ORIGINAL PAPER

Characterization of sugarcane bagasse by autofluorescencemicroscopy

Tiago A. Chimenez • Marcelo H. Gehlen •

Karen Marabezi • Antonio A. S. Curvelo

Received: 13 March 2013 / Accepted: 27 November 2013

� Springer Science+Business Media Dordrecht 2013

Abstract The spatial distribution of chemical com-

pounds in sugarcane bagasse is an important issue in

its use as a raw material for second generation ethanol

production from cellulose hydrolysis. Lignocellulosic

materials including whole bagasse, fiber, pith, and

respective samples obtained after chemical bleaching

were investigated using confocal fluorescence micros-

copy and spectroscopy with one and two-photon

excitation. Autofluorescence from unbleached sam-

ples revealed that emission from fiber walls containing

the lignin fraction was longitudinally oriented. After

bleaching treatment, the oriented emission was par-

tially disrupted. Autofluorescence from bleached

samples with a residual lignin content of about 1 %

was ascribed to improved dispersion of remaining

fluorophores throughout the samples inducing a con-

comitant reduction of fluorescence self-quenching in

the samples. The combination of autofluorescence

images with spectral emission and lifetime measure-

ments provides a tool for microscopic characterization

of natural bagasse samples. Moreover, the technique

allows monitoring bleaching processes related to

lignin removal.

Keywords Autofluorescence � Fluorescence

microscopy � Sugarcane bagasse � Lignin

Introduction

The global use of ethanol as a biofuel has expanded its

production from different natural resources (Farrell

et al. 2006). Sugarcane is one of the most common

feedstocks used to produce ethanol (Goldemberg

2007). In the production of ethanol from sugarcane

using fermentation, the total sugar represents only

12–17 % of the wet weight of the plant (Wheals et al.

1999). The solid material produced left after sugarcane

processing is called bagasse and it contains cellulose

(34–47 %), polyoses (24–29 %), and lignin

(18–28 %) (Nassar et al. 1996; Aguilar et al. 2002;

Gray 2007).

Bagasse is usually burned in boilers for steam

production and cogeneration of electricity, but it may

also be used for production of cellulosic ethanol

(Emsley 2008). The chemical or biochemical hydro-

lysis of cellulose from bagasse can be improved by

better knowledge of the micromorphology, chemical

composition and interaction of bagasse with different

biomacromolecules (Sant’Anna et al. 2013).

The dispersion of lignin and other related com-

pounds in the cellulose matrix can provide different

T. A. Chimenez � M. H. Gehlen (&) � K. Marabezi �A. A. S. Curvelo

Instituto de Quımica de Sao Carlos, Universidade de Sao

Paulo, Sao Carlos, SP 13560-590, Brazil

e-mail: marcelog@iqsc.usp.br

K. Marabezi � A. A. S. Curvelo

Laboratorio Nacional de Ciencia e Tecnologia do

Bioetanol, CTBE, Campinas, Brazil

123

Cellulose

DOI 10.1007/s10570-013-0135-9

features of the material including its recalcitrance and

the applications of the material. The distribution of

cellulose and lignin in plant tissues has been investi-

gated using different methods (Parameswaran and

Liese 1982; Daniel et al. 1991; Saka and Goring 1988;

Westermark et al. 1988; He and Terashima 1991; Li

et al. 2012).

There are also some specific methods to recognize

compounds within the cell walls of wood, such as

carbohydrate-binding modules (Joseleau et al. 2004),

most of which use molecular probes to stain specific

components (Vazquez-Cooz and Meyer 2002). Light

microscopy using dyes is a useful way to identify and

localize the presence of lignocellulosic compounds in

raw materials, and possesses the advantages of being

non-invasive and non-destructive (Konig 2000; Zhu

et al. 2011). In particular, the dye safranine has been

used to stain lignin in plant tissue and cellulose fibers

(Horobin 2002; Vazquez-Cooz and Meyer 2002; Bond

et al. 2008). Sugarcane bagasse is a complex material in

which different compounds should influence fluores-

cence its properties. Therefore studies using autofluo-

rescence has seldom been explored in conjunction or

with fluorescent dyes in order to get more information

about the system (Bond et al. 2008). On the other hand

authors have employed fluorescence lifetime imaging

microscopy (FLIM) related to lignin autofluorescence

to map its distribution in samples of sugarcane bagasse

and establishing relationship between lifetimes with

bleaching treatments (Coletta et al. 2013).

