ES2709476T3 - Dehydration methods in fermentation processes - Google Patents

Abstract

A method of dehydrating a whole stillage in a fermentation process, comprising: (a) saccharifying a cellulosic material with an enzymatic composition; (b) fermenting the saccharified cellulosic material with a fermentation organism to produce a fermentation product, wherein the fermentation medium comprises a xylanase and a pectinase; (c) distilling the fermentation product to form the whole stillage; and (d) separating the whole stillage into fine stillage and wet cake, whereby more liquid phase is transferred to the fine stillage, where the cellulosic material is corn and the fermentation product is ethanol.

Description

DESCRIPTION

Methods of dehydration in fermentation processes

Field of the invention

The present description relates to an improved process for dehydrating the entire vinasse in fermentation processes.

Background of the invention

Fermentation products, such as ethanol, are produced by first degrading the starch-containing material in fermentable sugars by liquefaction and saccharification, and then converting the sugars directly or indirectly into the desired fermentation product using a fermentation organism. The liquid fermentation products such as ethanol are recovered from the fermented dough (often referred to as "beer" or "beer dough"), for example, by distillation, which separates the desired fermentation product from other liquids and / or solids. The remaining fraction, known as "whole vinasse", is dehydrated and separated into a solid phase and a liquid phase, for example, by centrifugation. The solid phase is called "wet cake" (or "wet beans" or "WDG") and the liquid phase (supernatant) is called "fine vinasse". The dried dehydrated cake is dried to provide "dried distillers grains" (DDG) used as nutrients in animal feed. The fine vinasse typically evaporates to provide condensate and syrup (or "thick vinasse") or, alternatively, can be recycled directly to the porridge tank as "backset". The condensate can be sent to a metanator before being discharged or it can be recycled to the papilla tank. The syrup, which consists mainly of dextrins limit and non-fermentable sugars, can be mixed with DDG or added to the wet cake before drying to produce DDGS (dry grains of distillery with solubles).

Ethanol plants have struggled to maintain profitability, which is highly variable depending on the price of corn, the demand and the price of DDGS, tax credits, gasoline consumption, ethanol exports and changes in the standards of renewable fuels. (RFS). New technologies for energy saving, higher ethanol yield and higher value for co-products, as well as several oil separation technologies, contribute to the profitability of ethanol production.

WO2012 / 084225 and WO2007 / 056321 describe the treatment of fermented dough (beer) after fermentation with enzymes or enzymatic compositions to dehydrate the entire vinasse.

WO2011 / 126897 describes the use of an enzymatic composition comprising a hemicellulase, an endoglucanase and the GH61 polypeptide in a fermentation process to dehydrate the entire vinasse.

U.S. patent application No. 2005/0079270 A1 discloses a method for dehydrating maize vinasse solids comprising adding to the solids an anionic copolymer comprising sodium salt of acrylic acid, sodium salt of methacrylic acid or sodium salt of 2-acrylamido-2-methyl acid -1-propanesulfonic to form a mixture of water and coagulated and flocculated solids; and separating the water from the coagulated and flocculated solids using a dehydration device.

The dehydration of the entire vinasse requires a lot of energy and can consume up to a third of the energy needs of an ethanol producing plant or a similar fermentation product. Therefore, there is a need to improve the processes involved in the dehydration of the entire vinasse.

Compendium of the description

The present description refers to a method of dehydration of the vinasses obtained from a process for obtaining a fermentation product.

The present description relates to methods for dehydrating a whole vinasse in a fermentation process, comprising:

(a) saccharification of a cellulosic material with an enzymatic composition;

(b) fermentation of the saccharified cellulosic material, with a fermentation organism to obtain a fermentation product wherein the fermentation medium comprises a xylanase and a pectinase;

(c) distilling the fermentation product to form the entire vinasse; Y

(d) separating the whole vinasse into fine vinasse and wet cake, thereby transferring more liquid phase to the fine vinasse, where the cellulosic material is maize and the fermentation product is ethanol.

In a process for increasing the dehydration of the whole vinasse, the liquefied whole grain mass can be diluted by treatment with an efficient amount of enzymatic activity of a xylanase in combination with a pectinase.

