Bioresource Technology 102 (2011) 2897–2903

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Calculating sugar yields in high solids hydrolysis of biomass

Yongming Zhu a,⇑, Marco Malten b,1, Mads Torry-Smith a, James D. McMillan c, Jonathan J. Stickel c
a
Novozymes North America, Franklinton, NC 27587, United States
b
Novozymes China, Beijing 100085, China
c
National Renewable Energy Laboratory (NREL), Golden, CO 80401, United States

a r t i c l e i n f o a b s t r a c t

Article history: Calculation of true sugar yields in high solids enzymatic hydrolysis of biomass is challenging due to the
Received 7 July 2010 varying liquid density and liquid volume resulting from solid solubilization. Ignoring these changes in
Received in revised form 25 October 2010 yield calculations can lead to significant errors. In this paper, a mathematical method was developed
Accepted 26 October 2010
for the estimation of liquid volume change and thereafter the sugar yield. The information needed in
Available online 3 November 2010
the calculations include the compositions of the substrate, initial solids loading, initial liquid density,
and sugar concentrations before and after hydrolysis. All of these variables are measurable with conven-
Keywords:
tional laboratory procedures. This method was validated experimentally for enzymatic hydrolysis of
High solids
Cellulose enzymatic hydrolysis
dilute sulfuric acid pretreated corn stover at solid loadings up to 23% (w/w). The maximum relative error
Sugar yield of predicted glucose yield from the true value was less than 4%. Compared to other methods reported in
Biomass the literature, this method is relatively easy to use and provides good accuracy.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction sugars and should be incorporated in the yield calculation. In this

As hydrolytic enzymes are continuously improved to perform
well at high sugar concentrations, operating enzymatic hydrolysis
reactors at high levels of lignocellulosic solids becomes a natural
choice for reducing capital and energy costs (Hodge et al., 2009;
Kristensen et al., 2009a). In order to design and develop an efficient
high solids hydrolysis process, it is necessary to accurately quantify
the sugar yields, i.e., the production of soluble sugars from the
insoluble polysaccharides. A commonly used laboratory practice
is to take slurry samples and measure the sugar concentrations
in the liquid phase of the hydrolyzate with high performance liquid
chromatography (HPLC). The sugar yields are then calculated based
on the sugar concentrations using the following formula (taking
glucose yield as an example):
C g  C g0
Yg ¼
uG C is0 xG0
where all variables are defined in the Nomenclature. The denomina-
tor in Eq. (1) stands for the potential glucose input to the reactor
and the numerator is the production of glucose from enzymatic
hydrolysis.
If the substrate contains soluble sugar oligomers, these compo-
nents can also be hydrolyzed by enzymes to form fermentable
⇑ Corresponding author. Tel.: +1 919 4943402; fax: +1 919 4943422.
E-mail address: yggz@novozymes.com (Y. Zhu).
1
Present address: Novozymes A/S, 2880 Bagsværd, Denmark.
case, the yield (of glucose) would be expressed as
C g  C g0
Yg ¼ ð2Þ
uG C is0 xG0 þ ugos C gos0
In Eqs. (1) and (2), it is assumed that (i) the density of the
hydrolysis slurry is 1000 g/L, (ii) the volume of the liquid equals
to that of the hydrolysis slurry, and (iii) the volume of the liquid
remains unchanged during the hydrolysis. These equations could
give sufficient accuracy of yield prediction when the solid concen-
tration is very low, e.g., less than 2% (w/w). However, as the solid
concentration increases, the yield obtained from these equations
may deviate significantly from the actual value since none of these
three assumptions remains valid.
Several studies have emphasized the importance of using prop-
er methods for yield determination in high solids hydrolysis.
Hodge et al. (2009) modeled a fed-batch process for high solids cel-
ð1Þ
lulose hydrolysis where numerical computations were employed
to calculate glucose yield. The variations of liquid density and
insoluble solids weight (or liquid mass) were taken into account
and expressed as functions of glucose concentrations. Roche et al.
(2009) developed a method for calculating hydrolysis conversions
at high solids, in which the sugar concentrations in the liquid (g/L)
are converted to mass fractions of the hydrolysis slurry to facilitate
yield calculation. In another paper by Kristensen et al. (2009b), an
experimentally based method was proposed for yield determina-
tion. In this method, the slurry sample was diluted extensively
(e.g., 10 times) and the diluted sugar concentrations were
measured by HPLC. Due to the significant dilution, the volume

