Structural analysis for lignin characteristics in biomass ...€¦ · Hot-water soluble 14.6 7.2 18.9 13.2 Lignin 15.3 14.2 14.1 16.7 Hemicellulose 32.4 33.8 34.2 32.7 Cellulose 37.7

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Review

Structural analysis for lignin characteristicsin biomass straw

Seyed Hamidreza Ghaffar, Mizi Fan*

School of Engineering and Design, Brunel University, Uxbridge, Middlesex, UB8 3PH, United Kingdom

a r t i c l e i n f o

Article history:

Received 31 March 2013

Received in revised form

15 July 2013

Accepted 19 July 2013

Available online xxx

Keywords:

Straw lignin

Quantitative and qualitative anal-

ysis

NMR

FT-IR

Structural characteristics

Thermal analyses

* Corresponding author. Department of CivilE-mail address: [email protected] (M

Please cite this article in press as: Ghaffaand Bioenergy (2013), http://dx.doi.org/10

0961-9534/$ e see front matter ª 2013 Elsevhttp://dx.doi.org/10.1016/j.biombioe.2013.07.

a b s t r a c t

Agricultural by-products are the most promising feedstock for the generation of renewable,

carbon neutral substitutes for synthetic materials (e.g. biofuel, building materials). The

demand for efficient utilisation of lignin biomass has induced detailed analyses of its

fundamental chemical structures and development of analysing technologies. This paper

reviews the structural analysis techniques for straw lignin together with the morphology of

the lignin biomass and the study of the form and structure of organisms and their specific

structural features. The review showed that the studies on lignin could be divided into the

qualitative and quantitative analyses; different analytical methods could provide signifi-

cantly different results that are even sometimes not directly comparable. Among many

techniques reviewed, the magnetic resonance techniques have proved to be efficient

analytical tools for the structural elucidation of these complex biopolymers. Quantitative

and qualitative structural analysis of lignin indicated a great potential for industrial crops

optimisation due to in-depth microstructure interpretation, and detailed and accurate

chemical composition although the composition and structure of straw lignin have been

discovered highly complex and varied considerably within and among plants. The struc-

ture of lignin has remained one of the most difficult biopolymers to characterise, however

recent advances in analytical chemistry and spectroscopy have dramatically improved the

understanding of this natural resource, and further value added utilisations are being

expected for the lignin and its related biomass.

ª 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Research concerning lignin has significantly increased due to

its renewability and availability and the value added produc-

tion of lignin based products, such as biofuel and sustainable

construction materials for replacing petrochemicals based

products [1,2]. The structure of lignin is probably the single

most important parameter for understanding and hence uti-

lising lignin materials, and has been analysed by many

Engineering, Brunel Univ. Fan).

r SH, Fan M, Structural.1016/j.biombioe.2013.0

ier Ltd. All rights reserved015

research workers e.g. Refs. [3e5]. A number of chemical and

physical methods used for characterising lignin’s structure

are destructive. However there are non-destructive methods

too, such as FT-IR which is believed to be one of the most

informative methods of lignin investigation, ultraviolet (UV),

carbon-13 nuclear magnetic resonance spectroscopies (13C

NMR) and gas permeation chromatography (GPC) [6].

Lignin is an extremely complex three-dimensional poly-

mer (typically found in vascular plants) formed by radical

ersity, London UB8 3PH, United Kingdom.

analysis for lignin characteristics in biomass straw, Biomass7.015

.

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coupling polymerisation of p-hydroxycinnamyl, coniferyl and

sinapyl alcohols; these three lignin precursors monolignols

give rise to the so-called p-hydroxyphenyl (H), guaiacyl (G) and

syringyl (S) phenylpropanoid units, which show different

abundances in lignin from different groups of vascular plants,

as well as in different plant tissues and cell-wall layers [7].

These aromatic building units are linked with a variety of

ether and carbonecarbon bonds. The predominant linkage is

the so called b-O-4 linkage. About 40e60% of all inter-unit

linkages in lignin are via this ether bond. Different types of

linkage between phenyl propane units form three-dimen-

sional net structures and make it difficult to completely

degrade non-regular lignin macromolecules. Due to lignin’s

structural complexity it is hard to come to an agreedmodel for

their chemical and physical structure. Lignin is primarily a

structural material to add strength and rigidity to cell walls

and constitutes between 15 and 40% mass fraction of the dry

matter of plants, whether wood, straw or other natural woody

plants. Lignin is more resistant to most forms of biological

attack than cellulose and other structural polysaccharides

[8e10]. Lignin acts as a matrix together with hemicelluloses

for the cellulose microfibrials which are formed by ordered

polymer chains that contain tightly packed, crystalline re-

gions (Fig. 1). Covalent bonds between lignin and the carbo-

hydrates have been suggested to consist of benzyl esters,

benzyl ethers and phenyl glycosides [11e13].

