- Email: [email protected]
Semimicro determination of cellulose inbiological materials
A*N-ALYTICAL BIOCtIEIVIISTRY3:2,
420--424 (1969)
Semimicro Determination of Cellulose in Biological Materials DAVID M. UPDEGRAFF Chemical Division, Denver Research Institute, University of Denver, Denver, Colorado 80210 Received June 1I, 1969
The quantitative determination of cellulose in biological materials presents some difficult problems. The moisture con~ent of cellulose varies with the relative humidity, so that accurate analyses require that moisture determinations be made under carefully standardized conditions. Cellulose ordinarily occurs in association with lignin, polyuronide hemicelluloses, and xylan cellulosans. Matrone, Ellis, and Maynard (4), who were interested primarily in the nutrition of ruminants, pointed ou~ that the xylan cellulosans are probably digested to the same extent by animals as true cellulose, and therefore recommended a modification of the method of Norman and Jenkins (5), which determines true cellulose and xylan eellulosans together, over the method of Kiirschner and Hanak (3) or the Crampton and Maynard (1) modification of this method, in which xylan cellulosans are partially or completely removed, so that cellulose is determined alone. All of these methods are laborious gravimetrie procedures in which the sample, ground to 40 mesh or finer, is repeatedly extracted with a series of hot solvent mixtures which remove lipides, proteins, lignin, and hemicelluloses. The residue is then dried at 105 ° to 110°C, and weighed, and cellulose is determined by the weight~ loss on ignition. Our objective was to develop a simple, rapid, colorimetric method for the determination of cellulose in a large number of cultures of cellulolytic bacteria and fungi. Since the procedure of Crampton and Maynard required much less time for extractions than that of Matrone, Ellis, and Maynard, it was chosen as a starting point. The sedimentation of the cellulose, extraction with the acetic acid/nitric acid reagent of Crampton and Maynard to remove lignin, hemicellulose, and xylosans, and final solution of ~he sample in 67% tt~SQ (v/v) were all carried out in a single 15 ml centrifuge tube. Cellulose was then determined on an appropriate dilution of the sulfuric acid solution by the anthrone method of Scott and Melvin (7). 42O
CELLULOSE IN
BIOLOGICAL MATEBIALS
421
METHODS Procedure The exact procedure adopted is described below: 1. Homogenize sample of microbial culture in a Waring Blendor or Virtis homogenizer. A 100 ml sample containing from 0.5 to 2 gm of cellulose is ordinarily used, but small samples down to 10 ml with 0.05 to 0.2 gm of cellulose would be equally satisfactory. 2. Place 10.0 ml sample in 15 ml centrifuge tube. 3. Centrifuge 5 rain at 2000 to 3000 rpm in a clinical-type centrifuge. 4. Decant and discard supernatant. 5. Add 3.0 ml acetic nitric reagent; it is convenient to do this by adding 1.0 ml, mixing well on Vortex mixer, then adding the remaining 2.0 ml and remixing. 6. With a marble on top to reduce evaporation and create a refluxing action, place tubes in a boiling water bath for 30 rain. Maintain bath level at same level as the liquid in the tubes. 7. Centrifuge 5 rain at high speed. Decant and discard supernatant. 8. Add 10 ml distilled water wash in a manner similar to step 5. 9. Centrifuge 5 min at high speed. Decant and discard supernatant. 10. Add 10.0 ml 67% H2SQ (v/v) in a manner similar to step 5. 11. Let stand 1 hr. 12. Dilute 1.0 ml to 100 ml with distilled water. Centrifuge if any precipitate or turbidity is present. 13. Place 1.0 ml of this dilution in a 150 X 18 mm screw-cap type culture tube. Add 4.0 ml distilled water. 14. Place tubes in an ice bath to cool. 15. Add 10.0 ml cold anthrone reagent by layering with a pipet. 16. Mix well on Vortex mixer--a Parafilm cover is convenient to prevent splashing. Return to ice bath until all tubes are mixed. 17. Place a marble on top of each and place tubes in a boiling water bath for 16 rain. 18. Cool in ice bath 2-3 rain. Let stand at room temperature 5-10 rain. 19. Read each on a suitable spectrophotometer in 1-2 cm cells at a wavelength of 620 mtL against a reagent blank. Standard Curve To prepare the stock standard dissolve 50.00 mg pure cellulose (Nutritional Biochemicals Co., catalog No. 4427), dried for 6 hr at 105°C and cooled over anhydrous alumina, in 10.0 ml 67% H~SQ with gentle heat. Dilute this to 500 ml with distilled water to contain 100 ~g cellulose/ ml. Analyze 0.5, 1.0, and 1.5 ml stock standard, corresponding to 50, 100,
422
DAVID M. UPDEGRAFF
and 150 t~g of cellulose as in step 13 of the procedure. Add the appropriate quantity of distilled water to each to bring the final volumes to 5.0 ml.