0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.10.134
2898 Y. Zhu et al. / Bioresource Technology 102 (2011) 2897–2903
Nomenclature
ai amount of water used to form soluble sugar i from its
polysaccharide (grams per gram of sugar i)
qh density of hydrolyzate liquid at 25 °C (g/L)
qh,c calculated density of hydrolyzate liquid at 25 °C (g/L)
qh0 initial density of liquid at 25 °C (g/L)
qh0,c calculated initial density of liquid at 25 °C (g/L)
qw density of water at 25 °C (g/L)
ug molecular weight ratio of glucose to glucan monomer;
uG = 180/162 = 1.11
ugos ratio of glucose molecular weight to average monomer
weight of glucose oligomers; ugos = 180/166.5 = 1.08
(assuming glucose oligomers have an average DP of 4)
Cg concentration of glucose (g/L)
Cg0 initial concentration of glucose (g/L)
Cgos0 initial concentration of glucose oligomers (g/L)
Ci concentration of sugar i (g/L)
Cis0 initial concentration of insoluble solids (g/L)
Ci0 initial concentration of sugar i (g/L)
Cos cumulative concentration of soluble components other
than sugars (g/L)
Cos0 initial cumulative concentration of soluble components
other than sugars (g/L)
displacement by the solids in the diluted sample could be ignored
and the liquid density was very close to that of pure water. The su-
gar mass in the samples was thus obtained by multiplying the su-
gar concentrations and the volume of the diluted sample. Since the
total weight of the hydrolysis assay was known, the theoretical
maximum mass of sugars that can be produced from hydrolysis
could then be calculated. This method could work to determine su-
gar yields, but requires taking good representative samples, which
becomes increasingly difficult in high solids hydrolysis especially
in the early stage of the reaction.
In this paper, we report on an alternative, improved method for
estimating sugar yields in high solids enzymatic hydrolysis of cellu-
lose. This method uses sugar concentrations determined by HPLC to
calculate the change of liquid volume after enzymatic hydrolysis. In
case the biomass substrate contains significant amounts of soluble
components other than HPLC-detectable sugars, the measured ini-
tial liquid density is incorporated to reduce the error. Experiments
using dilute acid pretreated corn stover as substrates (water washed
or unwashed) showed that this method could accurately predict the
yield values at relatively high solids concentrations.
2. Theoretical analysis
2.1. Correlation of hydrolyzate liquid density (qh) with sugar
concentrations
Plenty of data plotting the density of pure glucose and xylose
solutions against sugar concentrations was reported by Ji et al.
(2007). Correlation of the densities (in g/L) of pure glucose and
xylose solutions ranging from 0 to 240 g/L showed that the densities
increased linearly with the increase of sugar concentrations. Further
examination in our laboratory showed that a linear relationship also
exists for glucose and xylose mixtures. A general correlation of the
density and sugar concentrations can be described as:
X
qh ¼ qw þ 0:35 Ci
where qw = 1000 g/L (25 °C). Eq. (3) implies that the contribution of
sugar concentrations to the liquid density is additive. Fig. 1 shows
that Eq. (3) can provide good prediction of the densities of glucose
fis0 initial mass fraction of insoluble solids in slurry
fts0 initial mass fraction of total solids (soluble sol-
ids + insoluble solids) in slurry
Xis0 initial mass fraction of insoluble solids in total solids
xG0 initial mass fraction of glucan in insoluble solids
MW molecular weight (Da)
Vh volume of hydrolyzate liquid (L)
Vh0 initial volume of liquid (L)
Vw volume of water input to hydrolysis reactor (L)
V w volume of water before ‘‘pre-hydrolysis’’ (L)
Wh0 initial mass of liquid (g)
Wis0 initial mass of insoluble solids (g)
Wt total weight of hydrolysis assay (g)
Ww the weight of water that is input to hydrolysis reactor
(g)
Ww,h the weight of water in the hydrolyzate (g)
Ww,p the weight of water that is consumed to hydrolyze poly-
saccharides (g)
Yg glucose yield (of theoretical maximum)
and xylose mixtures over a wide range of concentrations. Since glu-
cose and xylose are typically the most abundant sugars found in bio-
mass hydrolyzate, Eq. (3) will be used in the following calculations.
2.2. Estimating the change of liquid volume after hydrolysis
The change of liquid volume after hydrolysis can be estimated
by tracking the amount of water in the process. Assume one col-
lects the liquid phase of hydrolyzate after hydrolysis of lignocellu-
losic biomass. The volume of the liquid at 25 °C is Vh (in liter) and
the density qh (in g/L). The hydrolyzate liquid sample is analyzed
by HPLC for sugars (usually cellobiose, glucose, xylose, galactose,
arabinose, mannose, and possibly fructose). The cumulative con-
centration of the other soluble components, which is not usually
measured, is denoted as Cos (in g/L). The other soluble components
may comprise soluble salts, soluble lignin, sugar oligomers, acetic
acid, and so forth.
ð3Þ
Fig. 1. Predicted density using Eq. (3) vs. experimentally measured density of
solutions comprising glucose and xylose. The numbers in the parenthesis represent
glucose (g/L) + xylose (g/L).
 Y. Zhu et al. / Bioresource Technology 102 (2011) 2897–2903 2899