The autofluorescence of natural cellulose fibers has

been assigned to the presence of lignin fractions and

aromatic compounds, such as lignans, flavonoids and

tannins, found in the plant cell wall (Olmstead and

Gray 1993, 1997; Konya and Scaiano 1994; Albinsson

et al. 1999). Other researchers have ascribed this

fluorescence to the presence of nicotinamide adenine

dinucleotide in reduced form, flavins such as flavin

adenine dinucleotide, flavin mononucleotide and fla-

voproteins (Schwille et al. 1999). Studies of monocot

cells by ultraviolet fluorescence microscopy demon-

strated that the emission from natural phenolic acids in

the cell wall depends on sample pH (Harris and

Hartley 1976, 1980). Overall, several compounds may

be responsible for the autofluorescence of raw cellu-

lose fibers (Castellan et al. 2007).

Light microscopy has enlarged the scope for

investigation of cell walls (Perera et al. 2011; Harter

et al. 2012). In this context, confocal laser scanning

microscopy (CLSM) has allowed autofluorescence

images with resolution down to 400 nm and morpho-

logic features in two and three dimensions to be

obtained.

Studies exploiting the autofluorescence properties

of plant tissue and natural fibers have been performed

using fluorescence intensity imaging (Wuyts et al.

2010; Jaedicke et al. 2011). However, to identify

components using dye labeling or autofluorescence of

cellulosic materials, additional emission parameters

should be measured. Fluorescence intensity images

and local fluorescence decay profiles may be obtained

by multiphoton excitation using short laser pulses

(Cheng et al. 2001). Multiphoton excitation enhances

the depth resolution compared with that of normal

laser excitation (Denk et al. 1990). Such characteriza-

tion may allow sugarcane bagasse to be studied from a

microscopic point of view.

In this contribution, sugarcane bagasse, fiber and

pith are studied by confocal fluorescence microscopy

with one and two-photon excitation. The autofluores-

cence and dye emission from samples containing

added safranine are measured at steady-state and

under time-resolved conditions. Our goal is to evaluate

the efficiency of chemical bleaching to decompose and

remove lignin from natural cellulosic raw materials.

Experimental

Preparation of sugarcane bagasse samples

The bagasse was washed in distilled water to remove

impurities such as soil and dust. The same procedure

was also used to separate fiber and pith fractions by

wet depithing under a jet of water. A sieve system

consisting of 16 and 60 mesh screens was used to

separate the bagasse. Fiber was retained on the 16

mesh screen, while pith was washed through to the 60

mesh one. Extractives were removed by exhaustive

extraction in two sequential steps using cyclohexane/

ethanol (1:1, v/v) and then water.

Oxidative delignification was performed using dry

sample (whole bagasse, fiber or pith, 3 g) that was free

of extractives. The sample was placed in a 250 mL

three-neck round-bottomed flask containing distilled

water (160 mL). Glacial acetic acid (0.6 mL) and

sodium chlorite (1.88 g) were then added to the stirred

suspension. Delignification was performed at

Cellulose

123

70–80 �C and pH 4 for 3 h according to the optimal

parameters of Browning (1967). Further measures of

glacial acetic acid and sodium chlorite were added

every hour.

