Bioresource Technology 151 (2014) 244248

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

Lignin removal and benzenealcohol extraction effects on lignin

measurements of the hydrothermal pretreated bamboo substrate
X.J. Ma, S.L. Cao, X.F. Yang, L. Lin, L.H. Chen, L.L. Huang
College of Material Engineering, Fujian Agriculture and Forestry University, Fuzhou 350002, PR China

h i g h l i g h t s

 The extractives of the pretreated substrate increased with the reaction severity.
 Benzenealcohol extraction gave rise to a low lignin measurement.
 Tappi standard should be modied.
 Lignin removal was the results of lignin degradation and migration.

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

Article history: Lignin content of hydrothermal pretreated bamboo chips was determined by the two methods: TAPPI
Received 5 September 2013 standard method (222om-06) and TAPPI standard method without benzenealcohol extraction (BAE).
Received in revised form 21 October 2013 The results showed that including BAE resulted in lower Klason lignin (KL) and acid soluble lignin
Accepted 23 October 2013
(ASL) measurements in the prehydrolyzed substrate, that is to say, BAE removed parts of KL and ASL.
Available online 1 November 2013
Therefore, the TAPPI standard method should be modied by omitting the BAE for lignin measurements
of pretreated substrate. The following lignin removal analysis suggested that lignin was removed from
Keywords:
the bamboo substrate during pretreatment by a combination of degradation reaction and deconstruction;
Benzenealcohol extraction (BAE)
Lignin
thereafter the pseudo lignin generated in the hydrothermal pretreatment and condensation reaction
Pseudo lignin between the lignin fragments accounted for the later KL increase.
Hydrothermal pretreatment 2013 Elsevier Ltd. All rights reserved.

1. Introduction production. Lignin is a key factor that hinders enzymatic hydrolysis

To alleviate the energy crisis and climate change based on the
use of fossil energy, biofuel production from renewable resources
has received signicant attention (Ragauskas et al., 2006; Clark,
2007). For sustainable development, lignocellulosics (including
agricultural residues, forestry wood and energy crops) are regarded
as more appropriate feedstock for bioethanol production; lignocel-
lulosic ethanol is also called second-generation bioethanol (Blanca
and Juan, 2008). All lignocellulosic materials consist primarily of
cellulose, hemicelluloses, and lignin. In a heterogeneous enzymatic
reaction, the presence of hemicelluloses and lignin and complex
three-dimensional structure (called as recalcitrance) make the cel-
lulose inaccessible to hydrolytic enzymes (Himmel et al., 2007).
Pretreatment can remove hemicelluloses/lignin and disrupt the
structure of the lignocellulosic material to increase cellulose acces-
sibility to enzymes, and thus improve the hydrolysis and fermenta-
tion efciency. Employing pretreatment before enzymatic
hydrolysis is essential in a multi-step process for cellulosic ethanol
Corresponding author. Tel.: +86 15959041385; fax: +86 0591 83715175.
E-mail address: fafuhll@163.com (L.L. Huang).
through physical inhibition and adsorption. Lignin is subjected to
chemical reaction and physical changes during pretreatment. One
of the expected lignin chemical reaction pathways in dilute or
hydrothermal pretreatment is the cleavage of b-O-4 linkage (Cao
et al., 2012); the cleavage gives rise to a high content of lignin with
low molecular weight during hydrothermal pretreatment
(Leschinsky et al., 2008a). In addition, polymerization reactions
of lignin with itself or with carbohydrate degradation products also
occur depending upon the reaction conditions (Leschinsky et al.,
2008; Bujanovic et al., 2012). Moreover, as lignin melts, it migrates
through the cell wall and forms droplets or coalesces on the
biomass surface; this migration and redistribution of lignin has
been viewed as an obstacle for enzyme hydrolysis (Bujanovic
et al., 2012). Additionally, lignin degradation products in the
hydrolysate impede the dissolution of hemicelluloses because of
their tendency to form deposits (Leschinsky et al., 2008b). Further,
several studies have demonstrated that the Klason lignin (KL)
content of dilute acid pretreated biomass was higher than that of
the starting material (Mao et al., 2010; Chen et al., 2010; Jung
et al., 2010; Sannigrahi et al., 2008). It has been suggested that
aromatic materials formed by the combination of carbohydrate

