Methods for fermentation of xylose and hexose sugars

A method of isomerizing and fermenting one or more sugars, comprising:

• i) dispersing co-immobilized enzyme pellets in a mixture containing urea, an ionic borate source, and a substrate comprised of one or more of aldose and ketose sugars,
• wherein the co-immobilized enzyme pellets comprise a first immobilized enzyme comprising an xylose isomerase having a pH of about 6 or above that acts on the substrate in a first porous region and a second immobilized enzyme comprising urease in a second porous region,
• the first porous region having a pH higher than the pH of the mixture due to the presence of ammonia produced by the urease acting on urea that diffuses from the mixture into the second region of the pellet,
• the first immobilized enzyme catalyzing the conversion of aldose sugar to ketose sugar which forms a complex with the ionic borate source, the complex migrating from the higher first region of the pellet to the lower pH mixture where the complex dissociates into the ketose isomer of the substrate and borate;
• ii) isomerizing xylose into xylulose at a temperature range of about 30° C. to about 40° C. to produce xylulose at a xylulose:xylose ratio greater than 1:1;
• and
• iii) fermenting the isomers in the mixture to form ethanol.

A method for the simultaneous isomerization and fermentation of xylose to ethanol, comprising the steps of:

• i) mixing xylose, urea, and yeast cells having high ethanol productivity and ethanol tolerance in a reactor to form a mixture;
• ii) maintaining the pH of the mixture in the range of about 4.0 to about 5.5;
• iii) dispersing co-immobilized enzyme particles having at least two enzymes into the mixture, wherein the enzymes comprise at least xylose isomerase and urease;
• iv) adding a borate source to the mixture;
• v) allowing xylose to diffuse into the particles;
• vi) allowing the diffused xylose in the particles to isomerize to xylulose by activity of immobilized xylose isomerase;
• wherein the isomerization takes place at a temperature of about 30° C. to about 40° C.;
• vii) allowing the xylulose formed in the particles to diffuse out into the mixture;
• viii) maintaining the pH in the particle during isomerization in the range of about 7.0 to about 8.0 by allowing urea to diffuse into the particles, whereby the urea is hydrolyzed to ammonia by immobilized urease, and whereby the ammonia neutralizes hydrogen ions that diffuse into the particles;
• ix) optionally, agitating the mixture under substantially anaerobic conditions so as to allow the fermentation of xylulose to ethanol;
• and
• x) allowing xylulose to ferment into ethanol.

A method of isomerizing and fermenting one or more sugars, the method comprising:

• i) dispersing particles in a mixture, the mixture comprising an ionic borate source, urea, and xylose, the particles comprising:
• a first region having a first enzymatic activity comprising xylose isomerase (XI) at a pH of 6 or above;
• and
• a second region having a second enzymatic activity comprising urease at an acidic pH;
• ii) isomerizing xylose into xylulose at a temperature range of about 30° C. to about 40° C. to produce xylulose at a xylulose:xylose ratio greater than that produced at equilibrium by XI with no pH gradient;
• and
• iii) fermenting the mixture.