The process for the dehydration of a whole vinasse in a fermentation process can sequentially comprise the following steps: a) grind the whole grain; b) liquefying gelatinized ground whole grain, in the presence of an alpha-amylase; c) saccharify the liquefied material in the presence of a glucoamylase; d) ferment with a microorganism; e) distilling the fermented and saccharified material, providing a fraction of ethanol, wherein the liquefied mass is subjected to an effective amount of enzymatic activity of a xylanase and a pectinase during fermentation.

Brief description of the drawings

Figure 1 shows schematically an ethanol production process.

Description of the invention

The object of the present invention is to provide a method for the dehydration of whole vinasse. The addition to the fermentation medium of a xylanase in combination with a pectinase results in a wet cake with a drier mass. The advantage here is the lower consumption of energy in drying.

The present description relates to methods for dehydrating a whole vinasse in a fermentation process, comprising:

(a) saccharification of a cellulosic material with an enzymatic composition;

(b) fermenting the saccharified cellulosic material with a fermentation organism to obtain a fermentation product, wherein the fermentation medium comprises a xylanase and a pectinase;

(c) distillation of the fermentation product to form the entire vinasse; Y

(d) separating the whole vinasse into fine vinasse and wet cake, thereby transferring more liquid phase to the fine vinasse,

where the cellulosic material is maize and the fermentation product is ethanol.

The vinasse or whole vinasse is the product that remains after the dough has been converted into sugar, fermented and distilled in ethanol. The vinasse can be separated into two fractions, such as by centrifugation or screening: (1) wet sponge cake (solid phase) and (2) fine vinasse (supernatant). The solid fraction or wet distillate grain (DWG) can be pressed to remove excess moisture and then dried to produce dried grains of distillate (DDG). After the ethanol has been removed from the liquid fraction, the residual liquid can be evaporated to concentrate the soluble material in distilled condensed (DS) or dried and ground solids to create distilled dried solubles (DDS). DDS are often mixed with DDG to form dry distillate grain with solubles (DDGS). DDG, DDGS and DWG are collectively referred to as grain or grains of distillery.

The enzymes xylanase and pectinase are added during fermentation. The enzymes were able to degrade components in the fermentation medium.

The phrase "fermentation means" or "fermentation medium" refers to the environment in which the fermentation is performed and comprises the fermentation substrate, ie, the source of carbohydrates that is metabolized by the fermentation organism or organisms.

The fermentation medium can comprise other nutrients and growth stimulants for the fermentation organism. Nutrients and growth stimulators are widely used in the fermentation technique and include sources of nitrogen, such as ammonia, vitamins and minerals, or combinations thereof. After fermentation, the fermentation product can be separated from the fermentation medium. The fermentation medium can be distilled to extract the desired fermentation product, or the desired fermentation product can be extracted from the fermentation medium by microfiltration or membrane techniques. Alternatively, the fermentation product can be recovered by entrainment. The recovery methods are well known in the art.

The raw material to produce ethanol is maize.

The obtaining of a fermentation product is typically divided into the following main stages of the process:

a) reducing the particle size of the starch-containing material, for example, by dry or wet milling;

b) cooking the starch-containing material in an aqueous slurry to gelatinize the starch,

c) liquefying the gelatinized material containing starch to break the starch (by hydrolysis) into maltodextrins (dextrins);

d) saccharification of maltodextrins (dextrins) to produce low molecular weight sugars (eg, DP1-2) that can be metabolized by a fermentation organism;

e) fermentation of the saccharified material using a suitable fermentation organism that directly or indirectly converts the low molecular weight sugars into the desired fermentation product;

f) recovery of the fermentation product, for example, by distillation, to separate the fermentation product from the fermentation mass.

As also explained in the previous section "Background", the whole vinasse is a by-product consisting of liquids and solids that remain after the recovery (eg, by distillation) of a desired fermentation product of the fermented dough ( beer mixture). According to the invention, the fermentation product.

The entire vinasse contemplated according to the present description is the secondary product resulting from a process of preparation of the fermentation product, which includes the steps a) to c) mentioned above.

Dehydration of the entire vinasse in order to remove a significant portion of the liquid / water can be done using any suitable separation technique, including centrifugation, pressing and filtration. The whole vinasse can be heated to a temperature of about 20-60 ° C. The pH is in the range of 3 to 7, preferably pH of 3 to 6.

