorganisms, fragmentation, drying, hydrolysis of the total sugars produced by the algae, fermentation of the sugars resulting from the hydrolysis of the algae polysaccharides using a yeast capable of promoting the fermentation of galactosides and finally, the distillation of alcohol.

The physicochemical hydrolysis or treatment used in this process is acid hydrolysis at a temperature between 60 and 120 ° C and pressure between 1 and 3 atm. Preferably hydrolysis occurs at a pressure between 1 and 2 atm. The physicochemical process of acid hydrolysis employs a solution with an average content of 20% fermentable substrate, 5% acid in a concentration adjusted according to the type of polymer and 75% water. The acid may be hydrochloric acid or other acids that do not alter the chemical structure of the resulting monomers.

In this case, the macroalgae used are of the species Gelidium, Gigardina, Gracilaria, Encheuma and Pterocladia.

Within this context, patent application WO / 2009/058471 was also found, which describes a process for obtaining ethanol and biodiesel from cellulose comprising; to. Send a stream of cellulose to one or more containers and a mixing tank of cellulose and algae cellulose residues; b. hydrolyze cellulose to form hydrolyzed cellulose by the action of one or more fungi of the Neocallimastigomycota family; C. carry out a liquefaction of the hydrolyzed cellulose in order to separate the hydrolyzed hexoses and pentoses; d. separate the sugars to form xylitol and reduced sugars; and. ferment the reduced sugars to obtain fuel ethanol; F. Feed the algal culture with the xylitol stream and with carbon dioxide generated during the hydrolysis of the cellulosic material; g. Send the regenerated algal culture to a biodiesel reactor to produce biodiesel and obtain residual carbohydrate biomass for ethanol production.

In addition to the aforementioned documents, patent application WO / 2007/101 172 discloses a process for the production of ethanol from algae biomass starch, a process that begins with the rupture of biomass, then fermentation in the presence of yeast and the separation of the ethanol produced. The invention further relates to the processing of residual biomass from ethanol production for the recovery of biodiesel and / or generation of heat and carbon dioxide by combustion. The selected algae belong to the family Zygnemataceae, Cladophoraceae, Oedogoniales, Ulvophyceae, Charophyceae, or combinations thereof or selected algae from Spirogyra, Cladophora, Oedogonium, or combinations thereof.

Finally, WO2010046619A1 patent presents a process for the production of ethanol from cellulosic material. Said process comprises the following steps (i) hydrolysis of the cellulosic material with an aqueous solution of an acid to produce a hydrolyzate, (ii) extraction of the acid and water from the hydrolyzate with an organic solvent of water-miscible extraction to produce (a) an aqueous acid solution containing the extraction solvent and (b) a residue containing the sugars, (iii) a cleavage process of the residue containing the sugars to produce an aqueous solution of fermentable sugars, (iv) a fermentation step of the sugars and distillation of the alcohol produced from the fermented mixture, (v) a stage of evaporation of the solvent for extraction of the solution (a) and of the solution resulting from the fermentation process that does not have more than 10% by weight of the solvent of extraction, and finally (vi) a condensation stage of the extraction solvent for recycling. The preferred extraction solvent is dimethyl ether or ethanol. The process of hydrolysis, acid removal and solvent removal occurs in a single stage. Acid and water extraction solvent causes oligosaccharides to precipitate. The acid used for hydrolysis in this invention is preferably sulfuric acid. The acid and water can be added separately starting with the acid and then diluting the acid to the desired concentration. The hydrolysis process is done at a temperature between 50-55 ° C. The acid-cellulosic material solution ratio is between 2: 1 to 4: 1 by weight and the preferred duration is 120 minutes. Thus oligosaccharides produced by cellulose hydrolysis can be precipitated by the extraction solvent to produce sugar sludge / lignin. The sludge precipitated after solvent extraction and removal is passed through a second stage of hydrolysis of the oligosaccharides to a temperature of 140 C and a pressure between 5 - 6 bar for approximately 120 minutes, to finally go to the fermentation stage. The mentioned patent uses only lignocellulosic materials

As can be seen, the previously described processes of pretreatment and hydrolysis applied to macroalgae and microalgae, do not show tangible results of fermentation sugar extraction efficiencies, nor the use of methanol-acid mixtures in algal biomass. Therefore, there is a need in the state of the art to have new processes that in addition to reducing stages such as hydrolysis (required in lignocellulosic biomass), in turn increase the production of fuel alcohol without the degradation of carbohydrates with a Efficient pretreatment The applicant has managed to increase the total obtaining of the carbohydrates present in the micro and macroalgae with using mixtures of organic and inorganic solvents, which is the essence of the present invention.

