WO2010034219A1

WO2010034219A1 - Method for producing ethanol from raw material containing cassava residues

Info

Publication number: WO2010034219A1
Application number: PCT/CN2009/073362
Authority: WO
Inventors: Wenxin Liu; Jiashen Liu; Jingwei Yuan; Chengxiang Shang; Chunling Li; Ning Zhang; Kairong Jiang; Guozhong Wang; Xiaoming Hao; Hailong Lin; Likang Deng
Original assignee: Cofco Limited
Priority date: 2008-09-25
Filing date: 2009-08-20
Publication date: 2010-04-01
Also published as: CN101423848A; CN100588711C

Abstract

A method for producing ethanol from raw material containing cassava residues comprises the steps of: mixing raw material containing cassava residues with enzyme, and enzymolyzing the cellulose in the raw material to give enzymolysis product, wherein the mixing of the enzyme and the raw material comprises mixing the raw material and the enzyme in batches, the batch number is at least two, and each batch of the raw material is added at the amount of at most 60wt% of the total raw material; and fermenting the enzymolysis product. The inventive method can increase utilization rate of the raw material containing cassava residues, and has high ethanol yield.

Description

Method for producing ethanol from raw material containing cassava residues

Technical Field

The present invention relates to a preparation method of ethanol, and particularly relates to a method for producing ethanol from raw material containing cassava residues.

Technical Background

Presently, general cassava starch factories or cassava ethanol factories produce a large amount of waste cassava residues after starch or ethanol production. As the waste cassava residues contain a certain amount of starch, they are usually used as animal feeds.

For example, it is mentioned in "The Study of Cassava Residues Ferment to Produce Single Cell Protein" (Tang Yanhua, Xie Bifeng, College of Life Sciences, Fujian Normal University, Pharmaceutical Biotechnology, 2006, 13(1), p51-54) that Aspergillusniger and Saccharomyces cerevisiae are adopted to carry out mixed fermentation on cassava residues at appropriate conditions to increase protein content of the cassava residues; the treated cassava residues can be applied as yeast single cell protein feed for raising livestocks, and waste resource recovery can be realized.

In "Phytase Production by Enlarged Solid-State Fermentation of Cassava Residues" (Zhong Qiuping, Zhou Wenhua, Li Jianna, Li Meiqiu, Jiang Fangjun, Wu JianXiong, Food Science and Technology Department, South China University of Tropical Agriculture, Chinese Journal of Tropical Crops, 2004, 25(1), p45-48), the influence of solid-state fermentation conditions on phytase production from cassava residues using Aspergillus niger PD strain is studied. The results show that optimal conditions are moisture of 75%, material thickness of 5-10 cm, the inoculation amount of 1.5%, culturing temperature of 32-34°C , and culturing time of 6-8 days according to single factor analysis; under the optimal conditions, the maximum phytase activity is 4,189μmol/min»g.

In "Study on co -fermentation using cassava residue by mixed cellulolytic strains" (Lin Jie, Tan Zhaozan, Luo Weicheng, College of Food Science, College of Resource and Environment, College of Life science, South China Agricultural University, Journal of Safety and Environment, 2005, 5(6), p26-29), mixed fermentation using 6 cellulose-decomposing bacteria without mutual antagonism selected from nature is described, and the optimal enzyme production conditions for fermenting industrial waste cassava residues using the mixed bacteria are studied to be: medium composition including cassava residues of 4g, soybean powder of 0.75g, potassium dihydrogen phosphate of 0.2g, sodium chloride of 0.02g, MgSO4«7H₂O of 0.015g, and distilled water of 1,000ml; fermentation conditions including initial pH of 7.0, culturing temperature of 40 °C , culturing solution 140ml in each 250ml conical flask, inoculation amount of 3%, rotation speed of 140rpm, and fermentation time of 72hr. The result shows that fermentation using mixed bacteria can dramatically improve cellulose activity and tolerance to reducing sugar. It can be learned from the above description that the cassava residues are mainly used for producing feedstuff, or phytase, etc. Recently, they are also used as raw material for producing ethanol.

For example, CN101195836A discloses a novel process for producing ethanol from cassava residues, which comprises separating cassava residues from raw cassava or dry cassava pieces, cooking and saccharifying, subjecting to residue-water separation after saccharifying, directly using solid residues as feedstuff or fertilizer, and using the separated filtrate of the residue saccharification as slurry concocting liquor for raw cassava or dry cassava pieces. In this method, the cooking and saccharifying of cassava residues includes sieving cassava residues to give material with fineness below 1.8mm, delivering to storage pool in ethanol workshop, adding wastewater (yellow slurry) resulted from starch production at amount of 2-5 L per lkg of cassava residues, discharging to slurry concocting tank, adding high temperature resistance α-amylase at 5-15U/g, charging into heat exchanger to perform heat exchange with material discharged from cooking tank, liquefying when temperature is raised to 90-110°C , then introducing to cooking pot, cooking at temperature of 90-95 ^°C preferably to pale yellow or light brown, introducing the cooked liquor into saccharifying tank after vapor-liquid separation, cooling to 55-65 ^°C, adding β-amylase at 100-150 U/g for saccharifying for 20-60min, removing solid residues, and introducing the filtrate of cassava residue sacchrification to storage pool. In this method, the cooking and sacchrifying of raw cassava or dry cassava pieces includes adding the aforementioned filtrate obtained from cooking and saccharifying of cassava residues into pulverized raw cassava or dry cassava pieces at 2-4kg per kg of solid starch, adding high temperature resistant α-amylase, stirring, liquefying at 90-110⁰C, introducing to cooking tank, cooking for 40-60min at temperature of 90-95 ^°C, then introducing to saccharifying tank, cooling to 55-65 ^°C, adding β-amylase at amount of 100-150 U/g raw material, saccharifying for 20-60min, cooling via heat exchanger to 30-35 "C, introducing to fermentation tank, fermenting for 50-70hr until ethanol content is higher than 8.5%, and delivering to distillation workshop for distillation according to routine method. The method produces ethanol by enzymolyzing and saccharifying residual starch in cassava residues, and then mixing with fresh or dry cassava to carry out saccharifying again and fermenting. But as the method has low utilization rate of cassava residues, the ethanol yield is adversely affected.

Contents of the Invention

The object of the present invention is to overcome the disadvantages in available methods for preparing ethanol from raw material containing cassava residues, such as low utilization rate of cassava residues, and low ethanol yield, by providing a method for producing ethanol from raw material containing cassava residues which has high cassava residue utilization rate and high ethanol yield.