Reagents The acetic/nitric reagent is prepared by mixing 150 ml 80% acetic acid and 15 ml concentrated nitric acid. The anthrone reagent is 0.2 gm anthrone (Eastman, catalog No. 6432) in 100 ml concentrated H2S04, prepared fresh daily. Chill about 2 hr in refrigerator prior to use.
Sensitivity An absorbance of 0.34-0.37 should be obtained from 100 ug of cellulose using a 12 mm path length. RESULTS Some typical results of analyses carried out on sterile culture media containing 10 mg/ml of purified cellulose, ball-milled newsprint (Denver Post, without ink) or ball-milled newspaper (Wall Street Journal) are given in Table 1. The purified cellulose was dried at 105°C, as was the reference standard cellulose, but the newsprint and newspaper were dried at ambient temperature and humidity conditions. This procedure gives valid data on cellulose utilization from the paper, since the reference standard cellulose was dried under standardized conditions, and sterile control samples made from the same paper sample as the test sample were always analyzed to obtain the "initial cellulose concentration." This value was then subtracted from the "final cellulose concentration" to obtain the va~lue from which "% cellulose utilized" was calculated. TABLE 1 Analyses of Sterile Culture Media Containing Purified Cellulose, Ball-Milled Newsprint, a or Ball-Milled Newspaper b
Substrate Purified cellulose Newsprint Newspaper
Original conch., mg/Inl 10.0 10.0 10.0 10.0 10.0 10.0
Medium
Cellulose found~ rag/m1
~ cellulose recovered from substrate
Reese (6) Srinivasan (2) Reese Srinivasan Reese Srinivasan
10.2 10.6 5.4 5.6 6.1 6.1
102 106 54 56 61 61
Newsprint paper used for printing newspaper (without ink) Denver Post. bNewspaper--Wall Street Jmo'nal.
CELLULOSE IN BIOLOGICAL MATERIALS
423
The data indicate good quantitative recovery of purified cellulose (102 and 106%). T h e y also show t h a t the Denver Post newsprint contains 55% cellulose, while the Wall Street Journal paper contains 61% cellulose. A few selected cellulose values for cellulolytic fungus cultures grown on newsprint in Reese's medium are given in T a b l e 2. As before, the media contained 10 m g / m l of newsprint. TABLE 2 Analyses of Cellulolytic Fungus Cultures for Cellulose
Culture No. P9 P9 P 10 P 10 353 X 99 B 353 X 99 B C-264 C-264
Incubation Incubation time, temp., °C days 30 30 30 30 30 30 37 37
2 5 2 5 2 5 2 5
Initial cellulose conch., mg/ml 6.6 6.6 6.6 6.6 6.6 6.6 6.6 6.6
FinM cellulose conen., mg/ml 2.7 1.6 1.7 1.6 2.5 1.7 2.5 1.8
% cellulose utilized 59 75 74 75 62 75 63 73
E a c h of these four fungus cultures, which represent as y e t unidentified species of cellulolytic fungi, consumes cellulose from newsprint at a rapid rate. The original cellulose value found in the sterile control sample, 6.6 m g / m l , does not agree v e r y well with the mean value for newsprint of 5.5 m g / m l in T a b l e 1. This m a y have been caused by a difference in relative humidity, since the paper was dried at ambient t e m p e r a t u r e and humidity conditions at different times for these two experiments. As indicated earlier, the "initial cellulose concentration" was obtained on p a p e r treated exactly as t h a t for the "final cellulose concentration." SUMMARY A semimicro method is described for the determination of cellulose in microbial cultures, other biological materials, or pulp and p a p e r products. Lignin, hemicellulose, and xylosans are extracted with acetic acid/nitric acid reagent, and the remaining cellulose is dissolved in 67% II2S04 and determined by the anthrone reagent. T h e method gives quantitative recovery of purified cellulose from microbiological culture media, and also appears to be satisfactory for cellulose from p a p e r pulp. ACKNOWLEDGMENT The capable technical assistance of Mr. S. L. Braunstein and Mr. L. Griffin, who carried out mo~ of the analyses, is gratefully acknowledged. This work was supported