In a preferred embodiment, the dehydration is carried out by centrifugation. The preferred centrifuges in the current industry are decanter type centrifuges, preferably high speed decanter type centrifuges. An example of a suitable centrifuge is the Alfa Laval NX 400 conical series, which is a high performance decanter.

In another preferred embodiment, the separation is carried out using other conventional separation equipment, such as plate / frame filter presses, band filter presses, screw presses, thickeners and gravity covers, or similar equipment.

After having dehydrated, the wet cake, which contains about 30-35% by weight of dry solids, can be dried in a drum dryer, spray dryer, ring dryer, fluidized bed dryer, or the like, to produce "Destilena Dry Grains" (DDG). DDG is a valuable feed ingredient for livestock, birds and fish. It is preferred to provide the DDG with a content of less than about 10-12% by weight of moisture to avoid decomposition by molds and microbes, and to increase the shelf life. In addition, a high moisture content makes it more expensive to transport DDG. The wet cake is preferably dried under conditions in which the proteins of the wet cake are not denatured. The wet cake can be mixed with a syrup separate from the fine vinasse fraction, and dried in DDG with solubles (DDGS).

Maize is the raw material and the fermentation product is ethanol.

As mentioned above, beer (or fermented dough) comprises the fermentation product ethanol.

The fermentation organism can be a fungal organism, such as yeast, or bacterial. Suitable bacteria can be, for example, Zymomonas species , such as Zymomonas mobilis and E. coli. Examples of filamentous fungi include strains of Penicillium species . The preferred organisms for the production of ethanol are yeasts, such as, for example, Pichia or Saccharomyces. Preferred yeasts according to the description are species of Saccharomyces, in particular Saccharomyces cerevisiae or baker's yeast.

Furthermore, by adding the enzymes to the fermentation medium before the distillation step, an advantage is obtained since the enzymes in the enzymatic compositions are inactivated during the distillation.

The processes for producing ethanol from maize usually begin with grinding the maize in a dry milling or wet milling process. Wet milling processes involve splitting the maize into different components where only the starch fraction enters the fermentation process. The dry milling processes involve grinding the corn kernels in flour and mixing the flour with water and enzymes. In general, two different types of dry milling processes are used. The most commonly used process, often referred to as "conventional process", includes grinding the starch-containing material and then liquefying the gelatinized starch at an elevated temperature using typically a bacterial alpha-amylase, followed by simultaneous saccharification and fermentation (SSF) performed in the presence of a glucoamylase and a fermentation organism. Another well-known process, often referred to as the "starch hydrolysis process" (RSH process), includes grinding the starch-containing material, and then simultaneous saccharification and fermentation of the granular starch below the initial gelatinization temperature, Typically in the presence of a fungic acid alpha-amylase and a glucoamylase.

In a process to produce ethanol from maize, after the SSF or RSH process, the ethanol is distilled from the whole vinasse after fermentation. The resulting ethanol free slurry, generally known as whole vinasse, is separated into solid and liquid fractions (ie, wet cake and fine vinasse containing approximately 35 and 7% solids, respectively). The fine vinasse is often condensed by evaporation giving a thick vinasse or syrup, recombining with the wet cake and continuing drying in dry distiller's grains with soluble distilled dry grain, with solubles (DDGS) for use in animal feed. In the claimed method, SSF and RSH are not used.

The xylanase may preferably be of microbial origin, such as of fungal origin (eg, Aspergillus, Fusarium, Humicola, Meripilus, and Trichoderma) or of a bacterium (eg, Bacillus). In a preferred embodiment, the xylanase is derived from a filamentous fungus, preferably derived from an Aspergillus strain, such as Aspergillus aculeatus; or a strain of Humicola, preferably humicola lanuginosa. Examples of useful xylanases in the methods of the present invention include, but are not limited to, xylanase from Aspergillus aculeatus (GeneSeqP: AAR63790; WO 94/21785), xylanases from Aspergillus fumigatus (WO 2006/078256), and xylanases from Thielavia terrestris NRRL 8126 (WO 2009/079210). The xylanase may preferably be an endo-1,4-beta-xylanase, more preferably an endo-1,4-beta-xylanase from GH 10 or GH 11. Examples of commercial xylanases include SHEARZYME ™, BIOFEED WHEAT ™, HTec and HTec2 from Novozymes A / S, Denmark. Examples of beta-xylosidases useful in the methods of the present invention include, but are not limited to, beta-xylosidase from Trichoderma reesei (UniProtKB / TrEMBL registration number Q92458), Talaromyces emersonii (Swiss Prot registration number Q8X212) and Neurospora crassa (SwissProt registration number Q7SOW4).