The inventors of the present invention have found that the cassava residues mainly contain water, starch (on dry matter basis) 8-10wt%, cellulose 25-30wt%, hemicellulose 10-12 wt%, lignin 30-32wt%, crude protein 4-6wt%, crude fat 2-4wt%, and crude ash and inorganic salts 10-15wt%, wherein the contents of cellulose, hemicellulose, and lignin are relatively high. Moreover, the inventors have found that, although cassava residues can be called as cellulose-containing material, lignin, cellulose, and hemicellulose in cassava residues are in loosely combining state compared with those contained in other conventional cellulose-containing material (such as straw) which are in a structure that the cellulose is tightly wrapped by the lignin. Therefore, the inventors have tried to prepare ethanol by enzymolyzing cellulose and hemicellulose in cassava residues and fermenting the enzymolysis product; good effect is achieved, and more important, pretreatment of cassava residues is not required before enzymolysis, and the residues can be directly enzymolyzed and fermented to prepare ethanol. Additionally, the inventors of the present invention also have found that by adopting batch feeding method to mix raw material containing cassava residues with enzyme in batches, each batch of the raw material can be mixed well and fully contacted with the enzyme, and the enzyme concentration is kept at high level during reaction, such that the raw material containing cassava residues can fully react with the enzyme. Therefore, sugar conversion rate of the raw material is effectively raised to dramatically increase ethanol yield. The present invention provides a method for producing ethanol from raw material containing cassava residues, comprising the steps of: mixing raw material containing cassava residues with enzyme, and enzymolyzing the cellulose in the raw material to give enzymolysis product, wherein the mixing of the enzyme and the raw material comprises mixing the raw material and the enzyme in batches, the batch number is at least two, and each batch of the raw material is added at the amount of at most 60wt% of the total raw material; and fermenting the enzymolysis product.

The present invention adopts batch feeding method to mix the raw material containing cassava residues and the enzyme, each batch of the raw material can be mixed well and fully contacted with the enzyme, the enzyme concentration is kept at high level during reaction, such that the raw material containing cassava residues can fully react with the enzyme. Therefore, sugar conversion rate of the raw material is effectively raised to dramatically increase ethanol yield. For example, the ethanol yield in example 1 is 14.6%, which is increased by 25.9% compared with that in comparison example 1 (only 11.6%); the ethanol yield in example 6 is 18.6%, which is increased by 20.8% compared with that in comparison example 2 (only 15.4%).

Preferred Embodiments

According to the present invention, the method comprises the steps of: mixing raw material containing cassava residues with enzyme, and enzymolyzing the cellulose in the raw material to give enzymolysis product, wherein the mixing of the enzyme and the raw material comprises mixing the raw material and the enzyme in batches, the batch number is at least two, and each batch of the raw material is added at the amount of at most 60wt% of the total raw material; and fermenting the enzymolysis product. Herein, the raw material refers to the raw material containing cassava residues, unless otherwise specified.

Preferably, for improving production efficiency while ensuring full mixing of the raw material and the enzyme, high concentration of the raw material, and high enzymolysis speed, the amount of the first batch of the raw material may be 20-60wt%, more preferably 20-40wt% of the total raw material; the amount of the raw material mixed with the enzyme after the first batch may be 40-80wt%, more preferably 60-80wt% of the total raw material.

More preferably, the batch number is three, wherein the amount of the first batch of the raw material is 20-60wt%, more preferably 20-40wt% of the total raw material; the amount of the second batch of the raw material is 20-50wt%, more preferably 20-40wt% of the total raw material; and the amount of the third batch of the raw material is 20-40wt%, more preferably 20-30wt% of the total raw material.

For ensuring the enzyme has optimal reaction activity, the raw material is regulated to pH of 3-7, preferably 4-5.5, before mixing the first batch of the raw material with the enzyme, so as to ensure the enzyme has optimal reaction activity after mixed with the raw material. As pH fluctuation during enzymolysis is low, pH of enzymolysis can be regulated before enzyme addition according to routine method in the field, and pH regulation of the reactant material can be carried out according to various methods well known for those skilled in the art. For example, the raw material containing cassava residues can be firstly mixed with water (in case enzyme is added) or medium (in case enzyme-producing microbe is added), and then acidic or alkaline substances are added into the mixture according to pH of the mixture. For example, the acidic substance can be one or more of sulfuric acid, hydrochloric acid, or phosphoric acid, and the alkaline substance can be sodium hydroxide and/or potassium hydroxide.

Preferably, a time interval is included between two adjacent batches. For ensuring the enzyme can fully hydrolyze the raw material containing cassava residues to improve sugar conversion rate, the time interval is preferably 10-120min, more preferably 30-60min.

For achieving more uniform mixing of the enzyme and the raw material, the enzymolysis of the raw material is preferably carried out in presence of water, more preferably in suspension containing water and raw material. In other words, the present invention preferably comprises adding water during mixing of the first batch of the raw material and the enzyme to prepare suspension, mixing the total enzyme required by enzymolysis with the suspension, and then adding the rest raw material in batches according to the method in the present invention. The water addition amount can be regulated within wide range, preferably, the weight ratio of the water to the total raw material is 2-5:1. More preferably, the content of the first batch raw material containing cassava residues on dry matter basis is at least lwt%, preferably l-20wt%, more preferably 1.2-10wt%, based on the weight of the mixture of the first batch of the raw material, water, and the enzyme.

The inventor of the present invention have found that by controlling the content of the raw material on dry matter basis in the mixture of the first batch of the raw material, water, and enzyme during enzymolysis to be at least lwt%, preferably l-20wt%, more preferably 1.2-10wt%, the inventive method can ensure both short enzymolysis period, and high production efficiency, i.e. high monosaccharide yield, compared with the method which mixes total raw material containing cassava residues and enzyme in one time for enzymolysis.

According to the present invention, the raw material can be cassava residues or mixture of cassava residues and other cellulose-containing raw material, such as mixture of cassava residues and straw, the weight ratio of cassava residues to straw in the mixture containing cassava residues and straw for preparing ethanol through enzymolysis and fermentation can be regulated within wide range; preferably, based on total weight of the mixture of cassava residues and straw, the content of cassava residues is 40-90wt%, and the content of straw is 10-60wt%; more preferably, the content of cassava residues is 50-70wt%, and the content of straw is 30-50wt%. According to the present invention, the particle size of cassava residues is preferably 0.3-5mm, more preferably 0.5-3mm, for ensuring full contact between enzyme and cassava residues. Various methods well known for those skilled in the arts can be adopted to achieve particle size of cassava residues of 0.3-5mm, more preferably 0.5-3mm. For example, dry pulverizing or wet pulverizing can be adopted, the difference between the two pulverization methods is in that whether cassava residues are mixed with water. Wet pulverization comprises mixing cassava residues with water, and performing pulverization once ore more. The weight ratio of cassava residues to water can be 1: 0.2-5, preferably 1: 0.5-2. Various routine pulverizers can be used, such as SFSP series hammer-type pulverizers. The present invention has no special limitation on other steps in the method for producing ethanol from raw material containing cassava residues, which can refer to the conventional method for producing ethanol from cellulose-containing raw material.

As the structure of cellulose, hemicellulose, and lignin in cassava is not as firm and compact as that in straw, and cassava residues are usually final byproducts resulted from ethanol production using cassava, and have been subjected to high temperature treatment during production, lignin in cassava residues does not tightly wrap cellulose, and is in loose combination state with cellulose and hemicellulose; therefore pretreatment for separating cellulose and hemicellulose from lignin is not needed before enzymolysis, and cassava residues can be directly enzymolyzed and fermented for producing ethanol from cassava residues. The enzyme adopted for enzymolysis includes cellulase. The cellulose can be obtained by various means, which can be purchased commercially or obtained via secretion of enzyme -producing microbes.

As enzyme obtained via secretion of enzyme-producing microbes contains various byproducts, direct addition of enzyme is preferred. For the sake of cost, the usage amount of cellulase is preferably 10-25, more preferably 15-18 enzyme activity units per gram of the raw material containing cassava residues on dry matter basis. The enzyme activity of cellulase in the present invention is determined according to the standard method provided by National Renewable Energy Laboratory (NREL) of US, Measurement of Cellulase Activities NREL LAP-006; the said enzyme activity unit is the enzyme amount in μg required for converting Ig of Whatman No. 1 filter paper into glucose within one minute under the measurement conditions specified by the standard method.

