J. Chem. Tech. Biolechnol.

1982, 32, 1016-1022

Evaluation of the DNS Method for Analysing

Lignocellulosic Hydrolysates
Warwick L. Marsden, Peter P. Gray, Greg J. Nippard and Mark R. Quinlan
School of Biotechnology, University of New South Wales, Kensington, N.S. W., Australia
(Paper received 23 April 1982 and accepted 19 October 1982)

The dinitrosalicylic acid (DNS) method gives a rapid and simple estimation of the
extent of saccharification by measuring the total amount of reducing sugars in the
hydrolysate. However, it is subject to interference by citrate buffer and other substances
and by the differing reactivities of the various reducing sugars. These interferences
become more apparent when complex substrates such as sugar cane bagasse are
employed. The paper also shows how the DNS method can be adapted for use on a
Technicon Autoanalyser.

1. Introduction
The 3,5-dinitrosalicylic acid (DNS) method for determining reducing sugar concentration was
developed by Sumner with various co-worker~l-~ and later modified by Miller.4This latter paper has
become the basis for the widely used method, although an alternative method using a simpler reagent
. ~ the proliferation of research into the conversion of lignocellulosic
has been given by B r ~ n e rWith
materials (i.e. containing cellulose, hemicellulose and lignin) into sugars (and subsequently to
ethanol) the DNS method has achieved great prominence due to its simplicity. However, the method
has several limitations which are often overlooked when interpreting results of analyses performed
on sugar syrups produced from these lignocellulosic materials.
The colour forming reaction in the assay appears to be the reduction of the 3,5-dinitrosalicylic
acid to 3-amino-5-nitrosalicylic acid by the aldehyde groups on the reducing sugars.4 However,
different reducing sugars give different absorbances, not only on a mass basis, but also on a molar
b a ~ i s ~suggesting
-~ that the chemistry is more complex. The chemistry is further complicated by
interference from dissolved oxygen which introduces non-linearity into the absorbance versus
concentration plot at low con~entrations.~. * The reaction is also non-specific with all soluble
reducing substances, regardless of size or nature, potentially able to react with the DNS reagent. In
reporting results from hydrolysis of lignocellulosic materials there has been a tendency to quote the
DNS determined reducing sugar concentration as representing the total of the monosaccharides
(and disaccharides) present. The results are usually based on glucose standards. However, sugar
syrups derived from lignocellulosic materials contain monosaccharides other than glucose, disac-
charides, a range of soluble oligomers as well as other substances released during hydrolysis which
can produce colour with the DNS reagent.0
This non-specificity of the DNS method, coupled with the differing reactivities (and hence
absorbances) of the major reducing sugars derived from lignocellulosic materials, means that the
DNS method can only be used to determine a ‘relative’ measure of the amount of reducing sugars
formed during hydrolysis.
This paper seeks to outline these limitations, thus providing for more meaningful interpretation of
DNS results.
0142-0356/82/1100-1016 $02.00 0 1982 Society of Chemical Industry
1016
DNA method for analysing lignocellulosic hydrolysates 1017

2. Experimental
2.1. Substrate
The main substrate used was alkali treated bagasse (referred to as bagasse) prepared by digesting
25 g air dried bagasse with 500 cm3 0.5% (w/v) NaOH for 1 h at 100°C. The bagasse was then
washed with tap water until free of alkali and dried at 37°C for 3 days. The treated bagasse contained
31 % hemicellulose, 47% cellulose and 9 % lignin, determined using the method of Goering and
Van Soest.20
Sigmacell Type 20, a pure cellulose powder was used in one experiment.

2.2. Enzymic saccharifications

Most saccharifications were carried out over 24 h using 7.5% (w/v) solids and 1 FPU ~ m of- ~
Trichoderma viride cellulase;ll either QM 9414 cellulase obtained as a preparation from Novo
Industri A/S or C30 cellulaselz prepared in this School. All experiments were performed in shake
flasks at 200 rev min-I, 50°C and pH 4.8 (maintained by using 0.05 M sodium citrate buffer). An
experiment was conducted using 0.05 M acetate and phosphate buffers.

2.3. Reducing sugar analyses

2.3.1. Total reducing sugars-manual DNS method
In the manual method 3 cm3 of modified DNS reagent4 was added to 1 cm3 of sample plus 1 cm3 of
distilled water in a test tube and placed in a boiling water bath for 15 min and then cooled for at
least 15 min. The absorbance was then measured at 575 nm. Several variations in the volume of the
sample taken and wavelengths used were examined. Standard curves were prepared for glucose,
xylose and cellobiose (Figure la), while total reducing sugar values for saccharification samples were
read from the glucose curve.

0
-
0
W

Figure 1. Absorbance
versus concentration plots
for DNS Method: (a)
Manual; (b) Automated.
G, glucose; x , xylose;
a. cellobiose.
Sugor concentration (mg ~ r n - ~ )

2.3.2. Total reducing sugars-utomated DNS method

The automated method used a Technicon Autoanalyser as shown in Figure 2, with absorbances
being read at 600 nm. The standards range was 1 to 6 mg ~ m glucose,
- ~ but by changing the dilution
tubes this could be varied. Such a range reduced the laborious dilutions needed for the manual
methods where the range was 0.1 to 1.0 mg c w 3 glucose.