4~4
DAVID M, UPDEGRAFF
by a grant from the Office of Solid Wastes Management, Environmental Control Administration, Department of Health, Education, and Welfare. REFERENCES 1. 2. 3. 4. 5. 6. 7.
Cr~Mwo~, E. W., .~ND MAYNARD,L. A., J. Nutr. 15, 385 (1938). ttAN, Y. W., AND S RINIVASAN,V. R., Appt. Microbiol. 16, 1140 (1968). KimSCHNER, K., AND HANAX, A., Z. Untersuch. Lebensm. 59, 484 (1930). MAT~0NE, G., ELLIS, G. It., AND MAYNARD,L. A., J. Animal Sci. 5, 306 (1946). NO~MAN, A. G., AND JENKINS, S. ]~., Biochem. J. 27, 818 (1933). REUSE, E. T., SIU, R. G. H., AND LEVINSON, H. S., J. Bacteriol. 59, 485 (1950). SCOTT, T. A., Ja., aND MELVIN, E. It., Anal. Chem. "25, 1656 (1953).
420--424 (1969)
Semimicro Determination of Cellulose in Biological Materials DAVID M. UPDEGRAFF Chemical Division, Denver Research Institute, University of Denver, Denver, Colorado 80210 Received June 1I, 1969
The quantitative determination of cellulose in biological materials presents some difficult problems. The moisture con~ent of cellulose varies with the relative humidity, so that accurate analyses require that moisture determinations be made under carefully standardized conditions. Cellulose ordinarily occurs in association with lignin, polyuronide hemicelluloses, and xylan cellulosans. Matrone, Ellis, and Maynard (4), who were interested primarily in the nutrition of ruminants, pointed ou~ that the xylan cellulosans are probably digested to the same extent by animals as true cellulose, and therefore recommended a modification of the method of Norman and Jenkins (5), which determines true cellulose and xylan eellulosans together, over the method of Kiirschner and Hanak (3) or the Crampton and Maynard (1) modification of this method, in which xylan cellulosans are partially or completely removed, so that cellulose is determined alone. All of these methods are laborious gravimetrie procedures in which the sample, ground to 40 mesh or finer, is repeatedly extracted with a series of hot solvent mixtures which remove lipides, proteins, lignin, and hemicelluloses. The residue is then dried at 105 ° to 110°C, and weighed, and cellulose is determined by the weight~ loss on ignition. Our objective was to develop a simple, rapid, colorimetric method for the determination of cellulose in a large number of cultures of cellulolytic bacteria and fungi. Since the procedure of Crampton and Maynard required much less time for extractions than that of Matrone, Ellis, and Maynard, it was chosen as a starting point. The sedimentation of the cellulose, extraction with the acetic acid/nitric acid reagent of Crampton and Maynard to remove lignin, hemicellulose, and xylosans, and final solution of ~he sample in 67% tt~SQ (v/v) were all carried out in a single 15 ml centrifuge tube. Cellulose was then determined on an appropriate dilution of the sulfuric acid solution by the anthrone method of Scott and Melvin (7). 42O
CELLULOSE IN
BIOLOGICAL MATEBIALS
421