The standard method (Browning (1967) used to

evaluate total polysaccharides (holocellulose) in bio-

mass was modified to take into account the oxidation

step used to remove the lignin fraction from the natural

samples. The weight loss obtained by oxidation was

compared with the amount of lignin in unbleached

samples determined previously using the Klason

method. Typically, the percent fractions of holocellu-

lose and lignin in the natural samples were: whole

bagasse (79.9 and 20.1 %, respectively); fiber (76.7

and 22.3 %, respectively) and pith (79.2 and 20.8 %,

respectively). The content of polysaccharides obtained

after bleaching corresponds to the total polysaccharide

determined to be in the original samples. The bleach-

ing stage produced samples containing\1 % residual

lignin. The crystallinity of whole bagasse was deter-

mined by X-ray diffraction (XRD) on a Rigaku

UltraX-18HF rotating-anode generator with Cu Karadiation (k = 1.54 A). The experimental setup used

here was described by Driemeier and Calligaris

(2011), Driemeier et al. (2011). The crystallinity of

the bagasse sample was 0.55 g/g. Pulps derived from

bagasse using different pulping procedures are

reported to possess crystallinities of 0.59–0.78 g/g

(Driemeier et al. 2011). This increase in crystallinity is

caused by the removal of lignin enriching the fraction

of cellulose in the samples.

Safranine staining

Whole bagasse and bleached whole bagasse (BWB)

samples (5 g) were stained by immersion in an

aqueous solution of safranine (3 9 10-5 mol/L) for

5 min. Samples were then rinsed three times for

10 min each in warm water at 40 �C.

Scanning electron microscopy (SEM)

Sugarcane bagasse SEM images were obtained using a

microscope (LEO, 440) with an Oxford detector at an

accelerating voltage of 20 kV. The dried samples were

deposited on a carbon tape and covered with a 4 nm

gold layer.

Fluorescence microscopy

Fluorescence images were obtained using a plate

scanning confocal microscope setup. The custom-

made instrument was based on a microscope (Olym-

pus IX71) with a digital piezoelectric controller and

stage (PI, E-710.3CD and P-517.3CD) for nanometric

sample scanning. The excitation source was continu-

ous wave diode lasers (Coherent Cube, 405 nm), with

an operating at a low power producing \1 mW over

the samples. The excitation beam was converted into a

circularly polarized laser beam using zero-order

quarter-wave plates (Del Mar Photonics) and focused

on the sample with an objective (Olympus, UPLFLN

40X). The emission signal was separated from the

laser excitation beam using a dichroic cube (Chroma,

z405lp) and notch filter (Semrock, NF02-405U-25). In

two-photon excitation, an 800-nm mode-locked beam

with pulses of 150 fs from a Ti: sapphire laser (Mira

900) pumped by a coherent laser (Verdi, 5 W) was

used. The laser pulse frequency was reduced to 4 MHz

using a pulse picker (Conoptics). A dichroic cube

(Chroma, E750sp-2p with 700dcspxr) and notch filter

(Semrock, NF01-808U-25) were used in the confocal

line of the microscope in two-photon excitation

experiments. Photons were counted using an ava-

lanche photodiode point detector (Perkin Elmer,

SPCM-AQR-14) aligned with an 80-lm pinhole in

the confocal line. Transistor-transistor logic detector

signals were collected with a counter/timer peripheral

component interconnect card (NI 6601) and trans-

ferred to a personal computer for 2D plotting using a

scanning control program written in C#. Fluorescence

decays were measured by a single-photon timing

technique using a counting board (Becker and Hickl

140) with start laser pulses triggered by a photodiode

(PicoQuant TDA 200). Decays were analyzed by

multiexponential fitting without deconvolution using

the FAST software (Edinburgh Instruments).

