Limitations of the DNS Assay for Reducing Sugars from Saccharified Lignocellulosics

Douglas B. Rivers, Stephen J. Gracheck, Lindley C. Woodford, and George H. Emert Biomass Research Center, University of Arkansas, Fayetteville, Arkansas 72701 Accepted for Publication October 28, 1983

Great interest in cellulose as a renewable resource for the production of liquid fuels and chemicals developed as a consequence of the petroleum shortages of 1973 and 1979. Because the critical step in the production of liquid fuels or chemicals from biomass is the hydrolysis of cellulose to glucose, a highly dependable and accurate method of analysis is required. In our laboratory, as well as the 2,4-dinitrosalicyclic acid (DNS) assay for reducing sugars as glucose has been used according to the method of Miller.6 As research advances from the conversion of impractical, purified cellulosic substrates such as Avicel and Solka Floc to practical lignocellulosic substrates such as municipal solid waste (MSW), pulp and paper mill wastes (PMW), and agricultural wastes (AW), assays are required that are not easily influenced by a multitude of interfering chemicals present in the unpurified, native substrate. This communication reports the evaluation of two DNS assay procedures as well as high performance liquid chromatography (HPLC) and YSI Glucose Analyzer analyses of sugars resulting from enzymatic saccharification of lignocellulosics. MATERIALS AND METHODS
Trichoderma reesei QM 9414, obtained from the American Type Culture Collection (Rockville, MD), was used to produce the cellulase system containing endoglucanase, cellobiohydrolase, and cellobiase activities for saccharification. Permanent stock cultures were lyophilized and working stock cultures were maintained on potato dextrose agar plates at 4OC. Seed cultures of T. reesei were grown from spores in M3d medium8 containing 3% (w/v) glucose to increase biomass. This seed culture was used to initiate cellulase production in submerged culture according to the method of Gracheck ,8 Saccharifications were run in 250-mL shake flasks at 150 rpm with a 100-mL working volume. Temperature was controlled at 50C in an orbiting environmental shaker and pH was maintained at 5.0 with a 0.04M acetate buffer containing 0.004M NaN3. A 6% (w/v) concentration of
Biotechnology and Bioengineering, Vol. XXVI, Pp. 800-802 (1984) 0 1984JohnWiley&Sons, Inc.

cellulose was the substrate. The reaction was initiated with a whole culture T. reesei cellulose b r ~ t h . ~ ~ ~ Glucose, cellobiose, and xylose were determined with a Varian model 8520 HPLC and refractive index detector (Varian Instruments, Sunnyvale, CA) equipped with a Partisil PXS 10/25 PAC column (Whatman, Inc., Clifton, NJ), and 80% (v/v) aqueous Distilled in Glass Acetonitrile UV (Burdick and Jackson Laboratories, Muskeegon, MI). Peak areas as compared to external standards were used to quantitate the sugars present. Glucose was also determined with a model 23A Glucose Analyzer (Yellow Springs Instruments Co., Yellow Springs, OH). Total reducing sugars were determined by the assay method of Millet.6 and a modification of the Sumner and Somers method as glucose using a Bausch & Lomb Spectronic 710 spectrophotometer (Bausch & Lomb, Rochester, NY). RESULTS AND DISCUSSION Routine analysis for saccharides resulting from the enzymatic saccharifications of lignocellulosics in this laboratory have indicated both a lack of agreement and correlation between DNS and HPLC analyses. Although some degree of agreement was achievable when assaying pure sugar solutions containing glucose, cellobiose, and xylose, they are not representative of the analytical problems presented in cellulosic saccharifications intended to be analyzed on a regular basis. Therefore, saccharifications of the purified cellulose Avicel PH 105 (97.1% cellulose by dry weight) used both as a measure of T. reesei cellulase activity and as one type control case for saccharifications of other cellulosics were assayed by the two DNS assays and HPLC for comparison (Table I). As shown, there is a close correlation between the product yields when assayed by HPLC and modified DNS. Hydrolysates from lignocellulosics represented by pretreated MSW and PMW were analyzed for glucose production by DNS, HPLC, and YSI (Table 11). The data indicate a favorable comparison in the quantitation of glucose by HPLC and YSI. The DNS assay is not, however, in agreement. Instead, it resulted in data considerably
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T b e I Comparison of Miller DNS, modified DNS, and HPLC glual .

cose

+ cellobiose analyses of Avicel saccharification supernatants.