0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2013.10.078
 X.J. Ma et al. / Bioresource Technology 151 (2014) 244248
and lignin degradation products during acid pretreatment could be
responsible for the increase of KL content (Hu et al., 2012). Katah-
ira, Sannnigrahi and Hu veried that dilute acid hydrolysis of holo-
cellulose or cellulose could give rise to an increase of KL
measurements of the hydrolyzed substrate and the KL content in-
creased with increasing reaction severity (Sannigrahi et al., 2008;
Hu et al., 2012; Katahira et al., 2013). This gain of KL is termed
pseudo-lignin and could account for the surprising increase of KL
measurements and negatively affect enzymatic hydrolysis. As lig-
nin is an obstacle to the enzymatic saccharication of biomass,
accurate determination of lignin content is important to the quan-
titative analysis of lignin effects.
Generally, the TAPPI standard method is used to determine lig-
nin content of biomass. Briey, benzenealcohol extraction is the
rst step in order to decrease the interference of extractives. There-
after, extractives-free biomass is depolymerized by hydrolyzing
holocellulose with 72% sulfuric acid and followed by hydrolysis
of the dissolved polysaccharide in 3% boiling sulfuric acid. The
residual solids are considered as acid insoluble or Klason lignin
(KL) while the acid soluble lignin (ASL) in the ltrate is determined
by UVvisible spectroscopy at 205 nm.
Because of the complex chemical reactions and physical
changes that lignin undergoes during pretreatment, it is doubtful
that the TAPPI standard (222om-06) is applicable for lignin deter-
mination of pretreated substrate. Thus, a systematic hydrothermal
pretreatment of bamboo chips was performed. The TAPPI standard
(222om-06) and TAPPI standard without benzenealcohol extrac-
tion (BAE) were used to determine the lignin content (KL and
ASL) and the differences between the two methods were explored
by quantitative analysis of the lignin measurements. In addition,
lignin removal was thoroughly analyzed by combination of sub-
strate, hydrolysate liquor and precipitate.
2. Methods
2.1. Materials
Bamboo chips (Dendrocalamopsis oldhami) were provided by a
forest center (Fujian province, China, 2011). The bamboo chips
with size of (40 2)  (15 1)  5 mm were selected as the start-
ing materials.
2.2. Pretreatment experiments
18 experiments were carried out in an oil-bathed digester
which equipped with 10 cooking pots. The reactions were carried
out at 160 to 180 C with the liquor/solid ratio 3:1 (180 g bamboo
chips) for times up to 180 min. The cooking pots were put into the
oil-bathed digester starting the reaction when the oil temperature
reached the target temperature; the cooking pots started to rotate
while the rotate button was switched on. The schematic diagram of
the digester was shown in Fig. 1. At the end of the reaction, the
cooking pots were taken out and cooled in cold water. The solids
and hydrolysate were separated by ltration with 80 mesh bags.
The lignin content of the solid substrate was analyzed while the l-
trate was subjected to TEM analysis.
2.3. Holocellulose separation and hydrolysis
Holocellulose was obtained from bamboo powder following the
procedure described by Cao et al. (2012). The extractives-free pow-
der was subjected to oxidation reactions for removing lignin. Holo-
cellulose pretreatment was conducted in the oil-bathed 25 mL
hydrothermal reactor at 170 C for up to 240 min.
245
Controller
Thermometer for calibration
Oil bath digester sealing cover
Thermocouple
Heat conducting oil
Cooking-pot jacket
Cooking pot
Rotation axis
Heat coil
Fig. 1. Schematic diagram of prehydrolysis apparatus.
2.4. Analytical methods
The yields of pretreatment were determined by the weight and
moisture content of the starting bamboo chips and treated bamboo
chips.
The extractives of benzene95% alcohol (2:1) extraction were
determined by TAPPI standard method T264 cm-97.
KL content was determined by TAPPI standard 222om-06 and
the TAPPI standard method without BAE. In order to compare the
changes of KL removal, KL values were based on the original mate-
rial, i.e., the KL measurements were calculated by multiplying KL
measurements of the pretreated substrate with the pretreatment
yield. ASL in the ltrate was determined by UVvisible spectros-
copy at 205 nm. Multiple measurements (34 runs) were con-
ducted for all characterization techniques, and a mean value was
reported. The error margin of the KL and ASL measurements was
0.1% to 0.4% and 0.03% to 0.07%, respectively.
The pretreatment hydrolysate liquor was dripped onto a copper
mesh and vacuum dried for about 24 h. Images of the colloid par-
ticles in hydrolysate liquor were taken with a Transmission Elec-
tron MicroscopeTEM (JEM1010, Japan).
Bamboo and pretreated bamboo powder was characterized by
solid state cross-polarization magic angle spinning (CPMAS) 13C
NMR (Advance III500, Bruker, Switzerland) operating at a fre-
quency of 125.75 MHz.
Field Emission Scanning Electron Microscope (FE-SEM, FEI Nova
NANOSEM 230, operated at an accelerating voltage of 15 kV) was
used to characterize the morphology of the holocellulose and pre-
treated holocellulose samples.
3. Results and discussion
3.1. BAE effects on lignin measurements
From Fig. 2, the KL and ASL determined by the TAPPI standard
method showed that the KL decreased gradually at the initial stage
and then showed an increase in the later stage (P170 C). In con-
trast, ASL decreased rst and then plateaued at extended pretreat-
ment times. Furthermore, the KL and ASL content determined by
TAPPI standard method was 1035% and 1575% lower than that
determined without BAE. Increasing reaction severity increased
246 X.J. Ma et al. / Bioresource Technology 151 (2014) 244248