Examples of suitable bacterial xylanases include xylanases derived from a Bacillus strain, such as Bacillus subtilis, such as that described in U.S. Patent No. 5,306,633.

Commercially available contemplated xylanases include SHEARZYME ™, BIOFEED WHEAT ™ (from Novozymes AJS), Econase CE ™ (from AB Enzymes), Depol 676 ™ (from Biocatalysts Ltd.) and SPEZYME (TM) CP (from Genencor Int.).

The xylanase can be added in an effective amount in the range of 0.16 x 10 6 to 460 x 10 6 units per ton of beer mass or fermentation medium.

Determination of the activity of xylanase (FXU)

The activity of the endoxylanase is determined by an assay in which the sample of xylanase is incubated with a substrate of remazolxylan (4-O-methyl-D-glucurono-D-xylan taken with Remazol Brilliant Blue R, Fluka), pH 6 , 0. The incubation is carried out at 50 ° C for 30 min. The bottom of the substrate, which has not been degraded, is precipitated with ethanol. The remaining blue color in the supernatant is determined spectrophotometrically at 585 nm and is proportional to the endoxylanase activity. The endoxylanase activity of the sample is determined in relation to an enzymatic standard.

The pectinase used in the methods according to the present disclosure can be any pectinase, in particular of microbial origin, in particular of bacterial origin, such as a pectinase derived from a species within the genera Bacillus, Clostridium, Pseudomonas, Xanthomonas and Erwinia. , or of fungic origin, such as a pectinase derived from a species within the genera Trichoderma or Aspergillus, in particular from a strain within the species A. niger and A. aculeatus. Pectinases contemplated commercially include Pectinex Ultra-SPL ™ (from Novozymes), Pectinex Ultra Color (from Novozymes), Rohapect Classic (from AB Enzymes) and Rohapect 10L (from AB Enzymes). Pectinase can be added in an effective amount in the range of 1.4 x 109 to 23,500 x 109 Units per ton of fermentation medium.

Determination of the pectintranseliminase unit (PECTU)

The method is based on the enzymatic degradation of a pectin solution by a reaction with transeliminase, the double bonds formed produce an increase in absorption at 238 nm, which is followed by a spectrophotometer. Reaction conditions

Temperature: 30 ° C ± 0,5 ° C.

pH: 3.50 ± 0.02

Substrate: 0.24% Pectin (Obipektin, Brown Ribbon Pure, Art. No. 1.1B00.A. Lot No. 0304)

Enzyme concentration: 1.9-2.3 PECTU / mL

Reaction time: 6 minutes

Measurement time: 5 minutes

Wavelength: 238 nm

The activity is determined in relation to a PECTU standard. The result is given in the same units as for the standard, which is designated: PECTU- Pectintranseliminase Unit.

The term "alpha-amylase" means an alpha-1,4-glucan-4-glucanhydrolase (EC 3.2.1.1) which catalyzes the hydrolysis of starch and other linear and branched 1,4-glucosphide oligo- and poly-saccharides.

In one embodiment, the xylanase is added in an amount of 1-30, for example, 5-30, 7-25, 10-20, 10-17 or 12-15 micrograms / g of dry solids.

In one embodiment, the pectinase is added in an amount of 0.01-1.0, for example, 0.015-0.08, 0.015-0.06, 0.015 0.04, or 0.02-0.03 FXU / g of dry solids.

The saccharification and fermentation stages are carried out sequentially. Xylanase and pectinase are added during fermentation.

As mentioned above, the fermenting organism is preferably yeast, for example, a strain of Saccharomyces cerevisiae or Saccharomyces diastaticus. In an advantageous embodiment a yeast strain of Saccharomyces diastaticus (SIHA Amyloferm®, E. Begerow GmbH & Co, Langenlonsheim, Germany) is used since its exo-amylase activity can divide the liquid starch and also dextrin, maltose and melibiose.