DESCRIPTION OF THE FIGURES

Figure 1. Presents the flow chart of the stages that allow obtaining ethanol from the algae biomass through the improved process of the present invention, the process contemplates reaction stages, circulation, solvent recovery, residual biomass separation and fermentation of carbohydrate-rich liquor.

Figure 2. Shows the level contour obtained from the results of experimental designs, taking as a response variable the concentration of reducing sugars as a function of variables such as (a) acid vs methanol, (b) time vs methanol, (c) Time vs. Acid ; maintaining time, acid concentration, methanol concentration at the central point, respectively. Figure 3. Presents the level contour of the second design of experiments taking as a response variable the concentration of reducing sugars as a function of variables such as (a) acid vs methanol, (b) time vs methanol, (c) time vs. acid; maintaining time, acid concentration, methanol concentration at the central point, respectively.

Figure 4. Illustrates the cell count performed on algal biomass treated at different methanol concentrations, keeping the fixed acid concentration and time variables.

GENERAL DESCRIPTION OF THE INVENTION

The present patent application refers to a new improved process that maximizes the extraction of fermentable sugars present in biomass from unicellular microorganisms such as microalgae and macroalgae. The product obtained from the improved process is subsequently fermented for the production of alcohols. In general, the claimed process comprises the steps of:

to. Harvest or filtration of algal biomass

b. Treatment of algal biomass with mixtures of organic and inorganic solvents such as acids and alcohols.

C. Separation of liquor or input from residual biomass

d. Recovery and recirculation of the solvent used.

The product obtained by this process is the main input for the industries producing ethanol and / or other alcohols.

It is an object of the present invention to provide an improved process where the obtaining of reducing sugars from microalgae and macroalgae for the alcohol industry is maximized DETAILED DESCRIPTION OF THE INVENTION

The algae that can be used in the alcohol production process are microalgae and macroalgae, which are grown in an artificial or natural environment, preferably in a natural environment, diluted in liquid media in containers that maintain reduced culture media (Guillard F / 2) and a space of 1/3 free with air to allow the exchange of oxygen.

The strains already established in test tubes and agar plates are replicated to liquid culture media with Guillard F / 2 for indoor conditions and with Guillard medium modified with industrial grade reagents for outdoor tanks.

The composition of the macroalgae and microalgae used in this process can be altered with environmental conditions, such as: nutrient concentration, temperature, light intensity and physiological state. The carbohydrate composition can vary considerably between different types of algae, which are generally composed of a greater proportion of galactose followed by glucose, mannose and ribose. The algae that can be used in this process are microscopic and macroscopic, among them preferably are: Botryococcus braunni, Chlorella vulgaris, Scenedesmus sp, Chaetoceros gracillis, Chaetoceros calcitrans.

The first step of this improved algal biomass processing process involves the addition of mixtures of organic solvents and inorganic solutions, such as acids, methanol, ethanol, acetone, glycol, ethylene. Weak and / or strong acids such as hydrochloric acid, sulfuric acid, phosphoric acid are used. Sulfuric acid is used in concentrations of 0.05 to 3 M, preferably 0.05 to 1 M. The concentrations of organic solvents range from: 1 to 30% (v / v), preferably between 1 to 20%. The processing time is carried out in ranges of 10 to 300 min, preferably between 150 and 270 min. Figure 1 shows the flow chart of the steps that allow obtaining Ethanol from algae biomass by means of the improved process of the present invention that includes mixtures of methanol with acid and water that allow maximizing carbohydrate extraction of microalgae and macroalgae. The algal biomass (1) filtration product is subjected to an improved treatment of the present invention (2), the liquid obtained from this treatment is separated from the solid by conventional separation methods (3), part of the residual biomass from The separation stage with a certain carbohydrate content is recirculated by means of stream 101 to the treatment reactor (2), and the solvent is recovered by conventional solvent recovery methods and is recycled by stream 102 to the treatment reactor (2 ), the liquid of the separation stage can be used pure or it can be mixed mixed with carbohydrate-rich currents such as: cane juice, corn syrup, beet juices, among others and mixtures thereof (6) and taken to a fermentation process (4) where microorganisms such as fungi, bacteria, among others (5) are added, subsequently the fermented liquor is separated from the microorganisms isms by conventional separation means (7), eliminating through the current 103 the residues of the microorganisms (A), finally the distillation of the fermented liquor (8) is carried out to obtain ethanol, ascorbic acid or others (B ).