o
2.4
Temperature, C
30 BAE M, 160 2.1
BAE M, 170
27 BAE M, 180 1.8

24 1.5
KL (%)

ASL (%)
1.2
21
0.9
18
0.6
15 0.3

12
(a) 0.0
(b)
0 30 60 90 120 150 180 0 30 60 90 120 150 180
Pretreating time (min) Pretreating time (min)

Fig. 2. The effects of BAE on measured lignin content (BAE: lignin quantication by TAPPI method T 222om-06 with BAE; M: lignin quantication by modied TAPPI method
(no BAE); (a) KL; (b) ASL).

the difference between the two methods; the difference in mea-

surements of KL and ASL were as much as 14% and 2%, respectively.
Lignin is subjected to acidic homolytic cleavage during hydrother-
mal pretreatment resulting in a substantial decrease in the molec-
ular weight (MW) of the lignin in the residual substrate
(Leschinsky et al., 2008a). Lignin with a low molecular weight
(LMW) could be extracted by BA from the pretreated substrate
thus causing the discrepancy between the two analytical methods.
The increase of KL may be tied to carbohydrate and lignin deg-
radation products, or pseudo-lignin. The results from holocellulose
hydrolysis are presented in Table 1. It was easily observed that KL
increased with pretreatment time and as with bamboo, BAE re-
sulted in a lower KL measurement. From these results, BAE could
extract 5060% pseudo lignin from pretreated holocellulose but
only 1035% lignin from pretreated bamboo substrate. The MW
of lignin or pseudo lignin might account for this difference. The
droplets (pseudo-lignin) appeared on the surface of the pretreated
holocellulose substrate and the size of these droplets increased
with pretreatment time (Suppl. A). After the BAE procedure, the
majority of these droplets disappeared but a few still remained
on the most severely pretreated holocellulose (170 C, 240 min).
From Hus study, weight-average MW of pseudo-lignin extracted
from dilute acid pretreated poplar cellulose and holocellulose ran-
ged from 3440 to 5970 g/mol, depending on reaction time and the
original material (180 C, 4060 min, 0.10.2 M H2SO4); the MW of
pseudo lignin increased with the reaction severity (Hu et al., 2012).
Given Hus results, it may be that BAE can only remove low MW
pseudo-lignin.
3.2. Extractives and its probably origin
Generally, woody plant extractives are diverse substances, such
as resin acid, fatty acid, turpenoid compounds and alcohols; most
of these substances are soluble in water or neutral organic solvent
(Sheshmani, 2013). In bamboo (D. oldhami) material, there were
only 3.6 0.2% extractives. Fig. 3 showed that the extractives
Table 1
KL measurements of the pretreated holocellulose substrate.
Time (min) Yield (%) TAPPI standard
Extractives KL
0 100 Trace 1.1 0.2
30 79.5 0.2 1.0 0.2 0.6 0.3
90 73.3 0.2 1.3 0.3 0.8 0.3
150 71.7 0.2 2.2 0.3 1.2 0.3
240 72.3 0.2 3.6 0.4 2.8 0.3
21
18
15
Extractives (%)
12
9 o
Temperature, C
160
6
170
180
3
0 30 60 90 120 150 180
Pretreating time (min)
Fig. 3. Extractives content of pretreated bamboo as a function of pretreatment time
and temperature.
content of the pretreated substrate ranged from 5% to 20%, depend-
ing on reaction temperature and time. At 160 C the extractives
content increased linearly with reaction time, while at higher tem-
peratures (P170 C) the content rst increased linearly before
dropping gradually. When considered in tandem with the differ-
ences in measure lignin content described above, it is reasonable
to conclude that a fraction of these extractives are KL and ASL. High-