In the liquefaction stage, the gelatinized starch (downstream mass) is broken (hydrolyzed) into maltodextrins (dextrins). To achieve the hydrolysis of the starch, a suitable enzyme is added, preferably an alpha-amylase. The liquefaction can be carried out as a three-stage hot porridge process. The suspension is heated to a temperature between 60-95 ° C, preferably 80-85 ° C, and alpha-amylase can be added to initiate liquefaction (thinning). Then, the suspension may be fired in je t at a temperature between 95 and 140 ° C, preferably 105-125 ° C, for about 1-15 minutes, preferably for about 3-10 minutes, especially around 5 minutes. The porridge is cooled to 60-95 ° C and more alpha-amylase can be added to complete the hydrolysis (secondary liquefaction). The liquefaction process is generally carried out at a pH of 4.0 to 6.5, in particular at a pH of 4.5 to 6.

The saccharification stage and the fermentation stage are carried out as separate process steps. The saccharification is carried out in the presence of a saccharifying enzyme, for example a glucoamylase, a beta-amylase or a maltogenic amylase. Optionally, a phytase and / or a protease is added.

Saccharification can be carried out using conditions well known in the art, with a saccharifying enzyme, for example, beta-amylase, glucoamylase or maltogenic amylase, and optionally a debranching enzyme, such as an isoamylase or pullulanase. For example, a complete saccharification process can last from about 24 to about 72 hours.

The saccharification is typically carried out at a temperature of 20-75 ° C, preferably 40-70 ° C, typically around 60 ° C, and at a pH between 4 and 5, usually at about pH 4.5.

During the fermentation, the fermentation medium is subjected to an enzymatic composition according to the present description. The enzymatic composition comprises a xylanase and a pectinase.

The process of the present description may further comprise, before liquefying the starch-containing material, the steps of:

- reducing the particle size of the starch-containing material, preferably by grinding; Y

- forming a porridge comprising the material containing starch, and water.

The aqueous suspension may contain from 10 to 55% w / w of dry solids (DS), preferably 25-45% w / w of dry solids (DS), more preferably 30-40% w / w of dry solids (DS) of the material that contains starch. The suspension is heated above the gelatinization temperature and an alpha-amylase, preferably a bacterial alpha-amylase and / or fungic acid, can be added to initiate liquefaction (thinning). The porridge can be cooked in je t to further gelatinize the suspension before subjecting it to an alpha-amylase in step (a).

The starch-containing material is maize that can be milled and the methods comprise a maize milling step before step (a). In other words, the description also encompasses methods in which the starch-containing material can be obtained by a process comprising milling the maize, preferably dry milling, for example by hammer or roller mills.

Grinding is also understood as grinding, as is any process suitable for opening individual grains and exposing the endosperm for further processing. Two milling processes are normally used in the production of alcohol: wet and dry milling. The expression "dry milling" denotes the milling of the whole grain. In dry milling all the grain is milled and used in the remaining part of the dough formation process. The dough can be provided by forming a slurry comprising the ground material containing starch and working water. The preparation water can be heated to a suitable temperature before being combined with the ground starch-containing material to achieve a mass temperature of 45 to 70 ° C, preferably 53 to 66 ° C, more preferably 55 to 60 ° C C. The dough is typically formed in a tank known as a porridge tank.

After fermentation, the ethanol from the fermentation product can be separated from the fermentation medium. The porridge can be distilled to extract the ethanol from the fermentation medium by means of microfiltration or membrane. Alternatively, the fermentation product can be recovered by entrainment. The methods for recovering the fermentation products are well known in the art. Typically, ethanol is obtained with a purity of up to, for example, about 96 vol.%. of ethanol.

After the completion of the fermentation process, the remaining material is considered the entire vinasse. As used herein, the term "whole vinasse" includes the material left at the end of the fermentation process after the ethanol recovery. The fermentation product can optionally be recovered by any method known in the art. The whole vinasse is separated or divided into a solid phase and an ifquid phase by one or more methods to separate the fine vinasse from the wet cake. Such methods include, for example, centrifugation and decanting. The fermentation product can be recovered before separating the whole vinasse into a solid phase and a liquid phase.

Thus, the methods of the description comprise the distillation to obtain ethanol as a fermentation product. Fermentation and distillation are carried out sequentially, optionally followed by one or more process steps to continue the refining of the fermentation product.