Example 1. Improved treatment with mixtures of inorganic and organic solvents of algal biomass using methanol-sulfuric acid mixtures.

The following description is a typical example of the present invention:

The biomass used in this example is the Chaetoceros gracillis microalgae species, which was characterized in the main components, as shown in Table 1. Table 1 . Characteristics of the Chaetoceros gracillis microalgae.

Analysis Result

% Humidity 1 1 .53

% Ashes 61 .74

% Bulk, Total Rock NaCI, CaCI ₂

% Protein 9.19

% Nitrogen 1 .502

% Fat 0.061

% Calories 107.2

% Gross Fiber 0.18

% Carbs 17.47

The study of the release of sugars in algae was carried out using a composite central 2 ³ factorial design. This design raises a greater area of response with the use of axial points that allow to cover a greater range of study of the process variables such as: concentration of methanol in the liquor (% v / v), concentration H2S04 (M) and time of reaction (min). The ranges initially explored are: time: 60-180 min; Methanol concentration: 30-70% v / v; Acid concentration: 0.05-0.1 M. The axial points of the variables were: 19 and 221 min; 16 and 84% v of methanol; 0.03 and 012 M of H ₂ S0 ₄ .

Samples of 20 g of dry microalgae previously characterized (Table 1) were treated with 200 mL of a mixture of methanol-sulfuric acid and water at conditions provided for each experiment (according to table 2). This mixture was taken in an autoclave at 103.4 kPa and 121 ° C, during the reaction time specified for each run. After autoclaving, the carbohydrate-rich liquor and residual biomass were filtered off under vacuum.

To quantify the total reducing sugars (ART) in the carbohydrate-rich liquor, 0.5 mL of the different liquor samples were mixed with 0.5 mL of 3.5 Dinitrosalicylic acid (DNS) reagent in test tubes. This mixture was kept for 5 minutes in a 95 ° C bath. Subsequently immersed in an ice bath to stop the reaction and 5 mL of distilled water was added. Next, absorbance readings were performed on a spectrophotometer at 540 nm.

Following the quantification of ART, a response surface of the results obtained from the improved treatment was developed using the data of the central composite design (Table 2) where the percentage of ART was analyzed based on the operational variables through the STATISTIC computer program 9.0

Table 2. First Design of Experiments 2 ³ Central Compound

ART Performance Liquor

Methanol # H2S04 Time

Pretreatment Extraction Experiment [% v / v] [M] (min)

[gART / gBS *] * 100 (%)

1 30 0.050 0.54

60 3.13

2 30 0.050 1 .55

180 8.98

3 30 0.10 1 .84

60 10.65

4 30 0.10 1 .30

180 7.53

5 70 0.050 0.58

60 3.36

6 70 0.050 1 .25

180 7.24

7 70 0.10 0.95

60 5.50

8 70 0.10 0.77

180 4.46

9 16 0.075 2.60

120 15.06

10 84 0.075 0.39

120 2.26

11 50 0.03 0.62

120 3.59

12 50 0.12 1 .46

120 8.45

13 50 0.075 1 .04

19 6.02

14 50 0.075 3.41

221 19.75

15C 50 0.075 1 .08

120 6.25

16C 50 0.075 1 .01

120 5.85

17C 50 0.075 1 .04

120 6.02 According to the results obtained in the experimental matrix (Table 2), the contour surfaces are plotted (Figure 2), where the response variable was ART based on the variables time, methanol concentration and acid.

