A process for the isolation and acclimatization of bacteria for lignin degradation

Abstract

The invention provides a novel process of lignin degradation using a consortium of bacteria. To date, biodegradation of lignin has been centered to fungi only. Degradation of lignin by bacteria confer a new understanding that may be of tremendous industrial significance. This invention also discloses the isolation and acclimatization of ligninolytic bacteria from a specific site

1 - 9 . (canceled)  
   
   
       10 . A process of preparing a bacterial consortium comprising acclimatized ligninolytic bacterial isolates (CBTCC/52-03, CBTCC/53-03 and CBTCC/54-03) deposited at International Depository at IMTECH, Chandigarh, India as recognized by Budapest Treaty,  
     
       
         
               
               
               
             
                   
               
                   
               
                 S. No. 
                 Culture 
                 Deposition No. 
               
                   
               
                 1. 
                 CBTCC/52-03 
                 MTCC 5094 
               
                 2. 
                 CBTCC/53-03 
                 MTCC 5095 
               
                 3. 
                 CBTCC/54-03 
                 MTCC 5098 
               
                   
               
                   
               
           
              
              
              
              
             
             
              
              
              
              
              
             
          
         
       
     
     useful for lignin degradation using the said process comprising the steps of: 
 a) enriching bacterial flora of a site from where the bacteria is to be extracted;  
 b) isolating the ligninolytic bacteria using different media;  
 c) culturing the bacteria isolated in step (b) under defined conditions such as media, temperature, pH, carbon source etc.;  
 d) checking the lignin degrading capability of the isolated bacterial isolates by inoculating them in 10 ml of 0.4% lignin;  
 e) selecting the bacterial isolates which can decolorize the lignin effectively;  
 f) acclimatizing the short listed bacterial isolates for higher concentration of lignin to see the enhancement of their ligninolytic activity;  
 g) further short listing and formulation of bacterial isolates in order to see their synergistic effect for lignin degradation;  
 h) selecting from the short list of step (g), a microbial consortium comprising of three bacterial isolates exhibiting the lignin degradation up to 0.8%, and if desired;  
 i) culturing the bacterial isolates of the said consortium under defined conditions for the detection of enzyme activity in crude as well as in concentrated sample;  
 j) centrifuging the resulting culture after attaining the heavy growth O.D. (1.00-1.20);  
 k) collecting the supernatant and concentrating through ammonium sulfate precipitation followed by dialysis, and  
 l) assaying the lignin peroxidase in both the samples, crude as well as in concentrated sample by measuring the product (veratryldehyde) formation by a known spectrophotometric method at 310 nm.  
 
   
   
       11 . A process as claimed in  claim 10 , wherein the soil enrichment is done in 100 ml-soil extract and 0.3% lignin for the period of 96 hours.  
   
   
       12 . A process as claimed in  claim 10 , wherein the isolation of ligninolytic bacteria is carried out using four media, 0.3 lignin+agar; soil extract+agar; 50% soil extract+agar; 0.3% lignin+50% soil extract.  
   
   
       13 . A process as claimed in  claim 10 , wherein the culturing of the isolated bacterial isolates is carried out at pH 6.8-7.2 and at 30-35° C.  
   
   
       14 . A process as claimed in  claim 10 , wherein the lignin degradation is carried out by inoculating the isolated bacterial isolates in 10 ml of 0.4% lignin.  
   
   
       15 . A process as claimed in  claim 10 , wherein the short listed bacterial isolates are acclimatized to higher concentrations of lignin, having strength ranging from 0.5%-1% in 30 ml test tubes.  
   
   
       16 . A process as claimed in  claim 10 , wherein the said consortium comprising of three ligninolytic bacteria is obtained by testing different combinations of the isolated bacteria and selecting the consortium that has been able to degrade the lignin up to 0.8%.  
   
   
       17 . A process as claimed in  claim 10 , wherein the bacterial isolates of the said consortium are cultured in 1000 ml minimal salt medium having 1.0% glucose individually.  
   