The aqueous by-product (whole vinasse) of the distillation process is separated into two fractions, for example, by centrifugation: wet grain (solid phase) and fine vinasse (supernatant). The methods of the description also comprise the separation of the whole vinasse produced by distillation, in wet bean and fine vinasse; and may comprise recycling the fine vinasse to the starch-containing material before liquefying. The fine vinasse can be recycled to the ground whole grain porridge. The wet grain fraction can be dried, typically in a drum dryer. The dried product is known as dried grains of distillery, and can be used as mentioned above as high quality animal feed. The fine vinasse fraction can be evaporated providing two fractions (see Fig. 1 (i) a 4-6% SD condensate fraction (mainly starch, protein, oil and cell wall components), and (ii) a fraction of syrup, consisting mainly of dextrins limit and non-fermentable sugars, which can be introduced in a dryer along with the wet grains (from the stage of separation of the whole vinasse) to provide a product called dry grain of distillate with solubles , which can also be used as animal feed.Fine vinasse is the term used for the supernatant of the whole vinasse centrifugation.Typically, the fine vinasse contains 4-6% of DS (mainly starch and proteins) and has a temperature in about 60-90 ° C. In another embodiment, the fine vinasse is not recycled, but the condensate stream from the evaporated fine vinasse is recycled to the slurry containing the whole milled grain to be cooked in jet.

Those skilled in the art are aware of more details on how to carry out liquefaction, saccharification, fermentation, distillation and ethanol recovery.

Methods for dehydrating the vinasses of a fermentation product are known in the art. These methods include decanting or otherwise separating whole vinasse into wet cake and fine vinasse. See, for example, US patents. UU No. 6,433,146, 7,601,858 and 7,608,729, and United States Application Publication No. 2010/0058649.

The ethanol from the fermentation product can optionally be recovered from the fermentation medium using any method known in the art including, but not limited to, chromatography, electrophoretic procedures, differential solubility, distillation or extraction. For example, the alcohol is separated from the fermented cellulosic material and purified by conventional distillation methods mentioned above. Ethanol can be obtained with a purity of up to about 96% by volume which, for example, can be used as ethanol for fuel, ethanol for beverages, ie potable neutral liquors, or industrial ethanol.

The present invention is described in more detail by the following examples.

Examples

Dehydration of the whole vinasse

In the specification, a better dehydration capacity of the entire vinasse results in a wet cake with a drier mass. The advantage here is a lower energy consumption during drying.

For this example, the dewatering capacity of the entire vinasse during centrifugation was tested and determined.

Four different configurations were tested.

In the first fermentor (Fermentor # 1), the fermentation culture was not treated with enzymes (0 g / t of xylanase, 0 g / t of pectinase), in the second, third and fourth fermenters (Fermentor no. 4) the fermentation culture was treated with the enzymatic composition comprising a xylanase and a pectinase from the start of the fermentation in different concentrations (from 25 g / t to 200 g / t xylanase and from 25 g / t to 200 g / t of pectinase).

The tests were carried out on a fermentation scale of 2 L. After distillation, samples were taken from the fermenters with the whole vinasse and the whole vinasse was centrifuged to obtain a fine vinasse and the cake moist. Dehydration [%] was calculated from the fine vinasse mass [mg] divided by the whole vinasse mass [mg] multiplied by 100.

In one embodiment, the process of ethanol production from maize was carried out as follows:

A) Process for obtaining fermentation products

a) Reduction of the particle size of the starch-containing material by grinding

- the maize (Compama Pannonia, Hungna) was ground to a particle size of <2 mm (coffee grinder, Brunn company)

b) Formation of a porridge comprising the material containing starch, and water

- 1.5 kg of ground maize was added to 4.96 L mL of plant hot water (water hardness 3.57 mmol / L) at 35 ° C to obtain a solid solution of 25% with a final volume of 6 L in a Biostat C fermentor (comparM to Sartorius), which leads to a pH of about 5.6.

c) Liquefaction of starch-containing material

- the temperature is increased to 90 ° C

-1 mL of α-amylase "α-amylase VF-Kartoffel" (Schliessmann, Nr. 5049) was diluted in 10 mL of plant water and then the diluted amylase was added to the papilla.