er temperature and longer duration favor lignin migration and deg-
radation (Ma et al., 2013), which could enhance the BAE effect.
However, during longer pretreatment condensation reactions of lig-
nin fragments could become more signicant (Leschinsky et al.,
2008a) leading to less soluble lignin that cannot be extracted by BA.
Additionally, the extractives were higher than the differences of
lignin measurements between the two methods (Suppl. B); inferring
that in addition to lignin, extraction also removed other substances
from the pretreated substrate. In Yans study, monosaccharides ac-
counted for 56  60% of the extractives recovered by extraction with
95% ethanol (Yan et al., 2010). During hydrothermal pretreatment,
Without BAE the carbohydrates are degraded to produce monosaccharides such
KL as glucose and xylose. Although most of these monosaccharides
1.0 0.2 are dissolved in the liquor, a fraction may be retained in the pre-
1.5 0.2 treated substrate and subsequently removed by BAE.
2.0 0.3 In conclusion, due to the extractive effect of BA, the TAPPI stan-
3.1 0.2 dard method is not appropriate for lignin analysis of pretreated
6.2 0.4
bamboo therefore the BAE step should not be used.
 X.J. Ma et al. / Bioresource Technology 151 (2014) 244248
3.3. Lignin removal
Typically, hydrothermal pretreatment can cause lignin frag-
mentation and subsequent dissolution. Therefore, following hydro-
thermal pretreatment, lignin is partitioned among three locations:
(1) because hydrothermal pretreatment only causes slight deligni-
cation, most of lignin is retained in the pretreated substrate, (2) a
small amount of lignin is suspended or dissolved in the hydrolysate
liquor, and (3) a small amount of lignin precipitates onto the wall
of the reactor. It is apparent that the latter two parts are responsi-
ble for lignin removal.
Fig. 2b shows that the quantity of ASL of the pretreated sub-
strate was lower than that of the starting material and decreased
with pretreatment time until nally plateauing. ASL is basically
composed of low molecular weight degradation products and
hydrophilic derivatives of lignin (Yasuda et al., 2001). The lignin
degradation product with low MW dissolved in the hydrolysate li-
quor would account for the decrease of ASL in the substrate.
The KL decreased rst and then increased in the course of pre-
treatment (Fig. 2a). The phenol groups of lignin (assigned to
147 ppm) increased and non-phenolic groups decreased
(152 ppm) clearly showing cleavage of lignininter-unit linkages
(Suppl. C) (Wikberg and Maunu, 2004). Theoretically, the cleavage
of arylether bonds within the lignin structure resulted in an in-
crease of phenol groups and depolymerization of lignin macromol-
ecules. The cleavage of b-O-4 linkage together with other
degradation reaction probably resulted in lignin with low MW as
soluble species in the liquor.
Additionally, Selig and Donohoe suggested that thermalchem-
ical pretreatment at temperatures exceeding the melting tempera-
ture of lignin allow the lignin within the cell wall matrix to migrate
and to form spheres on contacting with water (Selig et al., 2007;
Donohoe et al., 2008). During the pretreatment, a portion of these
droplets could separate from the cell wall matrix by the washing
effect of the liquor as the reactor rotated and ultimately fall into
the hydrolysate liquor. The hydrolysate liquor could then be con-