~ ~ ~

Conversion of cellulose to glucose Miller DNS Theoretical conversion

(70)

Modified DNS Theoretical conversion ("lo) 35.6 38.9 46.4 47.6 46.4 46.4

HPLC Theoretical conversion

(70)

Sugars (g/L) 18.2 19.5 22.9 24.0 19.9 21.5

Sugars (g/L) 12.0 13.1 15.6 16.0 15.6 15.6

Sugars (g/L) 11.0 11.5 16.1 14.4 14.7 14.8

54.1 57.9 67.9 71.4 59.1 63.8

32.7 34.3 47.7 42.0 43.6 43.9

greater in quantity than either HPLC or YSI. No soluble oligomers above G2 were detectable by HPLC. Because DNS measures reducing sugars and HPLC measures specific sugars, absolute agreement was not, and should not, be expected. However, a reproducible correlation should be expected if the ratios of sugars present remain constant. This is a problem even when purified cellulosics are saccharified. A distinct difference is observed for MSW, PMW, and AW where pentoses are also produced. The HPLC and YSI assay specifically for the desired product of a lignocellulosic saccharification, glucose, which is expected to be in the highest proportion in the hydrolysate under normal conditions (adequate cellobiase). As expected, HPLC and YSI glucose analyses are in agreement. However, since the T. reesei cellobiase, which primarily hydrolyzes cellobiose into two glucose molecules, loses activity due to competitive end product inhibition, cellobiose also may be expected to accumulate. Therefore, cellobiose was also quantitated by HPLC. Higher soluble oligomers of glucose, G3-G6, have not been observed in sufficient quantities in our laboratory to be quantitated. The analysis of lignocellulosic hydrolysates by wet chemical assays is more complex than those of purified cellulosics due to the presence of compounds such as tannins and inks released as a consequence of lignocellulose hy-

drolysis resulting in color interferences with the DNS assay. It is difficult, if not impossible, to blank for these compounds in a wet chemical assay since the prehydrolysis supernatants contain little, if any, of these chemicals. Naturally, the greater the degree of hydrolysis, the greater the degree of interference due to these released chemicals. They are consequently determined as glucose and result in a falsely high analysis. This problem is prominent when potential saccharification substrates are MSW and PMW. Other chemicals, such as furfural and 5-hydroxymethyl furfural, results as by-products from pretreatment~~ utilizing either high temperatures and pressures or acid. They may interfere with DNS by supplying reactive reducing groups as well as a characteristic interfering color. Although this type error may seem minimal, the presence of a low-molecular-weight aldehyde can result in significant quantitative errors due to the much greater number of reactive reducing groups per weight unit.
CONCLUSIONS

These data suggest, then, that the DNS assay can be used as an accurate analytical method for the evaluation of reducing sugars both in pure solution as well as in supernatants from enzymatic saccharifications of purified cellulosics if glucose is the sole product. However, only specific assay methods, such as HPLC and YSI-type glucose analyzers, should be used for the analysis of saccharides produced from the hydrolysis of native or pretreated lignocellulosics since the DNS assay is susceptible to interferences and therefore results in inaccurate analyses. In fact, HPLC and appropriate glucose analyzers are the analytical methods of choice for all saccharifications regardless of the nature of the cellulosic substrate.

References
1. P. J. Blotkamp, M. Takagi, M. S. Pemberton, and G. H. Emert, AZChE Symp. Ser., 188, 74, 85 (1978). 2. S. Dhawan and J. K . Gupta, J. Gen. Appl. Microbiol., 23, 155 (1977). 3. B. F. Gallo, R. Andteotti, C. Roche, D. Ryu, and M. Mandels, Biotechnol. Bioeng. Symp., 8, 89 (1978).

T b e II. Comparative analyses of supernatants from saccharifications of pretreated lignocellulosics. al

Conversion of cellulose to glucose DNS Theoretical conversion
(70)

HPLC Theoretical conversion

(%)

YSI Theoretical conversion

(70)

Substrate Agitation bead-milled MSW P-irradiated MSW Agitation bead-milled PMW Thermomechanical pulped MSW Vibratory rod-milled MSW ND refers to not done.

Sugars (g/L) 35.0 23.4 35.1 19.6 20.6

Sugars (g/L) 23.5 14.4 14.5 13.6 11.1

Sugars (g/L)

103.9 69.5 104.4 58.2 61.2

69.8 42.7 43.2 40.5 33.0

ND ND 15.1 13.7 12.6 44.8 40.8 37.3

COMMUNICATIONS TO THE EDITOR

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4. M. N. Mangat and J. A. Howell, AIChE Symp. Ser., 174, 74, 77 (1978). 5. S. B. Shin, Y. Kitagawa, K. Suga, and K . Ichikawa, J. Ferment. Technol., 56,496 (1978). 6. G . L. Miller, Anal. Chem. ,31,425 (1959). 7. G . H. Emert, E. K . Gum, Jr., J. A. Lang, T. H. Kiu, and R. D. Brown, Jr., Adv. Chem. Ser., 136, 79 (1974).

8. S. J. Gracheck, K. E. Giddings, L. C. Woodford, and G . H. Emert, Roc. Agric. Energy, 2,305 (1980). 9. G. H. Horton, D. B. Rivers, and G. H. Emert, Ind. Eng. Chem. Prod. Res. Dev. , 19,422 (1980). 10. Enzymatic Hydrolysis of Cellulose, U.S. Patent No. 3,990, 945 (November 9, 1976). 11. J. B. Sumner and G . F. Somers, Laboratory Experiments in Biological Chemistry (Academic, New York, 1948).

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