- the temperature is increased to 90 ° C

- the fermenter is incubated for 90 minutes at 90 ° C and 450 rpm

- the porridge was cooled to 30 ° C, the pH was adjusted to ~ 4 with 30% H2SO4

d) Sacarification of the liquefied material obtained

- 1.5 mL of glucoamylase "Amylase GA 500" (Schliessmann, Nr. 5042) was diluted in 10 mL of sterile plant water and then the diluted glucoamylase, which is the liquefied saccharified material, was added to the papilla.

e) Fermentation

- 1.8 g of (NH 4) 2 SO 4 (ie, 300 ppm of ammonium sulfate) were added to the 6 L of saccharified liquefied material. - The saccharified liquefied material containing 300 ppm of ammonium sulfate was stirred in the Biostat C fermentor at 800 rpm for 5 minutes to distribute everything uniformly.

- the saccharified liquefied material containing 300 ppm of ammonium sulphate (mass) was distributed in individual portions of 1500 g in four Biostat B 2L fermenters (compar. Sartorius) containing a horseshoe mixer.

- Preparation of enzyme stock : 2.5 g of pectinase (Pec3) with 90349 U / mL and 2.5 g of xylanase (Xyl16) with 10027 U / mL were added to a 50 mL graduated cylinder, and filled up to 50 mL with plant water.

The enzyme stock was transferred to a 50 mL tube and then stored at 4 ° C until its use after one hour.

- The following volumes of the enzyme stock preparation were added to the 2L Biostat B fermenter containing 1500 g of the saccharified liquefied material containing 300 ppm of ammonium sulfate.

Fermentor # 1: 0 mL of the preparation of enzymatic stock leading to 0 g / t of pectinase and 0 g / t of xylanase Fermentor # 2: 0.75 mL of the enzyme preparation leading to 25 g / t of pectinase and 25 g / t of xylanase Fermentor # 3: 2.25 mL of the enzyme preparation leading to 75 g / t of pectinase and 75 g / t of xylanase Fermentor # 4: 6.00 mL of the enzyme preparation leading to 200 g / t of pectinase and 200 g / t of xylanase - Propagation of the yeast: 300 mL of medium YNB (nitrogen base of yeast) autoclaved, plus glucose with glucose medium 10 g / L resulting in a pH of 5.7 1 L culture flasks, which had been inoculated with 2 mL of yeast (Ethanol RED, compacta Fermentis) from a cryopreservation at -80 ° C containing 20% glycerol, were incubated for 23 hours (30 ° C) C, 150 rpm) leading to the yeast culture.

- Each of the four fermenters (Fermentor # 1 to # 4) was inoculated by 60 mL of the yeast culture.

- The culture of the fermenters was carried out at 30 ° C, at 150 rpm, without pH control for 92.5 hours.

- Samples of 7 mL were taken twice a day with a cut-off pipette to monitor the progress of the fermentation (ethanol concentration). The samples were transferred to 15 mL tubes and centrifuged at 4470 g for 10 minutes at 4 ° C, and stored at -20 ° C until further analysis.

f) Completion of the ethanol fermentation and distillation process

- To complete the fermentation process and distill the ethanol, the four fermenters were incubated at 80 ° C for 20 min and shaken at 150 rpm.

g) Sampling for analysis of dehydration

- After 20 minutes at 80 ° C and shaking at 150 rpm, 3 x 40 to 45 mL of sample from each fermentor were taken with a 50 mL pipette and filled into 50 mL tubes (whole vinasse samples).

B) Determination of the activity of the enzymatic product:

DNSA solution: For the DNSA solution the following compounds were used:

- 5.00 g of 3,5-dinitrosalidic acid (DNSA) were dissolved in 300 mL of distilled H2O

- add 50 mL of an NaOH / KOH solution (4M KOH 4M NaOH) dropwise

- 150 g of K-Na tetrahydrate tartrate are added

- the solution is cooled to room temperature

- Distilled H2O is added to 500 mL of final volume

- it is stored in the dark

a) Pectinase

Substrates: Polygalacturonic acid (Sigma 81325)

The substrates were dissolved in buffer to a concentration of 0.8% (w / v)

Tampon: 50 mM sodium acetate, pH 4.5

For the assay 96-well PCR microtiter plates (ComparMa Greiner) were used. The enzymes were diluted in buffer. 90 pL of substrate and 10 pL of enzyme solution were mixed. A blank was measured by replacing the enzyme solution with water. The incubation was carried out for 30 minutes at 37 ° C, followed by an enzymatic inactivation step of 5 minutes at 99 ° C and followed by cooling for 10 minutes at 4 ° C. In a second 96-well PCR microtiter plate (ComparM to Greiner), 50 pL of the incubated substrate-enzyme mixture was incubated with 50 pL of the DNSA solution at 98 ° C for 10 minutes and then cooled to 4 °. C and incubated for 5 minutes at 4 ° C.