Based on the above information, the weight of water in the
hydrolyzate liquid (Ww,h, in grams) can be calculated as follows:
 Vh
X 
W w;h ¼ qh V h  C os þ Ci V h
or
 and after enzymatic hydrolysis can be estimated using sugar
X 
W w;h ¼ qh  C os  Ci V h ð4Þ
During the hydrolysis, some water was consumed when the
polysaccharides were cleaved by enzymes to form shorter chains
and soluble sugars. For example, to produce 1 g of glucose from
glucan, 0.1 g of water was used (MW H2 O =MW glucose ¼ 18=180 ¼
0:1). If ai denotes the amount of water used to form soluble
sugar i, one will have acellobiose = 0.05, aglucose = agalactose = amannose =
0.1, and axylose = aarabinose = 0.12.
If the liquid contained no soluble sugars before hydrolysis, the
weight of water (g) that was consumed to hydrolyze polysaccha-
rides is
X In most enzymatic hydrolysis research on lignocellulosic bio-
W w;p ¼ ai C i V h ð5Þ
Based on the above analysis, the weight of water that was input
to the hydrolysis reactor is:
h X i contains no or little soluble oligomers before hydrolysis (as is the
W w ¼ W w;h þ W w;p ¼ ðqh  C os Þ  ð1  ai ÞC i V h ð6Þ
Because W w ¼ qw V w , Eq. (6) can be rewritten as
Vh qw
¼ P ð7Þ
V w ðqh  C os Þ  ð1  ai ÞC i
Eq. (7) applies to the situation where the liquid before enzy-
matic hydrolysis contains no soluble sugars. In the case that the li-
quid before hydrolysis contains soluble sugars [as is true when the
whole slurry from pretreatment is subject to enzymatic hydrolysis
(Schell et al., 1999; Kova et al., 2009)], the relationship of hydroly-
zate liquor volume to water volume is more complex. In this case,
let us assume that the sugars initially present in the hydrolyzate li-
quid have been produced through an additional, hypothetical,
‘‘pre-hydrolysis’’ step in which no soluble sugars were present at
the onset of pre-hydrolysis. If V w is used to denote the volume of
Substituting Eqs. (11) and (12) in Eq. (10) yields:
P
qh0;c  ð1  ai ÞC i0
¼ P ð13Þ
V h0 qh;c  ð1  ai ÞC i
Eq. (13) demonstrates that the ratio of liquid volumes before
concentrations measured by HPLC. In some cases, water is also
consumed during hydrolysis of acetyl groups from hemicellulose.
For example, in autohydrolysis pretreatment, a significant amount
of xylo-oligomers is present in the liquid phase. When the whole
slurry is subject to enzymatic hydrolysis, some of the acetyl groups
in the xylo-oligomers can be hydrolyzed. However, the liquid vol-
ume reduction due to the water consumption by the acetyl groups
is less than 1% of the total liquid volume (data not shown), and is
thus ignored in the calculation.
2.3. Calculation of sugar yield
mass, sugar yields are simply reported as the amount of ferment-
able sugar produced from insoluble polysaccharide, as described
by Eq. (1). This approach is reasonably accurate when the substrate
case using water washed substrates). However, under certain cir-
cumstances, considerable sugar oligomers may be found in the
substrate (for example, when unwashed steam exploded corn
stover is used for hydrolysis), and the oligomers can also be hydro-
lyzed by the enzymes to produce fermentable sugars. As such, the
initial sugar oligomers should be counted as a source of ferment-
able sugar production. Take the calculation of glucose yield for
example, the true value of which can be represented as
C g V h  C g0 V h0
Yg ¼
uG W t fts0 X is0 xG0 þ ugos C gos0 V h0
or
 