METHODS Procedure The exact procedure adopted is described below: 1. Homogenize sample of microbial culture in a Waring Blendor or Virtis homogenizer. A 100 ml sample containing from 0.5 to 2 gm of cellulose is ordinarily used, but small samples down to 10 ml with 0.05 to 0.2 gm of cellulose would be equally satisfactory. 2. Place 10.0 ml sample in 15 ml centrifuge tube. 3. Centrifuge 5 rain at 2000 to 3000 rpm in a clinical-type centrifuge. 4. Decant and discard supernatant. 5. Add 3.0 ml acetic nitric reagent; it is convenient to do this by adding 1.0 ml, mixing well on Vortex mixer, then adding the remaining 2.0 ml and remixing. 6. With a marble on top to reduce evaporation and create a refluxing action, place tubes in a boiling water bath for 30 rain. Maintain bath level at same level as the liquid in the tubes. 7. Centrifuge 5 rain at high speed. Decant and discard supernatant. 8. Add 10 ml distilled water wash in a manner similar to step 5. 9. Centrifuge 5 min at high speed. Decant and discard supernatant. 10. Add 10.0 ml 67% H2SQ (v/v) in a manner similar to step 5. 11. Let stand 1 hr. 12. Dilute 1.0 ml to 100 ml with distilled water. Centrifuge if any precipitate or turbidity is present. 13. Place 1.0 ml of this dilution in a 150 X 18 mm screw-cap type culture tube. Add 4.0 ml distilled water. 14. Place tubes in an ice bath to cool. 15. Add 10.0 ml cold anthrone reagent by layering with a pipet. 16. Mix well on Vortex mixer--a Parafilm cover is convenient to prevent splashing. Return to ice bath until all tubes are mixed. 17. Place a marble on top of each and place tubes in a boiling water bath for 16 rain. 18. Cool in ice bath 2-3 rain. Let stand at room temperature 5-10 rain. 19. Read each on a suitable spectrophotometer in 1-2 cm cells at a wavelength of 620 mtL against a reagent blank. Standard Curve To prepare the stock standard dissolve 50.00 mg pure cellulose (Nutritional Biochemicals Co., catalog No. 4427), dried for 6 hr at 105°C and cooled over anhydrous alumina, in 10.0 ml 67% H~SQ with gentle heat. Dilute this to 500 ml with distilled water to contain 100 ~g cellulose/ ml. Analyze 0.5, 1.0, and 1.5 ml stock standard, corresponding to 50, 100,
422
DAVID M. UPDEGRAFF
and 150 t~g of cellulose as in step 13 of the procedure. Add the appropriate quantity of distilled water to each to bring the final volumes to 5.0 ml.
Reagents The acetic/nitric reagent is prepared by mixing 150 ml 80% acetic acid and 15 ml concentrated nitric acid. The anthrone reagent is 0.2 gm anthrone (Eastman, catalog No. 6432) in 100 ml concentrated H2S04, prepared fresh daily. Chill about 2 hr in refrigerator prior to use.
Sensitivity An absorbance of 0.34-0.37 should be obtained from 100 ug of cellulose using a 12 mm path length. RESULTS Some typical results of analyses carried out on sterile culture media containing 10 mg/ml of purified cellulose, ball-milled newsprint (Denver Post, without ink) or ball-milled newspaper (Wall Street Journal) are given in Table 1. The purified cellulose was dried at 105°C, as was the reference standard cellulose, but the newsprint and newspaper were dried at ambient temperature and humidity conditions. This procedure gives valid data on cellulose utilization from the paper, since the reference standard cellulose was dried under standardized conditions, and sterile control samples made from the same paper sample as the test sample were always analyzed to obtain the "initial cellulose concentration." This value was then subtracted from the "final cellulose concentration" to obtain the va~lue from which "% cellulose utilized" was calculated. TABLE 1 Analyses of Sterile Culture Media Containing Purified Cellulose, Ball-Milled Newsprint, a or Ball-Milled Newspaper b
Substrate Purified cellulose Newsprint Newspaper
Original conch., mg/Inl 10.0 10.0 10.0 10.0 10.0 10.0
Medium
Cellulose found~ rag/m1
~ cellulose recovered from substrate
Reese (6) Srinivasan (2) Reese Srinivasan Reese Srinivasan
10.2 10.6 5.4 5.6 6.1 6.1
102 106 54 56 61 61
Newsprint paper used for printing newspaper (without ink) Denver Post. bNewspaper--Wall Street Jmo'nal.