Samples measured by fluorescence microscopy

were added to ethanol and briefly subjected to low

power ultrasound to disperse particles. Dry bagasse

samples were supported in a glass-bottomed Petri dish

(35 9 10 mm, 1.5, Ted Pella). Samples showed a

wide range in length (100–600 lm) and width

(20–100 lm). Fluorescence images covering a square

region of 60 9 60 lm with a pixel size of 200 nm

were recorded using false-color mapping, reaching the

best contrast enhancement according to the difference

Cellulose

123

in intensity of the fluorescence signal. Hereafter, the

position of a given emission spot was recovered for

spectral emission decay measurements. Only spots

with almost constant and high photon emission rate

indicating low or negligible damage caused by

photobleaching were further analyzed. Emission spec-

tra were obtained using a spectrometer (Maya 2000

Pro, Ocean Optics) coupled with a fiber to the

binocular side port of the Olympus IX71 microscope.

A long pass filter (Thorlabs, cut-off wavelength of

450 nm) was used to avoid the excitation light. The

emission spectrum of microcrystalline cellulose (Avi-

cel� PH-101, Sigma-Aldrich) was also measured for

comparison with those obtained for sugarcane

bagasse.

Results and discussion

SEM analysis of sugarcane bagasse samples (whole

bagasse, fiber and pith), and the corresponding

bleached samples (BWB, bleached fiber (BF) and

bleached pith (BP)) was used to determine the effect of

chemical bleaching. These regions were analyzed

because they present unique characteristics with

respect to their recalcitrance and morphology. Integral

bagasse consists of fiber and pith. The fiber surface is

formed by parallel stripes and has a high rate length/

diameter. In contrast, pith has a fragile and fragmented

structure correspondent to parenchyma cells designed

to store sucrose. These features are very important to

carry out hydrolysis and bleaching processes. In Fig. 1

SEM compares micrographs of unbleached and

bleached samples. Unbleached samples (Fig. 1a, b,

c) present a rigid and compact morphology. After

chemical bleaching, fiber and pith exhibited a more

disorganized morphology. The roughness increased in

the surfaces and some microsized splinters were

observed (Fig. 1d, e, f). Therefore, after bleaching,

samples showed most exposed structure, which allows

more accessibility hydrolysis reaction of lignocellu-

losic biomass (Novaes et al. 2012).

Fig. 1 SEM images of bagasse samples: a whole bagasse, b fiber, c pith, d BWB, e BF and f BP. Microsized splinters are indicated by

arrows in samples after bleaching process

Cellulose

123

Autofluorescence images of treated and untreated

sugarcane bagasse samples were performed to study of

morphological microenvironment in cellulose matrix

and to evaluate distribution of lignin fraction before

and after bleaching process.

Confocal fluorescence images of whole bagasse,

pith and fiber sample, and the corresponding bleached

samples BWB, BF and BP obtained by single-photon

excitation at 405 nm, are compared in Fig. 2. The

material absorbs light in a wide spectral range, from

the near ultraviolet to the visible, due to their

complexity and microenvironmental diversity. How-

ever, the samples showed higher emission intensity

when excited at 405 nm region. All samples exhibit

quite strong autofluorescence without fine structure. In

other words it is not possible shows the individual dots

that form the structure. Therefore, well oriented

straight row tracks structures formed by the longitu-

dinal organization of fibers do not appear in any region

observed in both natural and bleached samples.

Autofluorescence arises from compounds dispersed

in the walls of the plant cells (Harter et al. 2012),

mostly from the lignin fraction. Features related to

structure and thickness affect resolution and contrast

of fluorescence images. In addition, the excitation

light at UV–Vis region cause scattering in the samples,

and conventional confocal microscopy study of plant

tissues may be limited by the opacity of the medium.

(Cheng et al. 2000a; Benediktyova and Nedbal 2009).

This problem can be overcome by using two-photon

microscopy, because scattering and absorption are

substantially reduced in the near-infrared range com-

pared with in the visible (Denk et al. 1990; Cheng et al.

1998).

Images of sugarcane cellulose materials measured

by two-photon microscopy are illustrated in Fig. 3.

The images show greater structural detail compared

with images obtained using conventional confocal

microscopy. In addition, images generated from two-

photon excitation contained some microstructure

information and more detailed fluorescence mapping

than single-photon excitation images. Two-photon

Fig. 2 Fluorescence microscope images of a whole bagasse, b fiber, c pith, d BWB, e BF and f BP. Samples were excited at 405 nm.