C g VV h  C g0
h0
Yg ¼   ð14Þ
uG VW t fts0 X is0 xG0 þ ugos C gos0
h0

water input (liters) before this hypothetical ‘‘pre-hydrolysis’’ step In Eq. (14), the term Wt
can be evolved as below:
V h0
and Vh0 is used to denote the volume of hydrolyzate following
pre-hydrolysis, from Eq. (7) it follows that Wt Wt
¼ 
V h0 W h0
V h0 qw qh0
¼ P ð8Þ  
V w ðqh0  C os0 Þ  ð1  ai ÞC i0 Wt
¼ qh0
W h0
and  
Wt
Vh qw ¼ qh0
¼ P ð9Þ W t  W is0
V w ðqh  C os Þ  ð1  ai ÞC i !
1
¼ qh0
Dividing Eq. (9) by Eq. (8) gives 1  WWis0t
P
Vh ðq  C os0 Þ  ð1  ai ÞC i0
¼ h0 P ð10Þ or
V h0 ðqh  C os Þ  ð1  ai ÞC i
Wt qh0
It is noted that in Eq. (10), qh0 and qh are actual densities of the ¼ ð15Þ
V h0 1  fts0 X is0
liquid before and after hydrolysis. As mentioned earlier, since Cos is
usually not measured, the following assumptions are used to sim- Insertion of Eq. (15) to Eq. (14) gives:
plify the calculation:  
C g VV h  C g0
qh0  C os0  qh0;c ð11Þ Yg ¼   h0
qh0
uG 1fts0 Xis0 fts0 X is0 xG0 þ ugos C gos0
qh  C os  qh;c ð12Þ
or
where the subscript c means that the value of density was calcu- h   i 
Vh 1
lated using Eq. (3). Eqs. (11) and (12) apply when ‘‘the other soluble Cg V h0
 C g0 fts0 X is0
1
Yg ¼   ð16Þ
components’’ do not significantly contribute to the liquid density 1
uG qh0 xG0 þ ugos C gos0 fts0 X is0
1
and therefore can be neglected.
2900 Y. Zhu et al. / Bioresource Technology 102 (2011) 2897–2903
Table 1
Composition of corn stover pretreated by dilute sulfuric acid.a
fts0b Xis0c Insoluble solids (of dry weight)d
Glucan Xylan Galactan
Batch 1 0.328 ± 0.005 0.563 ± 0.001 52.9 ± 0.8% 2.4 ± 0.2% 0.0 ± 0.0% 0.2 ± 0.1% 31.8 ± 0.5% 8.0 ± 1.6% 4.7%
Batch 2 0.305 ± 0.003 0.606 ± 0.002 55.7 ± 0.4% 3.7 ± 0.2% 0.9 ± 0.1% 0.7 ± 0.0% 27.2 ± 0.3% 6.5 ± 0.6% 5.2%
a
Value are expressed as averages and standard deviations (n = 2 for fts0 and Xis0, n = 3 for compositional analysis). Pretreatment conditions: 1.1% (w/w) H2SO4, 190 °C, 1 min.
b
Initial mass fraction of total solids in slurry.
c
Initial mass fraction of insoluble solids in total solids.
d
The composition of insoluble solids are based on oven-dry weight.
e
The values in the parentheses are concentrations of total sugars (monomer + dimer + oligomers) and are expressed as of equivalent monomers.
If the sugar of interest is other than glucose, one needs to
replace the concentrations and mass fractions in Eq. (16) with
those of the corresponding sugar and adjust the u coefficients
accordingly.
3. Methods
3.1. Hydrolysis substrates
The substrates used in this work were dilute sulfuric acid pre-
treated corn stover generated from the NREL’s pilot demonstration
unit (Schell et al., 2003). The conditions for pretreatment were:
1.1% (w/w) sulfuric acid, 190 °C, 1 min. Two different batches were
used. The compositions of the pretreated corn stover are shown in
Table 1. It is noted that dilute acid pretreated corn stover slurry
contains substantial amount of sugars in the liquid (Table 1). Be-
fore loading to hydrolysis reactors, Batch 1 material was washed
extensively with de-ionized water on a Whatman GF/D microfiber
membrane until no detectable sugars were found in the washout
(HPLC detection limit 0.025 g/L for glucose), while Batch 2 material
was used as received.
3.2. Enzymatic hydrolysis
The enzymatic hydrolysis was carried out in 50 ml Oak Ridge
polypropylene copolymer centrifuge tubes (29  107 mm) with
caps and silicone sealing rings (LabSource Inc., Romeoville, IL).
The working weight of each assay was 20.0 g. All the hydrolysis
tubes were incubated in a rotisserie incubator (Model Combi-
D24, Finemould Precision Industrial Company, Seoul, Korea) set
at 50 °C and 12 rpm. All the assays were run in triplicate, and the
average values and standard deviations are reported.
To load the hydrolysis tubes, pre-determined amounts of sub-
strates, de-ionized water, 10% (w/w) NaOH (for neutralization of
the acid in the unwashed substrate), sodium citrate buffer
(0.05 M in hydrolysis) and penicillin (antibacterial, 2.5 ppm in
hydrolysis) were added. The content was mixed with a stainless
steel spatula and settled in a 5 °C refrigerator overnight. Then cel-