2.3.3. Xylose, glucose and cellobiose

The individual concentrations of these sugars were determined using a Waters Associates Model 440
h.p.1.c. fitted with a refractive index detector. A Radial Pak B cartridge was used with a mobile
phase of 75 % acetonitrile plus 0.1 % SAM 2 (an amine additive) and a flow rate of 2 cm3 min-1.
1018 W. L. Marsden et al.

Resample (0.60) Waste

98°C
I Oil or water bath
incubation coil
WC pull through (12.5 cm3)
(1.00) GryIGry
Waste

Convection cooling

Spectrophotometer

Figure 2. Diagram of the autoanalyser DNS method.

Samples were prepared by using Amicon Centriflow Ultrafiltration cones and then desalting in a
BTL desalting apparatus. Sucrose was used as an internal standard with concentrations being
calculated from peak heights.
Apart from one unidentified peak in the C30 hydrolysates, the only major identifiable peaks were
xylose, glucose, cellobiose, sucrose and glycerol (used in the QM 9414 preparation).
Other minor peaks were arabinose and mannose with traces of cellotriose found in some chromato-
grams.

3. Results
3.1. Variations in the method
3.1.1. Volume of Sample
In Miller’s method4 3 cm3 of DNS reagent was added to 3 cm3 of sugar sample, which gave a linear
range of approximately 0.1 to 0.6 mg (3117-3 glucose (Figure 3). By adding the 3 cm3 DNS reagent
to 1 cm3 sugar sample plus 1 cm3 distilled water this range is extended to about 1.0 mg ~ m - ~
glucose but more importantly it eliminates the depression of absorbance by citrate buffer (Figure 3),
which was discussed in Miller’s paper.4

3.1.2. Wavelength
The linear range could be increased by increasing the wavelength at which absorbances were read,
although there was a loss in sensitivity (Figure 4). Saccharification samples were found to give the
same DNS result provided samples and standards were read at the same wavelength. The method of
Bruner5 actually recommends the preparation of two standard curves; one at 540 nm and a second
at 590 nm.
DNS method for analysing IignoceUulosic hydrolysates 1019

' ,

buffer o n glucose standard curve. 0,1 cm3 sample

in distilled water; 0,1 cm3 sample in 0.05 M citrate
0.5 c
buffer; 0 , 3 cm3 sample in distilled water; H, 3 cm*
sample in 0.05 M citrate buffer.
O* 4 0.8 1.2
Sugar concentration (mg ~ m - ~ )

Figure 4. Effect of wavelength on glucose stand-

ard curve. U, 550 nm; 0,575 nm; 7, 600 nm. O0' I
Sugor concentration ( m g ~ r n - ~ )

3.1.3. Manual versus automated methods

Table 1 gives the average of duplicate samples for 24 h saccharifications using different cellulases
and three different buffers commonly used in enzymic saccharifications. No significant differences
between manual and automated methodswere observedwith acetate and phosphate buffers while with
citrate buffer the automated method was found to give results 3-5 % higher than the manual method;

Table 1. Manual versus automated DNS methods. (mg glucose ~ m - ~ )

QM 9414 C 30

Buffer Manual Autoanalyser Manual Autoanalyser

Acetate 38.1 38.6 39.1 38.7

Citrate 37.2 38.8 31.9 39.2
Phosphate 32.5 32.7 36.6 36.8

Saccharification conditions: 1 FPU cm-3 enzyme, 7.5% solids, 0.05 M buffer, pH 4.8,
S O T , 24 h.
1020 W. L. Marsden et ul.

a finding confirmed by repeated experimentation. Addition of citrate buffer to Autoanalyser

standards caused no significant change to the glucose standard curve, nor did increasing the residence
time in the heating bath. The reason for this interference by citrate buffer appears to lie in the
complex chemistry of the DNS reactions and the different reactivities of reducing sugars,fi as the
relative slopes for the three sugars shown in Figure 1 for the manual method, changed with the
automated method (Figure l(b)). This difference in slopes was independent of the wavelength used.

3.2. Reducing sugar mixtures

Hydrolysis of lignocellulosic material produces not only glucose but also significant quantities of
other reducing sugars such as xylose and cellobiose. Figure 1 shows quite clearly the different
absorbances produced by the three main products of saccharification. Depending on the relative
amounts of xylose and cellobiose. the DNS value may be inflated or deflated when expressed as
mg c m 3 glucose.

Table 2. Full analysis of hydrolysates (mg cm-s)

H.p.1.c. Manual .
@
H
Glucose Xylose Cellobiose Total DNS DNS

Sigmacell7.5%solids, 1 F P U ~ r n - ~ Q M 9 4 1 4 16.1 - 6. I 22.2 22.3 1 .oo

BagasseIO%solids, 1 . 5 F P U ~ m - ~ Q M 9 4 1 4 23.5 16.8 5.5 45.8 51.7 0.89
Bagasse 10 % solids, 1.5 FPU cm 3 C 30 26.4 6.4 3.9 36.7 47.9 0.77

Saccharification conditions: 0.05 M citrate buffer, pH 4.8, 50T, 24 h.

