Process for producing d-mannitol

Abstract

High concentration of free cells of heterofermentative lactic acid bacteria (LAB) in a resting or slowly growing state are used to convert fructose into mannitol. Efficient volumetric mannitol productivities and mannitol yields from fructose are achieved in a process applying cell-recycle, continuous stirred tank reactor and/or circulation techniques with native LAB cells or with LAB cells with inactivated fructokinase gene(s). Mannitol is recovered in high yield and purity with the aid of evaporation and cooling crystallization.

1 . A process for the production of mannitol by bioconversion, comprising the steps of 
 bringing a high initial concentration of free, mannitol-producing lactic acid bacterial cells into contact with a low-nutrient medium supplemented with a substrate convertible into mannitol, and a cosubstrate, in a bioreactor system,    performing the bioconversion under conditions suitable for converting said substrate into mannitol,    separating the bacterial cells from the medium by filtration to obtain a cell-free solution,    recovering from the cell-free solution the mannitol produced, and    reusing the separated bacterial cells in the bioreactor system.    
     
     
         2 . The process according to  claim 1 , wherein the bacterial cells are native lactic acid bacterial cells.  
     
     
         3 . The process according to  claim 2 , wherein the bacterial cells are of the strain  Leuconostoc mesenteroides  ATCC-9135.  
     
     
         4 . The process according to  claim 1 , wherein the bacterial cells are fructokinase-inactivated lactic acid bacterial cells.  
     
     
         5 . The process according to  claim 4 , wherein the cells are of the strain  Leuconostoc pseudomesenteroides  BPT143 (DSM 14613).  
     
     
         6 . The process according to  claim 1 , wherein the substrate is fructose.  
     
     
         7 . The process according to  claim 1 , wherein the cosubstrate is glucose.  
     
     
         8 . The process according to  claim 1 , wherein the bioconversion is performed until at least 70%, preferably 90% or more of the said substrate has been consumed.  
     
     
         9 . The process according to  claim 1 , wherein an average volumetric mannitol productivity of at least 10 g/L/h is achieved.  
     
     
         10 . The process according to  claim 1 , wherein the bioconversion is performed as a batch process in a bioreactor system comprising a bioconversion reactor unit and a filtration unit.  
     
     
         11 . The process according to  claim 10 , comprising the steps of separating the bacterial cells from the medium after the bioconversion step, and reusing said cells in successive bioconversions.  
     
     
         12 . The process according to  claim 1 , wherein the bioconversion is performed as a circulation process in a bioreactor system comprising a bioconversion reactor unit, a filtration unit and a mixing reactor unit.  
     
     
         13 . The process according to  claim 12 , wherein the bacterial cells are circulated between the bioconversion reactor unit and the filtration unit.  
     
     
         14 . The process according to  claim 12  or  13 , comprising the steps of leading the cell-free solution obtained by the filtration into the mixing reactor, and transferring the solution from the mixing reactor back to the bioconversion reactor unit.  
     
     
         15 . The process according to any one of the  claims 12  to  14 , comprising emptying the mixing reactor after the bioconversion step, and re-filling it with said low-nutrient medium supplemented with said substrate and co-substrate, to run successive bio-conversions reusing said bacterial cells.  
     
     
         16 . The process according to  claim 1 , wherein the bioconversion is performed as a continuous process in a bioreactor system comprising a mixing tank, a feed tank, a bioconversion reactor unit, a filtration unit and a recovery tank.  
     
     
         17 . The process according to  claim 16 , wherein the bacterial cells are circulated between the bioconversion reactor unit and the filtration unit.  
     
     
         18 . The process according to  claim 16  or  17 , comprising the steps of feeding the bioconversion reactor unit continuously with said low-nutrient medium supplemented with said substrate and co-substrate, and removing the cell-free solution gained by filtration from the bioreactor to withhold a constant volume in the bioconversion reactor unit.  
     
     
         19 . The process according to any one of the  claims 10  to  18 , wherein the filtration unit is a tangential flow filtration unit.  
     
     
         20 . The process according to any one of the  claims 10  to  18 , wherein said bioconversion is run in series or in parallel.  
     
     
         21 . A bacterial strain of the genus Lactobacillus or Leuconostoc, in which the fructokinase enzyme(s) is/are inactivated.  
     
     
         22 . The bacterial strain according to  claim 21 , in which the fructokinase enzyme(s) is/are inactivated by random mutagenesis.  
     
     
         23 . The bacterial strain according to  claim 22 , which is  Leuconostoc pseudomesenteroides  BPT143 (DSM 14613).  
     
     
         24 . The bacterial strain according to  claim 21 , in which the fructokinase enzyme(s) is/are inactivated by directed mutagenesis.  
     
     
         25 . Use of a bacterial strain of the genus Lactobacillus or Leuconostoc, in which the fructokinase enzyme(s) is/are inactivated, for producing mannitol.