According to Figure 2, the surface trend shows higher yields in red areas and low yields in green areas. The maximum ART yields obtained (2% ART) for this design are achieved with higher acid concentration and lower methanol concentration and also suggests that to improve yields, an increase in acid and a decrease in concentrations of the working ranges of the variables respectively mentioned. This behavior is the same when the response surfaces are plotted at the minimum and maximum reaction time point.

According to the results obtained in the first experimental design, a second design is made increasing the range of study, where: the time used for the treatment is in the range of 15-247min and the concentration of sulfuric acid is 0.1 - 0.6 M, and the range of methanol used decreased from 0 - 16.36% (v / v) as shown in table 3.

Table 3. Second Design of Experiments 2 ³ Central Compound

ART Performance Liquor

Methanol # H2S04 Time

Experiment Pretreatment [% v / v] [M] (min)

[gART / gBS ^* ] ^* 100 extraction

1 3.32 0.202 62 4.92 28.49

2 3.32 0.202 200 9.1 1 52.75

3 3.32 0.50 62 9.30 53.85

4 3.32 0.50 200 15.79 91 .43

5 13.04 0.202 62 4.32 25.01

6 13.04 0.202 200 8.48 49.10

7 13.04 0.50 62 6.90 39.95

8 13.04 0.50 200 14.85 85.99 9 0 0.35 131 9.74 56.40

10 16.36 0.35 131 10.45 60.51

11 8.18 0.1 131 3.41 19.75

12 8.18 0.6 131 10.39 60.16

13 8.18 0.35 15 2.62 15.17

14 8.18 0.35 247 14.03 81 .24

15C 8.18 0.35 131 7.74 44.82

16C 8.18 0.35 131 7.13 41 .29

17C 8.18 0.35 131 7.00 40.53

With the results presented in table 3, it can be inferred that the highest percentage of ART obtained in the improved treatment is recorded in trial 4, this value being 15.79% ART.

Comparing the results of test 2 with 4, where the time and methanol variable was set and the acid concentration varied, the high influence of the acid was observed, where the extraction yield was favored from 52% extraction to 91%.

When comparing the results obtained from tests 9 and 10, it can be observed that by decreasing the methanol concentration and keeping the acid concentration and time constant, lower percentages of ART are obtained (from 10.45 to 9.74%). This effect is less prevalent compared to the variable time and acid concentration.

According to Figure 3, the trend that presents the results in the contour graphs, corroborate the results obtained in the previous experimental design, in addition to showing the need to work the treatments in an acid concentration up to 0.6 M, times greater than 260 min and to be able to use methanol concentrations of 0 to 16% (v / v) since it has no influence on the range studied, and to achieve extraction yields greater than 90% Example 2. Effect of methanol concentration on microalgae pretreatment for sugar extraction.

According to the results of the tests carried out in example 1 (design of experiments 2) and with the contour graphs obtained, the study of the effect of methanol on the pre-treatment of microalgae was carried out, fixed values of time and concentration were established of acid for this study, where the values taken were those in which the trend showed greater extraction of sugars (247 min and 0.6 M). Methanol concentrations were varied from 0 to 100%.

The results are presented in table 4, for the methanol-acid mixture a higher percentage of reducing sugars is obtained compared to test 1 where only sulfuric acid was added. However, high concentrations of methanol decreases the production of ART. Under pretreatment conditions of 3 (% v / v) methanol, 0.6 MH ₂ S0 ₄ , during times of 247 min, pretreatment yields up to 100% are achieved.

Table 4. Percentage of ART in the pretreatment with a constant concentration of sulfuric acid and variation in the concentration of methanol for 247 minutes.