The enzymolysis temperature can be any appropriate action temperature for cellulase, usually 45-55 ^°C, preferably 48-52 ^°C . In theory, the longer the enzymolysis time, the better the effect. For the sake of equipment utilization rate, the enzymolysis time is preferably 25-48h, more preferably 30-4Oh. The enzymolysis time is timed from start of mixing of the enzyme and the raw material containing cassava residues; therefore, the time interval of two adjacent mixing batches is also included in the enzymolysis time.

According to one preferred embodiment in the present invention, the method for enzymolyzing the raw material containing cassava residues includes firstly mixing the first batch of the raw material with water, charging the mixture into enzymolysis tank, regulating the pH of the mixture to 3-7, elevating temperature to 45-55 °C , preferably 48-52°C , to reach optimal reaction activity condition of enzyme, then adding the enzyme into the enzymolysis tank while stirring, stirring for 30-60min; adding the second batch of the raw material, stirring for 30-60min, and then adding the third batch of the raw material. The weight ratio of the water amount to the total raw material containing cassava residues is 2-5:1; the amount of the first batch of the raw material mixed with the enzyme is 20-60wt% of the total raw material; the amount of the second batch of the raw material is 20-50wt% of the total raw material; and the amount of the third batch of the raw material is 20-40wt% of the total raw material.

The said cellulase is composite enzyme, at least including three kinds of enzymes, i.e., Ci cellulase, Cx cellulase, and cellobiase. Ci cellulase can convert crystalline cellulose into noncrystalline cellulose.

C_x cellulase is further classified into C_xl cellullase and C_x2 cellulase. C_xi cellulase is endocellulase, which can work on β-1,4- glucosidic bond from inside of hydrated noncrystalline cellulose molecule to generate cellodextrin and cellobiose. C_X2 cellulase is exocellulase, which can work on β-l,4-glucosidic bond from nonreducing end of hydrated noncrystalline cellulose molecule to cleave β-1,4- glucosidic bonds one by one to generate glucose. Cellobiase works on cellobiose to generate glucose.

The preferred enzyme for the enzymolysis further includes hemicellulase. As hemicellulase can degrade hemicellulose into water soluble xylose, in case that the enzyme for the enzymolysis includes hemicellulase, on one hand cellulose can be more fully exposed to increase contact chance of cellulose and cellulase, and on the other hand xylose as hemicellulose degradation product can be fermented by Pichia stipitis to generate ethanol; both resulting that the ethanol yield can be increased. The usage amount of hemicellulase is 4.0-8.0 enzyme activity units per gram of the raw material on dry matter basis. The enzyme activity unit (U) of hemicellulase of the present invention is the enzyme amount required for decomposing lwt% xylan solution to generate lμg of reducing sugar (calculated as xylose) in one minute at 50^°C and pH=4.8.

The activity of the hemicellulase of the present invention refers to the activity unit per gram of hemicellulase. The activity of hemicellulase is determined by using hemicellulase to hydrolyze lwt% xylan at 50 ^°C and pH=4.8 to generate reducing sugar (calculated as xylose), allowing the obtained reducing sugar to generate color reaction with excessive 3,5-dinitrosalicylic acid (DNS), and using spectrophotometer to measure absorption of the resulting solution at 550nm which is in positive relationship with generation amount of reducing sugar (calculated as xylose). The detailed determination method is as below: accurately weighing xylan 1.00Og, dissolving with 0.5ml of 0.1M acetic acid-sodium acetate buffer solution having pH 4.8, and diluting with deionized water to constant volume 100ml to give lwt% xylan solution; weighing 3Og of potassium sodium tartarate tetrahydrate, placing into 500ml conical flask, adding 16g of NaOH, then adding 50ml of deionized water, heating in water bath at 5°C/min until solid matter dissolves, adding Ig of 3,5-dinitrosalicylic acid, dissolving, cooling to room temperature, diluting with deionized water to constant volume 100ml to give 3,5-dinitrosalicylic acid (DNS) solution; drying xylose at 80 °C to constant weight, accurately weighing 1.00Og, dissolving into 1000ml of water, and adding lOmg of sodium azide for preservation to give lmg/ml standard xylose solution; accurately weighing 1.00Og solid hemicellulase or taking ImI of hemicellulase stock solution, dissolving with 0.5ml of 0.1M acetic acid-sodium acetate buffer solution having pH 4.8, and diluting with deionized water to constant volume 100ml to give to-be-detected enzyme solution having been diluted by 100 times; respectively using 50 ^°C water bath to heat 2ml of xylose gradient standard solutions (0. lmg/ml, 0.2mg/ml, 0.3mg/ml, 0.4mg/ml, and 0.5mg/ml, wherein the xylose gradient standard solutions are prepared from deionized water and lmg/ml standard xylose solution) or deionized water (used as control having no xylose) for 60min, mixing with 2ml of DNS, subjecting to boiling water bath for 5min, cooling, diluting with deionized water to constant volume 15ml, respectively testing absorption values of the xylose gradient standard solutions at 550nm after the reaction, and plotting standard curve by using absorption value as X-axis and xylose concentration as Y-axis. Regression equation y=bx+a can be obtained from the standard curve, wherein x is absorption value, y is xylose concentration, a is the intercept of the linear equation, and b is the slope of the linear equation; taking 0.2ml of to-be-detected enzyme solution, and 1.8ml lwt% xylan solution or 0.1M acetic acid-sodium acetate buffer solution (used as control having no xylose) with pH=4.8, and testing absorption value according to the same procedures as those for the xylose gradient standard solutions. The activity of hemicellulase is calculated according to the following equation:

_Λ • • , _u • _Ii ₁ (bx+α)xnx5xl000 _{τ τ}, _{t Λ S} Activity of hemicellulase= U/g(ml)