lulase enzyme was added to the tubes to start the hydrolysis and
the tubes were placed in the incubator. Since the high solid slurry
was very viscous, a 1 cm-diameter stainless steel ball was added to
each tube to improve the mixing during hydrolysis. The pH of the
assays was 5 before incubation. The enzymes used in the study
were Novozymes CellicÒ CTec cellulase produced from Trichoderma
reesei. The hydrolysis proceeded 2 or 4 days. Additional ‘‘dummy’’
tubes were constituted with the same contents as the hydrolysis
tubes and were sacrificed at time zero to get the initial data points.
3.3. Analysis
The fts0, fis0, and Xis0 of the dilute acid pretreated corn stover
slurry were determined following the NREL Standard Analytical
Pretreatment liquid (g/L)e
Arabinan Lignin Ash Others Cellobiose Glucose Xylose Arabinose
2.7 ± 0.1 15.8 ± 0.1 54.5 ± 0.5 10.7 ± 0.1
(18.5 ± 0.2) (62.5 ± 0.9) (10.1 ± 0.4)
4.9 ± 0.5 14.1 ± 0.9 55.3 ± 1.8 10.2 ± 0.1
(19.4 ± 0.5) (71.2 ± 1.8) (10.7 ± 0.2)
Protocols (Sluiter et al., 2008a,b). The fts0 were measured using
an IR-200 moisture analyzer (Denver Instrument Company, Den-
ver, CO). The sample was heated at 105 °C until less than 0.05%
of the initial weight was lost within 1 min. The fts0 was calculated
by dividing the final dry weight by the initial weight of the sample.
For fis0 measurement, a weighed slurry sample was washed on a
Whatman GF/D glass microfiber membrane until pH 5. The washed
solid cake was then dried in a 105 °C oven. The fis0 level was deter-
mined by dividing the dry weight of the washed solid cake by the
weight of the original slurry sample. The Xis0 was then calculated
from the fts0 and fis0, that is, X is0 ¼ ffts0
is0
. All fts0 and fis0 measurements
were made in duplicates (n = 2). The densities of the liquid before
enzymatic hydrolysis and the hydrolyzate liquid were measured
by taking 200 lL liquid samples with a HandyStepÒ repeating pip-
ette (BrandTech Scientific, Essex, CT) and weighing the samples on
an analytical balance (accuracy 0.1 mg). The density of the liquid
was obtained from the ratio of the sample weight to sample
volume.
To prepare HPLC samples, 1 g of slurry was extracted from each
hydrolysis tube with a cut-off pipette tip after the tube was vor-
texed. The weight of the remaining content in each sampled assay
was weighed for mass balance closure. The samples were acidified
to pH < 2 with 10 lL of 40% (w/w) H2SO4 (1% (v/w) of slurry sam-
ple) to stop the hydrolysis reaction. The hydrolyzate was filtered
through 0.45 lm syringe membranes and diluted with 5 mM
H2SO4 to prepare for HPLC analysis. An Agilent HPLC system
equipped with Aminex HPX-87H column (Bio-Rad Laboratories,
Hercules, CA) running at 0.6 mL/min of 5 mM H2SO4 (pH1.8)
was used for sugar quantification by integration of signal from a
refractive index detector. Xylose, mannose and galactose had the
same retention time on the column and co-eluted (Um et al.,
2003). As minor sugars in corn stover substrate, mannose and gal-
actose were combined with and counted as xylose in the yield cal-
culation. Carbohydrate analysis of the substrates was performed
following the NREL Standard Analytical Protocols (Sluiter et al.,
2008c). An Aminex HPX-87P (Pb2+ form cation) column running
at 0.6 mL/min of Milli-Q water was used for sugar quantification
on an Agilent HPLC system.
3.4. Quantification of actual sugar yields
After sampling, the remaining slurry in each assay was acidified
with 0.19 ml (1% (v/w) of the remaining slurry) of 40% (w/w) H2SO4
to stop the hydrolysis reaction. The acidified assays were then
washed five times each using either 60 mL (for washed substrates)
or 40 mL (for unwashed substrates) of de-ionized water through a
Whatman GF/D glass microfiber membrane. The reason for using
different water volumes was that the washed substrates contained
more insoluble solids than the unwashed substrates and thus re-
quired more water to wash out the sugars. In addition, the volumes
of the water were pre-determined before washing so that the con-
centrations of glucose in the washout fell in the HPLC linear range
 Y. Zhu et al. / Bioresource Technology 102 (2011) 2897–2903 2901