Test Methanol H ₂ S0 ₄ Time ART (%) Yield

(% V / V) (M) (min)

1 0 0.6 247 9.54 ± 0.04 54.99

2 3 0.6 247 17.21 ± 0.02 99.19

3 10 0.6 247 17.20 ± 0.00 99.14

4 16 0.6 247 16.02 ± 0.00 92.33

5 20 0.6 247 16.03 ± 0.08 92.39

6 40 0.6 247 15.73 ± 0.03 90.66 7 60 0.6 247 12.03 ± 0.09 69.34

8 80 0.6 247 1 1 .17 ± 0.08 64.38

9 100 0.6 247 10.80 ± 0.02 62.25

A stage of severe acid hydrolysis to the residual biomass of the improved treatment was performed, in order to quantify the residual sugars in the biomass. The hydrolysis was brought to standard conditions. Addition of 72% sulfuric acid, for 1 h at 30 ° C in a thermostated bath, subsequently the 4% acid is diluted and autoclaved for 1 h at 121 ^Q C. A liquor rich in residual carbohydrates is obtained, which It takes ART analysis. The results obtained in the hydrolysis with concentrated acid are presented in Table 5.

Table 5. Carbohydrates present in the microalgae after the improved treatment.

Lost Carbohydrate

Test

Residual (%) carbohydrates

1 7.2 ± 0.01 4.83

2 0.38 ± 0.05 0

3 0.27 ± 0.02 0.68

4 0.43 ± 0.06 6.48

5 0.53 ± 0.07 5.86

6 0.39 ± 0.05 8.36

7 0.12 ± 0.03 30.93

8 4.03 ± 0.02 13.59

9 4.50 ± 0.07 13.02

In trials 1, 8 and 9 there is still evidence of carbohydrate content in the residual biomass of the treatment stage with values of 7.2, 4.03 and 3.50% of ART, respectively. The lowest yield obtained in the pretreatment stage is for test 1, followed by tests 8 and 9 with values 55, 64 and 62% yield. Without wanting to stick to a particular theory we believe that: these low yields are due to: (1) in test 1 no methanol was added during the pretreatment stage, this caused a decrease in the efficiency of the process, resulting in a lower Release of carbohydrates and (2) in trials 6 and 7, a higher concentration of methanol was added causing greater degradation of carbohydrates in possible compounds such as: furfural, hydroxymethylfurfural and acetic acid. However, when concentrations higher than 80% of methanol are used, less degradation of carbohydrates and less extraction is observed, an explanation for this is the need to have sufficient water content in the mixture so that alcohols such as methanol are efficient in the breakdown of the cell wall.

To verify the greater degradation of the cell wall with methanol increments, the method of counting microalgae cells in Neubauer chamber was performed, as can be seen in Figure 4, this experiment allows to demonstrate the greater degradation of the cell wall with greater methanol concentrations, and consequently it could be affirmed that higher carbohydrate contents would be exposed to higher concentrations of acid (by not consuming acid in the cell wall rupture) thus achieving their degradation.

With this example it is possible to demonstrate the need to add small concentrations of methanol to the process of obtaining fermentable sugars from microalgae and macroalgae biomass to obtain yields close to 100% ART. In turn, it can be seen that excess methanol can cause degradation in carbohydrates, reaching degradation values of 30%, as was the case in trial 8.

Claims

one . An improved treatment process applied to biomass from microalgae and macroalgae, which includes a physical-chemical treatment of algal biomass through mixtures of organic solvents with inorganic acids, at a temperature between 100 ° C and 200 ° C, pressure between 101, 14 kPa and 303.42 kPa for a time between 10 and 300 minutes, characterized in that the organic solvent is methanol and the inorganic acid is sulfuric acid.

2. The process of claim 1 characterized in that the methanol concentration is between 1% and 30% v / v and the sulfuric acid concentration is between 0.05. M and 3 M.

3. The process according to claim 2 characterized in that the concentration of sulfuric acid is preferably between 0.075 and 1 M.

4. The process according to any of the preceding claims, characterized in that the concentration of methanol is preferably between 1 and 20% v / v.

5. The process according to claim 1, characterized in that the treatment time is preferably carried out at intervals of 150 and 270 min.

6. The process according to claim 1, characterized in that the temperature used is in the range of 100 ° C to 150 ° C.

7. The process according to claim 1, characterized in that the algal biomass is selected from the group consisting of Botryococcus braunni, Chiorella vulgaris, Scenedesmus sp, Chaetoceros gracillis, Chaetoceros calcitrans.

8. A fermentation process comprising the product of the process of claims 1 to 7 and bacteria, yeasts and fungi or mixtures thereof.