The first two results in Table 2 give a comparison between saccharifications of Sigmacell (con-
taining only cellulose) and bagasse (a complex mixture of cellulose, hemicellulose and other com-
pounds). The Sigmacell is hydrolysed to give mainly glucose with some cellobiose. The cellobiose
present would have depressed the DNS value making it appear that glucose and cellobiose were
the only substances with reducing ends present, while the literature13 and h.p.1.c. results suggest
that there was at least some cellotriose present.
With the saccharification of bagasse the h.p.1.c. total is consistently less than the DNS value, a
difference noted by other worker^.^. 23 This deficit is in part made up by other pentoses, but these
constitute only about 3 % of the potential reducing sugars in the bagasse.l5Jfi The remainder would
include some oligomers of glucose and the pentoses with the DNS value being inflated by xylose
(whose concentration was more than three times that of cellobiose) and by the reaction of other
compounds in the hydrolysate with the DNS reagent.
The saccharification results using the C30 cellulase had a much lower xylose concentration and a
lower ratio of h.p.1.c. to DNS which is consistent with the lack of inflation of the DNS value by the
xylose. However, the unidentified h.p.1.c. peak referred to in the section 2.3.3 appeared to
be xylose-related and was possibly xylobiose although it has not been possible to confirm this. As
this peak was slightly smaller than the xylose peak, correction of the h.p.1.c. total to include this
compound would bring the h.p.1.c. to DNS ratio for the C30 saccharification closer to that of the
QM 9414 saccharification. However, more than 11 % of the DNS value is unaccounted for using the
h.p.1.c. analysis of the three major products when bagasse was the substrate.
A comparison of the extent of saccharification between the two bagasse saccharifications on the
basis of the DNS result would show the QM 9414 as having the better cellulase. However, the full
analysis (or even just the glucose result, which can be obtained by the relatively simple and cheap
oxidase or hexokinase assays) shows that the C30 cellulase produced more glucose, the only product
fermentable by conventional yeasts. The low cellobiose value for the C30 result also indicates that
the C30 cellulase contains a more active 8-glucosidase component.
DNS method for analysing lignocellulosic hydrolysates 1021

While the DNS method can give a good relative measure of the overall degree of saccharification
care should be exercised when comparing results using differing substrates and cellulases.

4. Discussion
While the DNS method has been shown to be quite versatile as regards wavelength, sample volume
and buffers used, the differing reactivities of the saccharification products and possible interference
from citrate buffer and other compounds formed during saccharification must be considered when
interpreting results.
To help alleviate the problems caused by complex sugar mixtures, the ratio of the major sugars
could be determined and standards made to match, or alternatively adhere to the widely used
glucose equivalents and include the ratio when presenting the results. If component sugars can be
determined these can be combined with the DNS value in interpreting results.
It should be clear that to attempt to measure a specific reducing sugar concentration by sub-
tracting other, separately determined, reducing sugar concentrations from the DNS value17 will
lead to considerable errors as the DNS value includes an indeterminate amount of oligomer reducing
ends, and perhaps other undefined substances. Discrepancies of over 25 % between h.p.1.c. and DNS
results have been reported.2R
The area of greatest concern is the role played by unknown substances, possibly soluble lignin
derivatives, in the DNS reaction. In the early days of enzymic saccharification of cellulose, pure
cellulosic substrates were used, but as the process approaches economic feasibility more complex
substrates are being employed. When such substrates are used the DNS value becomes higher than
can be accounted for by the sugars p r e s e ~ i t . ~ , ~However,
~ ~ ~ * . ' the
~ degree to which the DNS result
is affected depends largely on the substrate used.lg
Citrate buffer is widely used, not only in saccharifications, but also in assaying for various cellulase
activities10920 which employ the DNS reagent as well. As acetate buffer appears to give similar
results to the citrate buffer it would be better to standardise on it for both saccharification experi-
ments and cellulase activity assays as has been done by some groups.21
Some concern still remains with assays in which the DNS reagent is used. In a recent paper22 it
was shown that the filter paper activity was linearly dependent on the concentration of 8-glucosidase
over a wide range of activity; a relationship which was attributed to differing reactivities of glucose
and cellobiose with the DNS reagent, as well as the removal of cellobiose inhibition.

5. Conclusions
The DNS method has been, and continues to be, a very simple, rapid method for measuring re-
ducing sugar concentrations, but results must be interpreted in the light of the method's limitations.
I t gives, at best, only a relative measure of the reducing sugar concentration. When combined with
other more specific analyses the method can help provide even greater insight into the sacchari-
fication process.
This paper has also shown that the normal method can be automated easily to enable more
efficient analysis using the DNS method.

Acknowledgement
The work presented here was carried out with a grant from Sugar Research Institute, Mackay,
Queensland.

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1022 W. L. Marsden ct al.

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