Table 2
Comparison of glucose yields (of theoretical maximum) obtained using different calculation methods.a
b
Substrate fts0 Xis0c Enzyme Hydrolysis Actual yield Yield Relative Yield using Relative Yield using Relative Yield using. Relative
dosage time using error (%) Eqs. (3), error (%) Eqs. (3), (13), error (%) Eq. (16), error (%)
(g/g-glucan) (hours) Eq. (1) (13), and (16) and measured
or (2) (16) measured qh0 but
qh0 ignoring
volume
change
Diluted acid 0.15 1.00 0.04 96 62.7 ± 0.3% 73.5 ± 0.7% 17.2 64.8 ± 0.7% 3.3 – – 62.2 ± 0.6% 0.9
pretreated 0.15 1.00 0.12 96 83.1 ± 0.6% 96.1 ± 0.8% 15.6 85.5 ± 0.8% 2.9 – – 81.3 ± 0.6% 2.2
corn stover 0.23 1.00 0.04 96 44.0 ± 1.2% 56.8 ± 1.8% 29.1 45.7 ± 0.7% 3.9 – – 43.6 ± 0.6% 1.1
(washed) 0.23 1.00 0.12 96 72.7 ± 0.1% 90.3 ± 0.6% 24.1 74.6 ± 0.5% 2.5 – – 69.2 ± 0.4% 4.9
Dilute acid 0.15 0.606 0.06 48 59.3 ± 0.3% 65.1 ± 0.4% 9.8 60.2 ± 0.4% 1.6 57.9 ± 0.3% 2.3 56.4 ± 0.3% 4.8
pretreated 0.15 0.606 0.18 48 82.0 ± 0.5% 89.9 ± 0.2% 9.5 83.7 ± 0.2% 2.1 80.4 ± 0.2% 1.8 77.8 ± 0.2% 5.2
corn stover 0.23 0.606 0.06 48 44.4 ± 0.2% 52.5 ± 0.1% 18.2 45.9 ± 0.4% 3.4 43.3 ± 0.1% 2.4 42.0 ± 0.1% 5.4
(unwashed) 0.23 0.606 0.18 48 64.8 ± 0.1% 76.3 ± 0.1% 17.5 67.7 ± 0.2% 4.4 64.1 ± 0.2% 1.0 61.2 ± 0.1% 5.6
a
Hydrolysis was done in triplicates (n = 3). Yield values are expressed as averages and standard deviations;
b
Initial mass fraction of total solids in slurry. fts0 in this table was reported as the ratio of dry weight of pretreated corn stover and total working weight of the assay. The
other soluble components introduced in assay preparation (sodium citrate, penicillin and NaOH) were not included.
c
Initial mass fraction of insoluble solids in total solids. Xis0 of the assays using unwashed corn stover was taken as the same as the pretreated slurry.