60 in which x is absorption value of the to-be-tested enzyme solution, b and a have same meanings as those in the regression equation of xylose concentration vs absorption value, n is dilution factor of the enzyme, 60 means that the enzymatic reaction time is 60min, and 5 is the sampling factor (herein 0.2 ml is taken from ImI of the to-be-tested enzyme solution for testing). Specific hemicellulase activity can be tested according to the above method, from which usage amount of hemicellulase can be calculated. Any microbes capable of fermenting pentose and/or hexose can be used in the fermentation process of the present invention. Saccharomyces cerevisiae is microbe capable of fermenting hexose which is widely used in brew industry, with ethanol resistance, less byproducts, and high ethanol yield; and Pichia stipitis is microbe capable of fermenting both pentose and hexose (Ethanol Continuous Fermentation of Pentose and Hexose by Pichia stipitis, Ji Gengsheng, etc., Journal of Nanjing Forestry University(Natural Science Edition), VoI 28, 3, p 9-13, 2004); therefore, the yeast for the fermentation is preferably Pichia stipitis and/or Saccharomyces cerevisiae. The inoculation amount of the yeast used in the fermentation is 10³-10⁸, more preferably 10 -10 colony forming units per gram of enzymolysis product. The yeast used for the fermentation in the present invention can be commercially purchased yeast solid formulation (such as dry yeast powder) or yeast strain (such as ATCC 2601 Brewer's yeast). The colony forming units of the yeast can be obtained by the method according to the methods well known for those skilled in the arts, such as plate count using methylene blue dye, and the detailed method is as below: dissolving dry yeast powder Ig in sterile water 10ml, or diluting strain activation liquid ImI with sterile water to 10ml, adding 0.1 wt% methylene blue 0.5ml, holding at 35°C for 30min, and counting viable bacteria (viable bacteria will not be dyed while dead bacteria will be dyed) in the solution by using blood cell counting plate and 10x optical microscope to give number of viable bacteria in Ig of the dry yeast or ImI of the strain activation liquid, i.e. number of colony forming units. The yeast can be inoculated by routine methods, such as adding seed liquid 5-15vol% into enzymolysis product. The seed liquid can be aqueous solution or culturing medium solution of dry yeast, or activation seed liquid of dry yeast or commercial strain. The fermentation temperature can be any temperature suitable for yeast growth, preferably 30-36 "C, more preferably 32-35 ^"C . The fermentation time is the time from inoculation to occurrence of decline phase of the yeast growth (i.e. the fermentation time is the sum of lag phase, log phase, and stable phase), which is preferably 32-48hr, more preferably 32-40hr. The ethanol as fermentation product can be subjected to separation and refinement, such as distillation, concentration, and dewatering, depending on requirement of different industrial products (for example fuel ethanol requires a purity of 99% or higher). Additionally, the water content of cassava residues can be tested by various methods. Unless specified otherwise, the water content of the cassava residues in the present invention is the ratio of difference between initial weight (Wl) of cassava residues and weight (W2) of cassava residues dried to constant weight at 70-100^°C to the initial weight (Wl) of cassava residues, i.e. water content (wt%) = (Wl -W2)/Wlxl00% .

According to the present invention, when the raw material containing cassava residues is a mixture of cassava residues and straw, the straw is preferably steam exploded straw and/or acid-treated straw. Crystalline structure of cellulose in straw is difficult to be destroyed, lignin in straw is nonpoly saccharide substance formed via polymerization of phenylpropane, in which aromatic hydrocarbons are crosslinked together via -C-C- bonds and -O- bonds, and the side chain is covalently bonded with hemicellulose to form a dense network structure which tightly wraps cellulose inside, makes cellulose hard to be attacked by cellulase, and makes cellulase and hemicellulase be unable to contact substrate. Therefore, straw has to be subjected to pretreatment, for example straw is allowed to contact cellulase and/or hemicellulase for cellulose hydrolysis after steam explosion or acid treatment.

The acid treatment or steam explosion can adopt methods well known for those skilled in the arts. For example, the acid treatment method comprises mixing straw and acid to dissolve lignin so as to make lignin separate from cellulose and hemicellulose. The acid species for dissolving lignin in the cellulose raw material are well known for those skilled in the arts, such as various routine acids in the field, for example the acid can be one or more selected from sulfuric acid, hydrochloric acid and phosphoric acid. There is no special limitation on usage amount and concentration of the acid, which can be regulated according to lignin amount in the straw to be dissolved. The said steam explosion method comprises mixing straw and water, and subjecting to steam explosion; or directly charging straw in steam explosion equipment for steam explosion. As steam explosion method is more favorable for destroying network structure among cellulose, hemicellulose, and lignin in straw to make cellulose be fully separated, benefit action of cellulase on cellulose surface, and improve hydrolysis rate of cellulose and sugar yield, the preferred straw pretreatment method in the present invention is steam explosion method.

The object of the present invention can be achieved by carrying out the steam explosion according to routine steam explosion conditions in available techniques, for example, the steam explosion temperature can be 180-200°C , the steam explosion pressure can be 1.4-1.8MPa, the holding time of the steam explosion pressure can be 3-7min. More preferably, the steam explosion temperature can be 185-195°C , the steam explosion pressure is 1.4-1.6MPa, and the holding time of the steam explosion pressure is 4-5min.

Moreover, sterilization can be realized at the steam explosion pressure and temperature; for preventing contamination of impurity strains (mainly bacteria) from generating toxins which affects enzyme activity in enzymolysis process, and from affecting yeast growth in fermentation process, antibiotics which has no effects on yeast but can inhibit growth of impurity strains, such as industrial penicillin, is preferably added before enzymolysis. The addition amount of the antibiotics is 1-10 units per mL of enzymolysis liquid or fermentation liquid. The enzymolysis liquid includes mixture of steam explosion products and cassava residues, enzyme, and water; and the fermentation liquid comprises enzymolysis products and inoculated yeast. As under the high temperature and high pressure conditions of the steam explosion, hemicellulose in straw can generate inhibitors such as furfural, or furan, etc., which will decrease activities of enzyme and yeast, the method further comprises water washing the steam explosion products after steam explosion and before enzymolysis of the mixture of steam explosion products and cassava residues. The water washing comprises mixing the steam explosion products with water, stirring to make the inhibitors dissolve in water, and separating by centrifuging to remove the inhibitors from the steam explosion products. In theory, the higher the water washing temperature, the more the dissolved inhibitors; for the sake of energy consumption, the water washing temperature is preferably at least 60⁰C, more preferably 60-80⁰C . The more the water addition for water washing, the more the solvent for dissolving the inhibitor; but for the sake of energy consumption of centrifugation separation, the water addition amount is preferably 2-1Og, more preferably 2-3g, per gram of straw on dry matter basis.

As sand/stone impurities and iron impurities may be blended in straw and may cause damage to steam explosion equipment, the method may further comprise subjecting straw to routine operation of stone removal and iron removal before steam explosion, such as air delivering straw while removing stone and iron via magnet attraction. As sand/stone can not be air delivered to the steam explosion equipment due to its large mass, and iron impurities can not enter into the steam explosion equipment due to attraction of magnet, stone removal and iron removal can be achieved. Additionally, straw is likely to block pipeline due to its own tangling, the straw is preferably processed to size of 0.5-3cm x 0.2-lcm x 0.2-lcm, more preferably l-2cm x 0.4-0.6cm x 0.5- lcm, before introduced to the steam explosion equipment.

The present invention will be further explained through the following examples.

Example 1

The present example is for describing the inventive method for producing ethanol from raw material containing cassava residues.

(1) Measurement of total weight of cellulose and total weight of hemicellulose in the raw material containing cassava residues