Scale bar 10 lm. The lateral color scale bars are related to the fluorescence intensity

Cellulose

123

Fig. 3 Fluorescence intensity image and decay traces of

indicated emission spots obtained by two-photon excitation at

800 nm of a whole bagasse, b fiber, c pith, and corresponding

bleached samples, d BWB, e BF and f BP. Scale bar 10 lm.

Decay fitting parameters are given in Table 1. The lateral color

scale bars are related to the fluorescence intensity. Oriented

fluorescence tracks are indicated by arrows in a whole bagasse,

b fiber

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123

microscopy can give supplementary characterization

of lignocellulosic materials (Cheng et al. 2000a, b),

because transitions between states of the same parity

are forbidden for one-photon excitation, but they are

allowed for the two-photon excitation (Callis 1993).

The fluorescence images in Fig. 3 show sequences

of small fluorescence spots that form well oriented

straight row tracks, which are related to lignin fraction,

especially from unbleached whole bagasse and fiber

samples. The average distance between two oriented

fluorescence tracks is about 12 lm, which is compa-

rable with the distance between cell walls determined

by SEM (see Fig. 1b). Fluorescence spots in pith

samples show a decrease in intensity and disordered

structure, it does not generate straight tracks format.

In contrast to untreated samples, images of

bleached samples do not contain emission tracks;

instead, some blurry emission spots are evident. This

result is consistent with disorder increasing in the

samples after chemical bleaching because of disper-

sion of crystalline cellulose domains.

Scanning the z position of the focal plane using two-

photon fluorescence microscopy allowed us to visu-

alize the architecture, fibril patterns, and other details

of the microstructure of cellulosic materials displaying

autofluorescence. 2D images at various depths of z-

axis from 0 to 10 lm are shown in Fig. 4. Orientation

of micrometer-sized emission tracks is closely depen-

dent of z-position. Each 1 micrometer change in depth

of the focal plane occur loss of orientation. This effect

may be related to the SEM images of the bagasse

sample in Fig. 1a, where oriented walls of the fiber are

located near its surface.

Lifetime measurements in combination with

microscopy techniques can be used to study biological

materials (Ulrich et al. 2004; Heskes et al. 2012).

Samples were investigated by fluorescence decay

measurements to identify lignin-rich regions. Fluores-

cence decay traces were recorded at a wavelength of

500 nm, as well as at 575 nm for samples with added

safranine, which corresponds to its emission maxi-

mum. Decay analyses were performed by FAST

software (Edinburgh Instruments) using multiexpo-

nential fitting without deconvolution. The fitting range

was chosen by considering the decay part of the time

dependent data according to optimal parameters.

Typical decay profiles of untreated and bleached

Fig. 4 Two-photon fluorescence microscope images of a whole

bagasse sample obtained at a focal plane of a 0 lm, b 5 lm and

c 10 lm. Scale bar 10 lm. The lateral color scale bars are

related to the fluorescence intensity

Cellulose

123

samples are illustrated in Fig. 3. The fluorescence

relaxation was fitted with a biexponential decay

function. The decay components, respective normal-

ized amplitudes and average values are presented in

Table 1. A fast decay component occurs in the range

of 0.4–1.4 ns, and a long-lived component takes more

than 2.0 ns to decay. Such biexponential decay

behavior with a short decay component of about

0.4 ns was first observed in the fluorescence emission

of Abies wood (Castellan and Davidson 1994).