100 pL of the reaction mixture was transferred to a well of a 96 well flat bottomed microtiter plate, and the adsorption was measured at 540 nm by a microtiter plate reader (Tecan M1000).

b) xylanase

Substrates: Xylan of birch wood (Sigma X0502)

The substrate was dissolved in buffer at a concentration of 1.5% (w / v)

Tampon: 100 mM sodium acetate, pH 5.0, containing CaCl220 mM and 0.4 g / L of Tween20

For the assay 96-well PCR microtiter plates (ComparM to Greiner) were used. The enzymes were diluted in buffer. 90 pL of substrate and 10 pL of enzyme solution were mixed. A blank was measured by replacing the enzyme solution with water. The incubation was carried out for 20 minutes at 40 ° C, followed by an enzymatic inactivation step of 5 minutes at 99 ° C and followed by cooling for 5 minutes at 4 ° C. 45.5 pL of the DNSA solution was added to the 96-well PCR microtiter plate by a multi-drop (comparM to Fisher-Scientific) and then the plate was incubated at 98 ° C for 10 minutes, cooled to 4 ° C and incubated for 5 minutes at 4 ° C.

100 pL of the reaction mixture was transferred to a well of a new 96 well flat bottom microtiter plate, and then the absorption at 540 nm was measured by a microtiter plate reader (Tecan M1000).

The activity is calculated as Units per pL or mg of enzymatic product. One unit is defined as the amount of reducing ends formed in pmol per minute. Enzyme activities are shown in Table 1.

Table 1

Example 1

After the fermentation process and the distillation of the ethanol, the following steps were taken to analyze the dehydration:

Determination of the mass of the whole vinasse

1. Approximately 40 mL of the whole vinasse was transferred, after distillation at 80 ° C, to a 50 mL Falcon tube (FT1) and the mass was determined (mass FT1sample). The mass of the FT1 ford was previously determined (mass FT1empty). The FT1 with the sample (FT1sample) was centrifuged at 800 g for 10 minutes.

Determination of the fine vinasse mass

2. The supernatant was decanted into a new Falcon tube (FT2) of 50 mL, which led to fine vinasse, and the mass was determined (mass FT2sample). The mass of FT2 was previously determined (mass FT2empty).

Calculation of the mass of the sample:

Whole vinasse mass [mg] = [mass of FT1sample - mass of FT1empty] Mass of fine vinasse (supernatant) [mg] = [mass of FT2sample - mass of FT2empty] Calculation of dehydration

Dehydration [%] = Fine vinasse mass [mg] / Whole vinasse mass [mg] * 100

Table 2

Table 2 shows the amount of fine vinasse (supernatant) in% of the whole vinasse removed after centrifugation at 800 rpm for 10 minutes. It was shown that a treatment with the enzymatic composition comprising a xylanase and a pectinase increases the dehydration capacity by 3.5% using 25 ppm of each enzyme, 4.0% using 75 ppm of each enzyme and 4 , 6% using 200 ppm of each enzyme.

Claims (2)

1. A method to dehydrate an entire vinasse in a fermentation process, comprising: (a) saccharifying a cellulosic material with an enzymatic composition; (b) fermenting the saccharified cellulosic material with a fermentation organism to produce a fermentation product, wherein the fermentation medium comprises a xylanase and a pectinase; (c) distilling the fermentation product to form the entire vinasse; Y (d) separating the whole vinasse into fine vinasse and wet cake, so that more liquid phase is transferred to the fine vinasse, where the cellulosic material is maize and the fermentation product is ethanol. 2. The method according to claim 1, wherein the fermentation organism is a bacterium, a yeast or a fungus.