The method comprises: pulverizing 3,00Og of waste cassava residues (with water content of 10%) resulted from ethanol fermentation to particle size of 3mm, taking 5g of pulverized resultant, drying at 45 °C to constant weight 4g, weighing 300.0mg of dried cassava residues, and placing into 100ml dry conical flask with weight of 80g; adding 3.00ml of 72wt% sulfuric acid solution into the conical flask, and stirring for lmin; placing the conical flask in 30 °C water bath for 60min, and stirring once every 5min to ensure uniform hydrolysis; diluting with deionized water to make the sulfuric acid concentration to 4wt%, and filtering with Buchner Funnel to give filtrate 84ml; transferring the filtrate 20ml to 50ml dry conical flask, regulating pH to 5.5 with calcium carbonate 2.5g, standing for 5hr, and collecting supernatant; filtering the supernatant with 0.2 micron filter membrane, and analyzing the filtrate with Biorad Aminex HPX-87P HPLC at conditions as below: sample size: 20μl, mobile phase: HPLC ultrapure water filtered with 0.2 micron filter membrane and degassed via ultrasonic oscillation, flow rate: 0.6ml/min, column temperature: 80-85 °C , detector temperature: 80-85°C, detector: refractive index detector, and running time: 35min. Wherein lxlθ^"6-4.θmg/ml D-(+) glucose and lxl0^~6-4.0mg/ml xylose are adopted as standard samples. HPLC analysis result shows the glucose concentration in the cassava residues acid hydrolysis solution is 0.00112mg/ml, and it can be calculated that glucose 0.283g can be obtained by acid hydrolysis of the cassava residues Ig. As 72wt% sulfuric acid solution can completely hydrolyze cellulose of cassava residues into glucose, the obtained glucose weight is 1.11 times of the cellulose in cassava residues, i.e. Ig of the cassava residues contain cellulose 0.255g, and 3,00Og of the cassava residues totally contain cellulose 764.1g. HPLC analysis result shows that the xylose concentration in the cassava residues acid hydrolysis solution is 0.00045mg/ml, and it can be calculated that xylose 0.114g can be obtained by acid hydrolysis of the cassava residues Ig. As 72wt% sulfuric acid solution can completely hydrolyze hemicellulose in cassava residues into xylose, the xylose weight is 1.14 times of the hemicellulose in the cassava residues, i.e. Ig of the cassava residues contain hemicellulose O.lOg, and 3,00Og of the cassava residues totally contain hemicellulose 302.4g. (2) Enzymolysis

The enzymolysis comprises: adding water into enzymolysis tank, adding first batch cassava residues at the amount of 35wt% of the total cassava residues left after sample test in step (1) while stirring, regulating pH to 4, heating to 52°C , totally adding cellulase (Imperial jade bio-technology Co. Ltd.) 40,500 enzyme activity units (about 27Og) into 2,70Og cassava residues (on dry matter basis) according to addition amount of cellulase 15 enzyme activity unit per gram of cassava residues on dry matter basis, and mixing for 30min while holding the temperature at 52⁰C ; then adding second batch cassava residues at the amount of 35wt% of the total cassava residues left after sample test in step (1), and mixing for 50min while holding the temperature at 52°C ; further adding the third batch cassava residues at the amount of 30wt% of the total cassava residues left after sample test in step (1), and enzymolyzing for 40hr while holding the temperature at 52°C . Wherein the weight ratio of water addition amount to total cassava residues is 3:1. The enzymolysis product is filtered by Buchner funnel, 20ml of the filtrate is transferred to dry 50ml conical flask and allowed to stand for 5hr, and then the supernatant is collected. The supernatant is filtered via 0.2micron filter membrane, and subjected to HPLC analysis according to the conditions in step (1). The glucose in the enzymolysis product is determined to be 773.8g. The weight of the glucose resulted from enzymolysis is divided by 1.11 to obtain weight of enzymolyzed cellulose in cassava residues 697.1g, cellulose conversion rate and monosaccharide yield are calculated according to the following equations, and the calculation result is shown in Table 1. The amount of cellulose enzymolyzed per hour is 16.9g. (The first batch cassava residues (on dry matter basis) count for 9.2wt% of the obtained mixture.)

Cellulose conversion rate = 100% x weight of enzymolyzed cellulose /total weight of cellulose Monosaccharide yield = 100% x weight of glucose obtained via enzymolysis/ weight of cassava residues on dry matter basis (3) Fermentation The enzymolysis product is cooled to 35 °C , and inoculated with Saccharomyces cerevisiae (Angel super alcohol active dry yeast, Hubei Angel Yeast Co., Ltd) at 10⁵ colony forming units per gram of enzymolysis product . The resultant mixture is cultivated at 32 °C in fermentation tank for 40 hr while stirring; the fermentation product is distilled at 100 °C, and then the obtained distillation fraction is redistilled at 78.3 °C to obtain ethanol 395.2g. The ethanol yield can be calculated according to the equation as below, and the calculation result is shown in Table 1.

Ethanol yield = 100%xethanol weight/weight of cassava residues on dry matter basis

Comparison example 1

The present comparison example is for describing reference method for producing ethanol from raw material containing cassava residues.

Ethanol is produced according to the same method as example 1, except that the total cassava residues left after sample test in step (1) are charged into enzymolysis tank containing water in one time in the enzymolysis step (2), then uniformly mixed with enzyme (weight ratio of total cassava residues and water is 3:1) to carry out enzymolysis for 41 hours and 20 minutes. HPLC analysis is carried out according to the conditions in step (1) in example 1, the glucose in the enzymolysis product is determined to be 643.7g, the weight of the glucose resulted from enzymolysis is divided by 1.11 to give weight of enzymolyzed cellulose in steam explosion product 58Og, amount of cellulose enzymolyzed per hour is 14g, and ethanol resulted from redistillation is 312.7g. Cellulose conversion rate, monosaccharide yield, and ethanol yield are calculated according to the method and equation in example 1, and the calculation result is shown in Table 1.

Example 2

The present invention is for describing the inventive method for producing ethanol from raw material containing cassava residues.

Ethanol is produced according to the same method as example 1, except that the cassava residues are mixed with the enzyme in three batches in enzymolysis step, wherein the first batch cassava residues are added at the amount of 60wt% of the total cassava residues left after sample test, then mixed for lOOmin at 50 °C ; then the second batch cassava residues are added at the amount of 20wt% of the total cassava residues left after the sample test, and mixed for 50min at 50 °C ; the third batch cassava residues are added at the amount of 20wt% of the total cassava residues left after the sample test, and enzymolyzed for 42hr at 50 °C . HPLC analysis is carried out at the conditions in example 1, glucose in the enzymolysis product is determined to be 757.4g, the weight of glucose resulted from enzymolysis is divided by 1.11 to give weight of enzymolyzed cellulose 682.3g, and amount of cellulose enzymolyzed per hour is 16.2g. Fermentation is carried out according to the method in example 1, and ethanol resulted from redistillation is 387.3g. Cellulose conversion rate, monosaccharide yield, and ethanol yield are calculated according to the method and equation in example 1 , and the calculation result is shown in Table 1. (the first batch cassava residues (on dry matter basis) count for 14.6wt% of the obtained mixture.)

Example 3

Ethanol is produced according to the same method as example 1, except that the cassava residues are mixed with the enzyme in three batches in enzymolysis step, wherein the first batch of the cassava residues are added at the amount of 20wt% of the total cassava residues left after sample test, then mixed for 20min at 52°C ; then the second batch cassava residues are added at the amount of 40wt% of the total cassava residues left after the sample test, and mixed for 40min at 52°C ; the third batch cassava residues are added at the amount of 40wt% of the total cassava residues left after the sample test, and enzymolyzed for 42hr at 52⁰C . HPLC analysis is carried out at the conditions in example 1, glucose in the enzymolysis product is determined to be 784.5g, the weight of glucose resulted from enzymolysis is divided by 1.11 to give weight of enzymolyzed cellulose 706.8g, and amount of cellulose enzymolyzed per hour is 16.8g. In fermentation, the difference between examples 3 and 1 is that the enzymolysis product is cooled to 32 °C, and inoculated with Saccharomyces cerevisiae (Angel super alcohol active dry yeast, Hubei Angel Yeast Co., Ltd) 10⁶ colony forming units per gram of enzymolysis product; the resultant mixture is cultivated at 32 °C in fermentation tank for 36 hr while stirring; the fermentation product is distilled at 100°C , and then the obtained distillation fraction is redistilled at 78.3 °C to obtain ethanol 401.2g. Cellulose conversion rate, monosaccharide yield, and ethanol yield are calculated according to the method and equation in example 1, and the calculation result is shown in Table 1. (the first batch cassava residues (on dry matter basis) count for 5.5wt% of the obtained mixture.)