CELLULOSE IN BIOLOGICAL MATERIALS
423
The data indicate good quantitative recovery of purified cellulose (102 and 106%). T h e y also show t h a t the Denver Post newsprint contains 55% cellulose, while the Wall Street Journal paper contains 61% cellulose. A few selected cellulose values for cellulolytic fungus cultures grown on newsprint in Reese's medium are given in T a b l e 2. As before, the media contained 10 m g / m l of newsprint. TABLE 2 Analyses of Cellulolytic Fungus Cultures for Cellulose
Culture No. P9 P9 P 10 P 10 353 X 99 B 353 X 99 B C-264 C-264
Incubation Incubation time, temp., °C days 30 30 30 30 30 30 37 37
2 5 2 5 2 5 2 5
Initial cellulose conch., mg/ml 6.6 6.6 6.6 6.6 6.6 6.6 6.6 6.6
FinM cellulose conen., mg/ml 2.7 1.6 1.7 1.6 2.5 1.7 2.5 1.8
% cellulose utilized 59 75 74 75 62 75 63 73
E a c h of these four fungus cultures, which represent as y e t unidentified species of cellulolytic fungi, consumes cellulose from newsprint at a rapid rate. The original cellulose value found in the sterile control sample, 6.6 m g / m l , does not agree v e r y well with the mean value for newsprint of 5.5 m g / m l in T a b l e 1. This m a y have been caused by a difference in relative humidity, since the paper was dried at ambient t e m p e r a t u r e and humidity conditions at different times for these two experiments. As indicated earlier, the "initial cellulose concentration" was obtained on p a p e r treated exactly as t h a t for the "final cellulose concentration." SUMMARY A semimicro method is described for the determination of cellulose in microbial cultures, other biological materials, or pulp and p a p e r products. Lignin, hemicellulose, and xylosans are extracted with acetic acid/nitric acid reagent, and the remaining cellulose is dissolved in 67% II2S04 and determined by the anthrone reagent. T h e method gives quantitative recovery of purified cellulose from microbiological culture media, and also appears to be satisfactory for cellulose from p a p e r pulp. ACKNOWLEDGMENT The capable technical assistance of Mr. S. L. Braunstein and Mr. L. Griffin, who carried out mo~ of the analyses, is gratefully acknowledged. This work was supported
4~4
DAVID M, UPDEGRAFF
by a grant from the Office of Solid Wastes Management, Environmental Control Administration, Department of Health, Education, and Welfare. REFERENCES 1. 2. 3. 4. 5. 6. 7.
Cr~Mwo~, E. W., .~ND MAYNARD,L. A., J. Nutr. 15, 385 (1938). ttAN, Y. W., AND S RINIVASAN,V. R., Appt. Microbiol. 16, 1140 (1968). KimSCHNER, K., AND HANAX, A., Z. Untersuch. Lebensm. 59, 484 (1930). MAT~0NE, G., ELLIS, G. It., AND MAYNARD,L. A., J. Animal Sci. 5, 306 (1946). NO~MAN, A. G., AND JENKINS, S. ]~., Biochem. J. 27, 818 (1933). REUSE, E. T., SIU, R. G. H., AND LEVINSON, H. S., J. Bacteriol. 59, 485 (1950). SCOTT, T. A., Ja., aND MELVIN, E. It., Anal. Chem. "25, 1656 (1953).