   
       18 . A process as claimed in  claim 17 , wherein the culture flask is incubated at 30-34° C. at 100-120 rpm for 3 days to obtain heavy growth.  
   
   
       19 . A process as claimed in  claim 10 , wherein the cultures are centrifuged at 10,000 rpm for 30 minutes at 4° C.  
   
   
       20 . A process as claimed in  claim 10 , wherein the collected supernatant is concentrated through 80% ammonium sulphate precipitation followed by dialysis.  
   
   
       21 . A process as claimed in  claim 10 , wherein the lignin peroxidase is assayed in both the samples, crude as well as in concentrated sample by measuring the product (veratryldehyde) formation spectrophotometrically at 310 nm.  
   
   
       22 . A process as claimed in  claim 10 , wherein the sawdust soil is enriched before extraction to get the desired ligninolytic bacterial flora.  
   
   
       23 . A process as claimed in  claim 10 , wherein the sawdust soil is enriched by: 
 a) enriching the said soil in soil extract and 0.3% lignin;    b) incubating the enrichment flask at 100-120 rpm/30-32° C.    
   
   
       24 . A process as claimed in  claim 23 , wherein the 5-7 gram of said soil is enriched in 100-120 ml soil extract and 0.3% lignin.  
   
   
       25 . A process as claimed in  claim 23 , wherein the enrichment is done at 100-120 rpm/30-32° C. for a period of 96 hours.  
   
   
       26 . A process as claimed in  claim 10 , wherein the ligninolytic bacteria is isolated from the saw-dust site using the following media: 
 a) 0.3 lignin+agar;    b) soil extract+agar;    c) 50% soil extract+agar;    d) 0.3% lignin+50% soil extract.    
   
   
       27 . A process as claimed in  claim 10 , wherein the isolated ligninolytic bacterial stains are acclimatized to higher concentrations of lignin by: 
 a) inoculating the isolated bacterial strains in lignin ranging from 0.5% to 1.00%, and    b) incubating the tubes at 30-32° C. for a long period.    
   
   
       28 . A process as claimed in  claim 27 , wherein the ligninolytic bacterial isolates are inoculated in lignin ranging from 0.5% to 1.00%.  
   
   
       29 . A process as claimed in  claim 27 , wherein the inoculated tubes of lignin are kept at 30-32° C. for a period of 3 months.  
   
   
       30 . A process of lignin degradation, said process comprising inoculating the bacterial consortium comprising bacterial strains CBTCC/52-03, CBTCC/53-03 and CBTCC54-03 deposited at International Depository at IMTECH, Chandigarh, India, and having accession numbers MTCC 5094, MTCC 5095 and MTCC 5098 in an solution containing lignin for 1 to 5 days at temperature between 25 to 35° C.  
   
   
       31 . A process as claimed in  claim 30 , wherein the bacterial consortium comprises whole cell bacterial isolates of bacterial strains MTCC 5094, MTCC 5095 and MTCC 5098.  
   
   
       32 . A process as claimed in  claim 30 , wherein the consortium comprises bacterial strains MTCC 5094, MTCC 5095 and MTCC 5098.  
   
   
       33 . A process as claimed in  claim 30 , wherein the consortium comprises bacterial strains MTCC 5094, MTCC 5095 and MTCC 5098 in the range of 20 to 40% by wt. each.  
   
   
       34 . A process as claimed in  claim 30 , wherein the consortium comprises bacterial strains MTCC 5094, MTCC 5095 and MTCC 5098 in equal proportions.  
   
   
       35 . A process as claimed in  claim 30 , wherein the bacteria are isolated from a mixture of sawdust and soil.  
   
   
       36 . A process as claimed in  claim 30 , wherein the bacteria are isolated from a mixture of sawdust and soil from Roorkhee, India.  
   
   
       37 . A process as claimed in  claim 30 , wherein the consortium exhibits lignin degradation of up to 0.8%.