Example 4 The present invention is for describing the inventive method for producing ethanol from raw material containing cassava residues.

Ethanol is produced according to the same method as example 1, except that, the cassava residues are mixed with the enzyme in three batches in enzymolysis step, wherein the first batch cassava residues are added at the amount of 10wt% of the total cassava residues left after sample test, then mixed for lOmin at 52 °C ; then the second batch cassava residues are added at the amount of 40wt% of the total cassava residues left after the sample test, and mixed for 50min at 52 °C ; the third batch cassava residues are added at the amount of 50wt% of the total cassava residues left after the sample test, and enzymolyzed for 42hr at 52 °C . HPLC analysis is carried out at the conditions in step (1) of example 1, glucose in the enzymolysis product is determined to be 760.8g, the weight of glucose resulted from enzymolysis is divided by 1.11 to give weight of enzymolyzed cellulose 685.5g, and amount of cellulose enzymolyzed per hour is 16.3g. The fermentation is carried out according to the same method as example 1 , and ethanol resulted from redistillation is 388.8g. Cellulose conversion rate, monosaccharide yield, and ethanol yield are calculated according to the method and equation in example 1, and the calculation result is shown in Table 1. (the first batch cassava residues (on dry matter basis) count for 2.8wt% of the obtained mixture.)

Example 5

3,25Og of cassava residues with water content of 15.1wt% is pulverized to particle size of 5mm; the cassava residues hydrolyzed by sulfuric acid is analyzed according to the HPLC method in example 1 to obtain that glucose concentration in cassava residues hydrolysis liquid is 0.00108mg/ml, and it can be calculated that 0.259g of glucose can be obtained through acid hydrolysis of water washed cassava residues Ig. As 72wt% sulfuric acid solution can completely hydrolyze cellulose of the cassava residues into glucose, the obtained glucose weight is 1.11 times of the cellulose weight in cassava residues, i.e Ig of water washed cassava residues contain cellulose 0.233g, and 3,25Og of cassava residues totally contain cellulose 757.25g. HPLC analysis shows that xylose concentration in cassava residues acid hydrolysis liquid is 0.00048mg/ml, and it can be calculated that Ig of the cassava residues can be acid hydrolyzed to give xylose 0.115g. As 72wt% sulfuric acid solution can completely convert hemicellulose in the cassava residues into xylose, the xylose weight is 1.14 times of the weight of the hemicellulose in cassava residues, i.e. Ig of the water washed cassava residues contain hemicellulose O.lOlg, and 3,25Og of cassava residues totally contain hemicellulose 328.4g.

(2) Enzymolysis

The enzymolysis comprises adding water into enzymolysis tank, adding first batch cassava residues at the amount of 60wt% of the total cassava residues left after sample test in step (1) while stirring, regulating pH to 5, heating to 50 °C, adding total cellulase (Imperial jade bio-technology Co. Ltd.) 33,000 enzyme activity units (about 220g) and hemicellulase (Beijing Chemical Reagents Company) 16,560 enzyme activity units (about 22Og) into 2,76Og cassava residues (on dry matter basis) according to addition amount of cellulase 12 enzyme activity units and hemicellulase 6 enzyme activity units per gram of cassava residues on dry matter basis, and mixing for 80min at 50 °C ; then adding second batch cassava residues at the amount of 40wt% of the total cassava residues left after sample test in step (1), and enzymolyzing for 42hr at 50°C ; wherein the weight ratio of water addition amount to total cassava residues is 3.5:1. HPLC analysis is carried out at the conditions in step (1) of example 1, glucose in the enzymolysis product is determined to be 714.5g, the weight of glucose resulted from enzymolysis is divided by 1.11 to give weight of enzymolyzed cellulose 643.7g, xylose in the enzymolysis product is determined to be 283g, the weight of xylose resulted from enzymolysis is divided by 1.14 to give weight of enzymolyzed xylose 248.3g, the total amount of cellulose and xylose enzymolyzed per hour is 20.6g. Cellulose conversion rate, hemicellulose conversion rate, and monosaccharide yield are calculated according to the following equations, and the calculation result is shown in Table 1. (the first batch cassava residues (on dry matter basis) count for 12.0wt% of the obtained mixture.)

Cellulose conversion rate = 100% x weight of enzymolyzed cellulose /total weight of cellulose Hemicellulose conversion rate = 100% x weight of enzymolyzed hemicellulose /total weight of hemicellulose Monosaccharide yield = 100% x (weight of glucose via enzymolysis + weight of xylose obtained via enzymolysis)/ weight of cassava residues on dry matter basis (3) Fermentation

The enzymolysis product is cooled to 34⁰C , and inoculated with Saccharomyces cerevisiae (Angel super alcohol active dry yeast, Hubei Angel Yeast Co., Ltd) 10⁴ colony forming units and Pichia stipitis 10⁴ colony forming units per gram of enzymolysis product. The resultant mixture is cultivated at 33 °C in fermentation tank for 37 hr while stirring; the fermentation product is distilled at 100⁰C, and then the obtained distillation fraction is redistilled at 78.3 °C to obtain ethanol 365.4g. The ethanol yield can be calculated according to the equation as below, and the calculation result is shown in Table 1. Ethanol yield = 100%xethanol weight/weight of cassava residues on dry matter basis

Example 6

The present example is for describing the inventive method for producing ethanol from raw material containing cassava residues. (1) Pretreatment of straw

The pretreatment comprises cutting l,504g of impurity-free corn straw with water content of 10% into segments of 1.2cm x 0.5cm x 1.0cm, holding under pressure of 1.6MPa at 195 °C for 5min, and releasing the pressure to finish steam explosion; mixing the resultant with 70 °C water at mass ratio of 1:3, stirring for 30min, and subjecting to solid-liquid separation at 900rpm by using LW400 horizontal helical centrifuge (Jiangsu Huada Centrifuge Manufacturing Co. Ltd.) to give

4,51Og of water- washed steam explosion product with water content of 70wt%.