Departure from single exponential decay behavior

is expected for a complex matrix like bagasse that

contains multiple intrinsic fluorophores such as aro-

matic compounds with phenolic and acid groups from

the lignin fraction (cinnamic residues), and traces of

endogenous biomolecules such as enzymes and

cofactors, chlorophyll, xanthophylls, coumarin and

flavonoid derivatives (Olmstead and Gray 1997;

Schwille et al. 1999; Albinsson et al. 1999; Billinton

and Knight 2001). Hafren and Oosterveld-Hut (2009)

studied wood fibers using fluorescence lifetime imag-

ing. Biexponential fitting at two emission regions of

436 and 480 nm resulted in wavelength-independent

decay times of about 0.7 and 4.3 ns. These values are

within the range of decay times observed for our

samples. For bagasse samples without added safra-

nine, the decay times were almost independent of

wavelength.

Analysis of decay parameters in this system is a

difficult task because of its complexity. Nevertheless,

some important issues were identified. In general, the

fluorescence decay of untreated and bleached samples

is nonexponential and occurs in\20 ns, as illustrated

in Fig. 3. The calculated average decay time increases

by about 68 % from fiber to BF, 47 % from whole

bagasse to BWB, and 41 % from pith to BP (see

Table 1). Samples stained with safranine show

slightly different decay behavior, as illustrated in

Fig. 5. First, unbleached samples like whole bagasse

show a third decay component with a lifetime of

4.8 ns. This component is ascribed to the decay of the

singlet excited state of safranine formed mainly

through energy transfer from the natural compounds

to the added dye. In addition, the fluorescence lifetime

of safranine in other solvents was also measured. In a

polyvinylpyrrolidone film, a single component with a

lifetime of about 4 ns was recorded, while the

fluorescence lifetime of safranine in propanol was

3 ns. Therefore, the observed increase of safranine

fluorescence lifetime in bagasse samples is a result of

absorption of the dye in lignin-rich regions. The

second difference is the moderate quenching of the

second decay component of the whole bagasse sample,

which is reduced from 1.9 to 1.3 ns. This result is

consistent with fluorescence quenching of excited

chromophores of the lignin fraction by energy transfer

to the adsorbed safranine. Fluorescence behavior is

closely linked to the chemical environment of the

cellulosic matrix (Castellan et al. 2007). Chemical

bleaching causes lignin components to be decomposed

and dispersed. The bleaching reaction also decreases

the amount of organic materials and intrinsic fluoro-

phores in the sample. Therefore, bleaching reduces

energy migration and fluorescence self-quenching in

Table 1 Decay times (si) and normalized amplitudes (ai) of micrometer-sized emission spots from cellulose samples isolated from

sugarcane bagasse obtained by multiexponential decay fitting

Sample s1 (ns) s2 (ns) s3 (ns) a1 a2 a3 hsi v2

Whole bagasse (500 nm) 0.4 1.9 0.43 0.57 1.7 1.15

Whole bagasse/safranine (500 nm) 0.8 2.8 0.71 0.29 2.0 1.20

Whole bagasse/safranine (575 nm) 0.4 1.3 4.8 0.73 0.23 0.04 2.0 1.05

BWB (500 nm) 0.9 3.1 0.58 0.42 2.5 1.03

BWB/safranine (500 nm) 0.8 3.3 0.56 0.44 2.7 1.13

BWB/safranine (575 nm) 1.1 2.8 0.44 0.56 2.2 1.00

Fiber (500 nm) 0.7 2.4 0.63 0.37 1.6 1.18

Pith (500 nm) 0.7 2.2 0.61 0.39 1.7 1.16

BF (500 nm) 0.9 3.4 0.60 0.40 2.7 1.12

BP(500 nm) 0.7 2.7 0.39 0.61 2.4 1.05

hsi = Amplitude-weighted average decay time

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123

the samples. Self-quenching is mainly observed when

the lignin matrix is intact and its intrinsic fluorophores

are closely confined. The emission intensity of the

bleached samples is still strong because most of the

remaining chromophores are partially isolated or

dispersed in the cellulosic matrix, reducing excited-

state deactivation by self-quenching. These results are

in agreement with those obtained by Coletta et al.

(2013) using fluorescence lifetime imaging (FLIM) to

map the distribution of lignin in sugarcane bagasse.