(0.5–15 g/L). The use of the excessive amount of water for washing
ensured a nearly complete removal of the soluble components
from the remaining insoluble solids, as no sugars were detected
by the HPLC in the last droplets of the filtrates. After washing,
the volumes (mL) of the collected washout were measured and
the sugar concentrations (in g/L) were analyzed by HPLC equipped
with Aminex HPX-87H column (Bio-Rad Laboratories, Hercules,
CA) running at 0.6 mL/min of 5 mM H2SO4 (pH1.8). The soluble
sugars recovered from each hydrolysis assay were then quantified
taking into account of the losses of mass by sampling before wash-
ing. Due to the small sample size (1 g of slurry sample vs. 20 g of
total) and vortex homogenization before sampling, the change in
insoluble solids concentration of the hydrolysis slurry, if any, was
negligible after sample withdrawal.2
4. Results and discussion
Table 2 compares the glucose yields calculated using Eqs. (1)
and (2) with those using the methods developed in this study.
Two different levels of total solids (fts0 = 0.15 and 0.23) were em-
ployed in the hydrolysis of both washed and unwashed substrates.
It is noted that the fts0 of the washed substrate equals to the fis0 (or
Xis0 = 1) since all the soluble components were removed during
washing. However, the fts0 of unwashed substrate were higher than
the fis0. The fraction of insoluble solids of the total solids, Xis0, in un-
washed substrate (Batch 2) was 0.606. From Table 2, it can be seen
that ignoring the liquid density and volume changes in yield
initial liquid density (Eq. (16)). Table 3 summarizes the liquid den-
sities predicted using Eq. (3) and measured experimentally. It is
seen that when washed substrate was used for hydrolysis, the li-
quid densities after hydrolysis could be accurately predicted with
HPLC data, which suggested that only small quantity, if any, of
non-sugar substances was solubilized during hydrolysis. However,
the prediction of liquid density was much less accurate for un-
washed substrate apparently because of the presence of a variety
of non-sugar components in the liquid. The inaccuracy of density
estimation could largely explain the increasing errors in the yield
prediction of unwashed substrate hydrolysis when using Eqs. (3),
(13), and (16). In order to improve the accuracy of prediction, a
modified method is proposed here. In this modification, the liquid
volume ratio, VV h , was still calculated based on HPLC data using
h0
Eqs. (3) and (13), but in Eq. (16), the measured initial density of
liquid was assigned for qh0. This modification further reduced the
error of the yield prediction particularly at the higher solids con-
centration of unwashed substrate. As can be seen in Table 2, the
relative error of calculated yield of unwashed corn stover hydroly-
sis at fts0 of 0.23 declined from 3.4–4.4% to 1.0–2.4%. Measuring the
density of initial liquid is a simple operation in the laboratory. One
just needs to pipette a small volume of the liquid sample (the same
as for HPLC sample preparation) and weigh the sample on a bal-
ance. The density of the liquid can thus be calculated by dividing
the sample weight by the volume.
As can be seen in Eq. (16), liquid volume ratio before and after
hydrolysis (VV h ) plays an important role in the yield calculation. For
h0

calculation (using Eqs. (1) and (2)) led to significant overestima- the hydrolysis in our experiment, the liquid volumes were esti-
tions of the yields (relative error 15.6–29.1% for washed substrate mated to increase by 2–8% (Table 3). Ignoring the volume changes,
and 9.5–18.2% for unwashed substrate), the error generally that is, assuming VV h ¼ 1, would lead to considerable underestima-
h0

increasing with solids loading. However, the relative errors
dramatically dropped after using the equations from this study
(Eqs. (3), (13), and (16)), decreasing to only 2–4% and 1–5% for
washed and unwashed substrates, respectively. Kristensen et al.
(2009b) also reported an overestimation of 23% in the yield calcu-
lation when using an equation similar to Eq. (1) for enzymatic
hydrolysis of pretreated straw (washed).
Proper estimation of liquid density is important for the predic-
tion of sugar yields, as a direct relation exists for the yield and the
tions of the yields (Table 2). Roche et al. (2009) account for the
change in the mass of insoluble solids, and conversely the liquid
mass, during enzymatic hydrolysis of washed substrate. However,
there was an error in their equations that is important for the case
of unwashed substrates (see Appendix). After correcting for the
error, and with appropriate substitutions and rearrangements,
the same equation depicting the liquid volume change can be
successfully reached (neglecting soluble components other than
HPLC-detectable sugars).

2
For example, 1 g of slurry sample is taken from 20 g assay containing 23% 5. Conclusions
insoluble solids. Because of insufficient vortexing before sampling, this sample
contains 25% of insoluble solids, which is 8.7% higher than that of the assay. However,
this imperfect sample withdrawal results in only 0.11% of absolute drop, or 0.46% of An improved, mathematically-based method was developed for
relative decrease, in the insoluble solids concentration in the bulk slurry. calculating sugar yields in high solids hydrolysis of lignocellulosic
2902 Y. Zhu et al. / Bioresource Technology 102 (2011) 2897–2903

Table 3
Liquid density (g/L) and volume change in enzymatic hydrolysis of dilute acid pretreated corn stover.