Measurement of total weight of cellulose and total weight of hemicellulose in solid steam explosion product:

The method comprises: taking 1Og of the above water-washed steam explosion product, drying at 45 °C to constant weight 3g, weighing 300. Omg of dried steam explosion product, and placing into 100ml dry conical flask with weight of 8Og; adding 3.00ml of 72wt% sulfuric acid solution into the conical flask, and stirring for lmin; placing the conical flask in 30 °C water bath for 60min, and stirring once every 5min to ensure uniform hydrolysis; diluting with deionized water until concentration of sulfuric acid is 4wt%, and filtering with Buchner Funnel to give filtrate 84ml; transferring the filtrate 20ml to 50ml dry conical flask, regulating pH to 5.5 with calcium carbonate 2.5g, standing for 5hr, and collecting the supernatant; filtering the supernatant with 0.2 micron filter membrane, and analyzing the filtrate with Biorad Aminex HPX-87P HPLC. The conditions for HPLC are set as below: sample size: 20μl, mobile phase: HPLC ultrapure water filtered with 0.2 micron filter membrane and degassed via ultrasonic oscillation, flow rate: 0.6ml/min, column temperature: 80-85 °C, detector temperature: 80-85°C , detector: refractive index detector, and running time: 35min. 0.14.0mg/ml D-(+) glucose and 0.1-4.0mg/ml D-(+)xylose are adopted as standard sample. HPLC analysis result shows the glucose concentration in the steam explosion product acid hydrolysis solution is 1.6mg/ml, and it can be calculated that glucose 0.134g can be obtained by acid hydrolysis of the steam explosion product Ig. As 72wt% sulfuric acid solution can completely hydrolyze cellulose of the steam explosion product into glucose, the obtained glucose weight is 1.11 times of the cellulose in the steam explosion product, i.e. Ig of the water washed steam explosion product contains cellulose 0.121g, and 4,50Og of the steam explosion product totally contains cellulose 544.5g. HPLC analysis result shows that the xylose concentration in the steam explosion product acid hydrolysis liquid is 0.4mg/ml, and it can be calculated that xylose 0.034g can be obtained by acid hydrolysis of the water washed steam explosion product Ig. As 72wt% sulfuric acid solution can completely hydrolyze hemicellulose in the steam explosion product into xylose, the xylose weight is 1.14 times of the hemicellulose in the steam explosion product, i.e. Ig of the steam explosion product contains hemicellulose 0.029g, and 4,50Og of the steam explosion product totally contains hemicellulose 130.5g. (2) Enzymolysis

The enzymolysis is carried out according to the same method as example 1, except that the raw material is a mixture of 1,50Og of the cassava residues with water content of 10% in example 1 and 4,50Og steam explosion product (with water content of 70wt%) obtained by pretreatment of 1,50Og straw (with water content of 10%).

The enzymolysis product is filtered by Buchner funnel, 20ml of the filtrate is transferred to dry 50ml conical flask and allowed to stand for 5hr, and then the supernatant is collected. The supernatant is filtered via 0.2micron filter membrane, and subjected to HPLC analysis according to the conditions in step (1). The glucose in the enzymolysis product is determined to be 942.4g. The weight of the glucose resulted from enzymolysis is divided by 1.11 to obtain weight of enzymolyzed cellulose in the cassava residues and the steam explosion product 849g, cellulose conversion rate and monosaccharide yield are calculated according to the following equations, and the calculation result is shown in Table 1. The amount of cellulose enzymolyzed per hour is 20.5g. (the first batch mixture of the cassava residues and steam explosion product (on dry matter basis) counts for 9.2wt% of the obtained mixture of the first batch mixture of steam explosion product and cassava residues, enzyme, and water)

Cellulose conversion rate = 100% x weight of enzymolyzed cellulose /total weight of cellulose Monosaccharide yield = 100% x weight of glucose obtained via enzymolysis/ total weight of cassava residues and straw on dry matter basis (3) Fermentation The enzymolysis product is fermented according to the method in example 1, the fermentation product is distilled at 100°C, the obtained distillation fraction is redistilled at 78.3 °C to give ethanol 502.2g, the ethanol yield is calculated according to the following equation, and the calculation result is shown in Table 1. Ethanol yield = 100% x ethanol weight / total weight of cassava residues and straw on dry matter basis

Comparison example 2

Ethanol is produced according to the same method as example 6, except that all the mixture of the steam explosion product and the cassava residues left after sample test in step (1) is charged into enzymolysis tank containing water in one time in the enzymolysis step (2), then uniformly mixed with enzyme (weight ratio of total mixture to water is 3:1) to carry out enzymolysis for 41 hours and 20 minutes. HPLC analysis is carried out according to the conditions in step (1) in example 1, the glucose in the enzymolysis product is determined to be 777.6g, the weight of the glucose resulted from enzymolysis is divided by 1.11 to give weight of enzymolyzed cellulose in steam explosion product 700.5g, amount of cellulose enzymolyzed per hour is 16.9g, and ethanol resulted from redistillation is 415.8 g. Cellulose conversion rate, monosaccharide yield, and ethanol yield are calculated according to the method and equation in example 6, and the calculation result is shown in Table 1.

Example 7

The example is for describing the method for producing ethanol from raw material containing cassava residues.

Ethanol is produced according to the method in example 6, except that the steam explosion product is not water-washed in the straw pretreatment step.

After emzymolysis, the glucose in the enzymolysis product is determined to be 933.8g, the weight of the glucose resulted from enzymolysis is divided by 1.11 to give weight of enzymolyzed cellulose in the mixture of steam explosion product and cassava residues 841.3g. Cellulose conversion rate, monosaccharide yield, and ethanol yield are calculated according to the method and equation in example 6, and the calculation result is shown in Table 1. The amount of cellulose enzymolyzed per hour is 20.3g. (the first batch mixture of the cassava residues and steam explosion product (on dry matter basis) counts for 9.2wt% of the obtained mixture of the first batch mixture of steam explosion product and cassava residues, enzyme, and water). The ethanol resulted from redistillation is 470. Ig. Ethanol yield can be obtained by the method and equation in example 6, and the calculation result is shown in Table 1.

Example 8 The example is for describing the inventive method for preparing ethanol from raw material containing cassava residues.

Ethanol is produced according to the method in example 6, except that during enzymolysis, the raw material is a mixture of 2,100g of the cassava residues with water content of 10% and 2,70Og steam explosion product (with water content of 70wt%) obtained by pretreatment of 90Og of straw (with water content of 10%); during enzymolysis, the mixture of cassava residues and steam explosion product is mixed with enzyme in three batches, wherein the first batch mixture of cassava residues and steam explosion product is added at the amount of 20wt% of the total mixture of the steam explosion product and the cassava residues left after sample test in step (1), regulated to pH 5, and heated to 50⁰C, total cellulase (Imperial jade bio-technology Co. Ltd.) 35,640 enzyme activity units (about 237 g) and hemicellulase (Beijing Chemical Reagents Company) 9,900 enzyme activity units (about 132g) are added into 1,98Og mixture (on dry matter basis) according to addition amount of cellulase 18 enzyme activity unit and hemicellulase 5 enzyme activity units per gram of the mixture on dry matter basis, mixed for 20min at 50 °C ; then second batch mixture is added at the amount of 40wt% of the total mixture of the steam explosion product and the cassava residues left after sample test, mixed for 40min at 52°C (wherein ratio of water addition amount and total mixture weight is 4:1); and then the third batch mixture is added at the amount of 40wt% of the total mixture of the steam explosion product and the cassava residues left after sample test, and enzymolyzed for 42hr at 52 °C . HPLC analysis is carried out according to the conditions in step (1) in example 1, the glucose in the enzymolysis product is determined to be 876.8g, the weight of the glucose resulted from enzymolysis is divided by 1.11 to give weight of enzymolyzed cellulose in the mixture 789.9g, the xylose in the enzymolysis product is determined to be 248.1g, the weight of the xylose resulted from enzymolysis is divided by 1.14 to give weight of enzymolyzed xylose in the mixture 217.6g, total amount of cellulose and xylose enzymolyzed per hour is 23.4g, and cellulose conversion rate, hemicellulose conversion rate, and monosaccharide yield are calculated according to the following equation, and the calculation result is shown in Table 1. (the first batch mixture of the cassava residues and steam explosion product (on dry matter basis) counts for 4.2wt% of the obtained mixture of the first batch mixture of steam explosion product and cassava residues, enzyme, and water) Cellulose conversion rate = 100% x weight of enzymolyzed cellulose /total weight of cellulose Hemicellulose conversion rate = 100% x weight of enzymolyzed hemicellulose /total weight of hemicellulose

Monosaccharide yield = 100% x (weight of glucose +xylose obtained via enzymolysis)/total weight of cassava residues and straw on dry matter basis In fermentation, the enzymolysis product is cooled to 32°C, and inoculated with Saccharomyces cerevisiae (Angel super alcohol active dry yeast, Hubei Angel Yeast Co., Ltd) 10⁶ colony forming units per gram of enzymolysis product. The resultant mixture is cultivated at 32⁰C in fermentation tank for 36 hr while stirring; the fermentation product is distilled at 100°C, and then the obtained distillation fraction is redistilled at 78.3 °C to obtain ethanol 574.5g. The ethanol yield can be calculated according to the equation as below, and the calculation result is shown in Table 1.