They also obtained two components related to

fluorescence decay, one with the fast and another

with the slow decay time. The faster decay is

associated to dense lignin regions and becomes slower

after samples undergo pretreatment to remove lignin.

Emission spectra of the fiber samples were also

measured under the same conditions; the results are

depicted in Fig. 6. Untreated fiber exhibited high

intensity emission with a maximum at about 500 nm,

consistent with the emission spectra reported for wood

samples (Donaldson et al. 2010). When safranine was

added to the fiber sample, the emission was quenched

Fig. 5 Fluorescence intensity images and decay of each

indicated emission spot obtained by two-photon excitation at

800 nm of samples stained with safranine: a whole bagasse and

b BWB. Scale bar 10 lm. The lateral color scale bars are

related to the fluorescence intensity

Cellulose

123

and a new band appeared at about 575 nm corre-

sponding to the fluorescence of the added dye.

Safranine has negligible absorption at an excitation

wavelength of 405 nm, so dye emission may occur

after energy transfer from the intrinsic fluorophores to

safranine. The BF sample exhibited a similar emission

profile to that of the untreated fiber, but its intensity

was reduced by half. The reduction in intensity

confirms that chemical bleaching removed the lignin

fraction, so autofluorescence from the sample

decreased. However, the intensity is much higher than

would be expected from a sample containing a lignin

fraction of about 1 % after chemical bleaching. This

means that intrinsic fluorophores remained in some

regions of the sample, and these showed higher

emission efficiency than in the unbleached sample

because of matrix dispersion and less self-quenching.

Chemical treatment cleaves the lignin macrostructure

into smaller components, which then disperse over the

solid matrix, reducing self-quenching. For the BF

sample stained with safranine, the dye emission still

appears but at lower intensity, which agrees with

energy transfer occurring from the fluorophores

remaining in the sample to the added safranine dye.

The reference Avicel sample exhibited very weak

emission under the same conditions compared with

that from bagasse samples.

Conclusions

We used two-photon fluorescence microscopy to

characterize the autofluorescence from micro-regions

of sugarcane bagasse samples. Fluorescence images

show that fibril packages of bagasse sample partially

lose their longitudinal orientation after chemical

bleaching. Extraction of lignin allows the remaining

natural fluorophores to disperse in the sample, which

reduces self-quenching. The presence of highly emis-

sive spots after chemical bleaching indicates that

fluorescent lignin derivatives remain in some regions

of the lignocellulosic matrix. This result may indicate

the region where autocatalytic acid hydrolysis of

cellulose could start (Gehlen 2009, 2010).

Our results are able to provide information related

to the morphological properties and lignin distribu-

tion. In addition, it is possible to understand how these

parameters are affected by the bleaching process.

Therefore, this study provides support for future site-

specific morphological analyses of the effects of using

different pretreatments on cellulose hydrolysis. Con-

sequently, it acts in the field of second generation

bioethanol production.

Acknowledgments The authors thank the Conselho Nacional

de Desenvolvimento Cientıfico e Tecnologico and Coordenacao

Fig. 6 Fluorescence

spectra from (solid curve)

fiber, (long dashed curve)

fiber/safranine, (two dotted

dashed curve) BF, single

dotted dashed curve) BF/

safranine and (shorted

dashed curve) Avicel.

Samples were excited at

405 nm and spectral

intensity was recorded using

a linear CCD spectrometer

fiber coupled to the

binocular side port of the

microscope

Cellulose

123

de Aperfeicoamento de Pessoal de Nıvel Superior Brazilian

research funds. M.H.G. thanks Fundacao de Amparo a Pesquisa

do Estado de Sao Paulo for financial support that allowed a

fluorescence confocal microscope to be built. We thank

Professor Dr. Johan Hofkens and Dr. Kris P. F. Janssen for the

use of SIS software from the MDS Laboratory of the

Department of Chemistry of Katholieke Universiteit Leuven.

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