Substrate fts0a Xis0b Enzyme dosage Hydrolysis Liquid density before hydrolysis Liquid density after hydrolysis Estimated liquid
(g/g-glucan) time (h) c c volume ratio before
Measured Calculated Relative Measured Calculated Relative
and after hydrolysis
with Eq. (3)c error (%) with Eq. (3)c error (%) Vh
V h0 using Eq. (13)

Dilute acid 0.15 1.00 0.04 96 995 ± 1 – – 1016 ± 1 1032 ± 1 1.5 1.042
pretreated 0.15 1.00 0.12 96 995 ± 0 – – 1026 ± 1 1032 ± 1 0.5 1.052
corn stover 0.23 1.00 0.04 96 998 ± 1 – – 1028 ± 2 1030 ± 1 0.2 1.050
(washed) 0.23 1.00 0.12 96 998 ± 0 – – 1044 ± 0 1030 ± 0 1.3 1.078
Dilute acid 0.15 0.606 0.06 48 1056 ± 1 1014 ± 0 4.0 1089 ± 2 1028 ± 0 5.6 1.023
pretreated 0.15 0.606 0.18 48 1056 ± 0 1015 ± 0 3.9 1084 ± 2 1034 ± 0 4.7 1.031
corn stover 0.23 0.606 0.06 48 1086 ± 1 1023 ± 0 5.8 1101 ± 1 1040 ± 1 5.6 1.027
(unwashed) 0.23 0.606 0.18 48 1082 ± 2 1024 ± 0 5.4 1111 ± 2 1048 ± 1 5.6 1.042
a
Initial mass fraction of total solids in slurry. fts0 in this table was reported as the ratio of dry weight of pretreated corn stover and total working weight of the assay. The
other soluble components introduced in assay preparation (sodium citrate, penicillin and NaOH) were not included.
b
Initial mass fraction of insoluble solids in total solids. Xis0 of the assays using unwashed corn stover was taken as the same as the pretreated slurry.
c
The values are expressed as averages and standard deviations (n = 3).

biomass. With this method, the sugar yields can be directly calcu- After making the correction noted above, the equation that re-
lated from the sugar concentrations that are usually determined by lates the fraction of insoluble solids to the soluble sugar concentra-
HPLC. This method was experimentally validated for relatively tions (Eq. (5) of Hodge et al. (2009) and Eq. (4) of Roche et al.
high solids hydrolysis of dilute acid pretreated corn stover (washed (2009)) can also be corrected to be:
and unwashed). At 23% of solids loading, the maximum relative er- P P
fis0 ð1  r i C i0 =qh0 Þ  r i ðC i =qh  C i0 =qh0 Þ
ror of predicted glucose yield from the true value was less than 4%. fis ¼ P ðA:3Þ
When used for other substrates, this method may need further val- 1  r i C i =qh
idations due to the variation of biomass materials. where r i is the molecular weight (MW) ratio of an polysaccharide
monomer to its soluble monomer i. Since ai is defined as amount
Acknowledgements of water used to form soluble sugar i from its polysaccharide (grams
per gram of sugar i), it is easy to see that r i ¼ 1  ai .
We thank Lindsay Jones for laboratory assistance and Prashant By putting Eq. (A.3) in terms of the mass fraction of hydrolyzate
Iyer and Hui Xu for their discussions in this work. liquor (fh = 1  fis) we arrive at the more compact expression
P
fh0 ð1  r i C i0 =qh0 Þ
fh ¼ P ðA:4Þ
Appendix 1  r i C i =qh
By making the further substitution of fh = qhVh/Wt and some
Rather than tracking the change in the volume of hydrolyzate
rearrangement, we can arrive at
liquid, Hodge et al. (2009) and Roche et al. (2009) accounted for P
the changes in the solid and liquid phases by deriving equations Vh q  ri C i0
¼ h0 P
in terms of the mass fraction of insoluble solids. However, their V h0 qh  r i C i
equations relating the change in mass fraction of a soluble species
or
to the change in its concentration in the liquid phase (Eqs. (3) and
P
(4) of Hodge et al. (2009) and Eq. (2) of Roche et al. (2009)) only Vh q  ð1  ai ÞC i0
apply to the case where no soluble sugars are present at the start ¼ h0 P ðA:5Þ
V h0 qh  ð1  ai ÞC i
of enzymatic hydrolysis (e.g., washed solids) and were mistakenly
represented to also apply to the case when soluble sugars are pres- which we recognize to be exactly Eq. (10) when the presence of sol-
ent (e.g., whole slurry). The incorrect equation was given as: uble components other than HPLC-detectable sugars are neglected.

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