Ethanol yield = 100% x ethanol weight/total weight of cassava residues and straw on dry matter basis

Example 9

The example is for describing the inventive method for producing ethanol from raw material containing cassava residues.

Ethanol is produced according to the method in example 6, except that the straw is acid treated straw. The method comprises mixing 1,203 g of corn straw (water content 10%) with 2wt% sulfuric acid 250ml at 140 °C under 0.06MPa for 40min to give acid treated product, filtering, mixing with water at ratio of 1:3, stirring for 30min, and subjecting to solid-liquid separation at 900rpm by using LW400 horizontal helical centrifuge (Jiangsu Huada Centrifuge Manufacture Co. Ltd.) to give acid treatment product 4,33Og (with water content of 75wt%).

HPLC analysis is carried out according to the method for analyzing sulfuric acid hydrolyzed steam explosion product, in which 1Og of the acid treated product is sampled for test; the result shows that the glucose concentration in acid hydrolysis liquid of acid treatment product is 1.8mg/ml, 0.126g of glucose is obtained by acid hydrolyzing water -washed acid treatment product Ig. As 72wt% sulfuric acid can completely hydrolyze the cellulose in the acid treatment product into glucose, the weight of glucose is 1.11 times of the weight of the cellulose in acid treatment product, i.e. Ig of water-washed acid treatment product contains cellulose 0.114g, and 4,32Og of acid treatment product contains cellulose 490.4g. The xylose concentration in acid hydrolysis liquid of acid treatment product is 0.8mg/ml, 0.057g of xylose is obtained by acid hydrolyzing water-washed acid treatment product Ig. As 72wt% sulfuric acid can completely hydrolyze the hemicellulose in the acid treatment product into xylose, the weight of xylose is 1.14 times of the weight of the hemicellulose in acid treatment product, i.e. Ig of the water-washed acid treatment product contains hemicellulose 0.05Og, and 4,32Og of acid treatment product contains hemicellulose 217.9g.

The enzymolysis is carried out according to the method in example 6, and the raw material is a mixture of l,800g of the cassava residues with water content of 10% in example 1 and 4,32Og acid treatment product (with water content of 75wt%) obtained by acid treatment of l,200g corn straw (with water content of 10%). The enzymolysis product is filtered by Buchner funnel, 20ml of the filtrate is transferred to dry 50ml conical flask and allowed to stand for 5hr, and then the supernatant is collected. The supernatant is filtered via 0.2micron filter membrane, and subjected to HPLC analysis according to the conditions in step (1). The glucose in the enzymolysis product is determined to be 899.5 g. The weight of the glucose resulted from enzymolysis is divided by 1.11 to obtain weight of enzymolyzed cellulose in the mixture of the cassava residues and the acid treatment product 810.4g, cellulose conversion rate and monosaccharide yield are calculated according to the equations in example 6, and the calculation result is shown in Table 1. (the first batch mixture of the cassava residues and acid treatment product (on dry matter basis) counts for 9.2wt% of the obtained mixture of the first batch mixture of acid treatment product and cassava residues, enzyme, and water)

The fermentation method is same as that in example 6, the weight of obtained ethanol is 458.7g, ethanol yield is calculated according to the equations in example 6, and the result is shown in Table 1.

Table 1

It can be learned from the results in Table 1 that the ethanol yield and monosaccharide yield of the inventive method for producing ethanol from raw material containing cassava residues are both high, which means the utilization rate of the raw material containing cassava residues is significantly improved.

Claims

1. A method for producing ethanol from raw material containing cassava residues, comprising the steps of: mixing raw material containing cassava residues with enzyme, and enzymolyzing the cellulose in the raw material to give enzymolysis product, wherein the mixing of the enzyme and the raw material comprises mixing the raw material and the enzyme in batches, the batch number is at least two, and each batch of the raw material is added at the amount of at most 60wt% of the total raw material; and fermenting the enzymolysis product.

2. The method according to claim 1, wherein the amount of the first batch of the raw material is 20-60wt% of the total raw material, and the amount of the raw material mixed with the enzyme after the first batch is 40-80wt% of the total raw material.

3. The method according to claim 1, wherein the batch number is three, the amount of the first batch of the raw material is 20-60wt% of the total raw material, the amount of the second batch of the raw material is 20-50wt% of the total raw material, and the amount of the third batch of the raw material is 20-40wt% of the total raw material.

4. The method according to claim 2 or 3, wherein when the first batch of the raw material is mixed with enzyme, water is added in such an amount that, the content of the first batch of the raw material on dry matter basis is at least lwt% relative to the total weight of the obtained mixture.

5. The method according to claim 4, wherein the content of the first batch raw material on dry matter basis is 1.2 to 10wt% relative to the total weight of the obtained mixture.

6. The method according to claim 1, wherein a time interval is included between two adjacent batches.

7. The method according to claim 6, wherein the time interval is 10-120min.

8. The method according to claim 1, wherein the enzyme used in the enzymolysis includes cellulase, the usage amount of cellulase is 10-25 enzyme activity units per gram of the raw material on dry matter basis, the enzymolysis temperature is 45-55 °C, the enzymolysis time is 25-48hr, and the enzymolysis pH is 3-7.

9. The method according to claim 8, wherein the enzyme used in the enzymolysis further includes hemicellulase which is used for enzymolyzing hemicellulose in the raw material, and the usage amount of hemicellulase is 4-8 enzyme activity units per gram of the raw material on dry matter basis .

10. The method according to claim 1, wherein the yeast for the fermentation is Pichia stipitis and/or Saccharomyces cerevisiae, the inoculation amount of yeast used in the fermentation is 10³-10⁸ colony forming units per gram of enzymolysis product, and the fermentation is performed at temperature of 30-36 °C for 32-48hr.

11. The method according to claim 1, wherein the raw material is cassava residues or mixture of cassava residues and straw.

12. The method according to claim 1 or 11, wherein the raw material is the mixture of cassava residues and straw, and the contents of the cassava residues and the straw are respectively 40-90 wt% and 10-60 wt% based on total weight of the mixture of cassava residues and straw.

13. The method according to claim 12, wherein the contents of the cassava residues and the straw are respectively 50-70 wt% and 30-50 wt% based on total weight of the mixture of cassava residues and straw.

14. The method according to claim 11, wherein the straw is steam exploded straw and/or acid-treated straw.

15. The method according to claim 11, wherein the particle size of cassava residues is 0.3-5mm.