sidered as a colloid containing lignin droplets as colloidal particles.
It was suggested that droplets were only present in the liquor dur-
ing the initial stages of pretreatment (Suppl. D). The occurrence of
the droplets in the liquor likely depended on the characteristics of
the liquor (pH, zeta potential) (Yasarla and Ramarao, 2012); the de-
gree of hemicelluloses degradation and condensation reactions of
lignin fragments may also contribute to this phenomenon. From
the Leschinsky study, treatment of prehydrolysate liquor under
hydrothermal pretreatment conditions in the absence of wood res-
idue led to formation of deposits on the wall of the reactor; further
analysis conrmed that the deposit consisted mainly of lignin deg-
radation products with a higher average MW than that of the de-
posit separated directly from the hydrolysate by centrifugation
(Leschinsky et al., 2008b). Condensation reactions between the lig-
nin droplets and subsequent precipitation might be the reason for
the disappearance of the droplets at the later stages of pretreat-
ment. Also, deposits formed from droplets or dissolved lignin frag-
ments would precipitate back onto the substrate surface and thus
partially cause the increase of KL content. It could be concluded
that lignin removal not only depends on lignin dissolution as solu-
ble species, but also on the lignin migration as colloidal particles in
the hydrolysate liquor. An earlier study by Liu and Wyman found
that lignin removal was limited to less than 35% for batch runs
but ow-through operation increased lignin removal to as high
as 75% when a high ow rate was used (Liu and Wyman, 2003).
Although the signicant effects of lignin removal were observed,
the relationship between lignin removal and ow rate were not
fully addressed. Given recent studies, lignin migration combined
with the washing effect of hot water at high ow rate probably ac-
247
counts for the signicant lignin removal observed by Liu and
Wyman.
At the later stage of hydrolysis, the KL content exceeded the ori-
ginal KL, indicating that, some acid insoluble substance formed
during pretreatment. The gain of KL probably originated from
pseudo-lignin, which is the product of reactions between carbohy-
drate and lignin degradation products. From Table 1, it is observed
that pseudo-lignin formation increases with pretreatment time.
4. Conclusions
BAE has a signicant effect on the lignin measurements of pre-
treated substrate. Benzenealcohol extraction removed a fraction
of lignin from the pretreated substrate; therefore lignin measure-
ments determined by TAPPI standard method (222om-06) are low-
er than measurements made without extraction. Therefore, TAPPI
standard without BAE should be used for lignin determination of
pretreated substrate. KL decreased rst due to the depolymeriza-
tion reaction and migration of lignin; KL in the pretreated substrate
then increased due to pseudo lignin produced from degradation
products and condensation reaction between dissolved lignin
fragments.
Acknowledgements
The authors gratefully acknowledge the support from Natural
Science Foundation of Fujian Province, China, 2012J01065 and For-
estry Department of Fujian Province, China, [2012]2 and Scientic
Research Foundation of Graduate School of Fujian Agriculture and
Forestry University, 324-1122yb002.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2013.10.
078.
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