There is a little study of lignin native (in situ)which does not

change the structure of lignin, such as, chemically unmodified

lignin by using ionic liquid 1-ethyl-3-methylimidazolium ace-

tate [Emim] (CH3COO) as a pre-treatment solvent [14]. How-

ever, there has been much interest for industrial lignin due to

the existence of large scale manufacturing processes, i.e. wood

pulping, one of the largest industries in the world in which the

main objective is to separate individual fibres from wood and

to do so ligninmust firstly be removed, and lignin from various

resources and industries were studied through elemental

analysis in order to understand the fundamentals of lignin and

their properties for value added applications [15]. Huge quan-

tities of lignin are extracted by wood pulping and other in-

dustries annually, though the bulk of which is still either

burned to recover energy or otherwise considered waste. Only

about 1e2% of this lignin is used to make other products [16].

Despite lignin’s unique characteristics as a natural product

Fig. 1 e Cellulose strands surrounded by hemicellulose and

lignin [1].

Please cite this article in press as: Ghaffar SH, Fan M, Structuraland Bioenergy (2013), http://dx.doi.org/10.1016/j.biombioe.2013.0

with multiple chemical and biophysical functionalities, it is

largely underutilised due to its image as low quality and low

value addedmaterial. Current work of researchers has focused

on expanding the frontiers of application of NMR to straw

lignin analysis in order to explore lignin’s potential [4,17,18].

The main goal of this paper is to study in details the

structure of straw lignin and to review the analytical tech-

niques up to date for the analysis of lignin; this will then

contribute to a better understanding of lignin and hence could

lead to the optimisation of lignin for specific and value added

industrial utilisations.

2. Composition and morphology of straw

Straw consists mainly of three groups of organic compounds:

cellulose, hemicellulose and lignin which account for more

than 80% of the dry matter of cereal straws such as oats,

barley and wheat [19]. Straw also contains various other

organic compounds including protein, small quantities of

waxes which protect the epidermis of the straw, sugars, salts

and insoluble ash including silica. Different fractions across

the straw vary in chemical composition which is also affected

by soil type and fertiliser treatment. Overall, there is a higher

concentration of cellulose in internode, of ash (in which silica

is the main constituent) in leaf and leaf base and of lignin in

the node cored compared to the other parts of straw (Table 1)

[20,21].

Similar to wood, straw is considered a natural composite

material because of its composition of polysaccharides (cel-

lulose and hemicelluloses) and lignin. The former two com-

ponents are hydrophilic and the latter is hydrophobic.

However, they are practically insoluble in water due to

hydrogen bonding and covalent bonding with lignins [6]. The

ultrastructure of straw fibres, like wood tracheids, consists of

middle lamella (ML), primary wall (P) and secondary walls (the

outer S1, the middle S2, the inner S3). The percentage volume

fraction (PVF) and thickness of variousmorphological layers of

wheat straw can be different from those of wood (e.g. spruce)

(Table 2) [22], especially the S1 layer of wheat straw fibre is

thicker than that of the spruce tracheid and PVF of ML and cell

corner (CC) in wheat straw fibre is greater than that of spruce

tracheid.

When it comes to anatomical structure of straw, normally

the sclerenchyma cells in the bundle itself have a small

diameter and a thick fibre wall while the extra vascular fibres

often have a much larger diameter and a very variable wall

Table 1 e Chemical components of wheat strawdetermined by gravimetric analysis [20].

Mass fraction of dry material (%)

Leaf Internode Leaf base Node core

Hot-water soluble 14.6 7.2 18.9 13.2

Lignin 15.3 14.2 14.1 16.7

Hemicellulose 32.4 33.8 34.2 32.7

Cellulose 37.7 44.8 32.7 37.5

Ash 11.5 4.7 12.4 6.3




Table 2 e The thickness and PVF of wheat straw [22].

Morphological layers Wheat straw Spruce tracheid

Thick fibre Thin fibre

Thickness (mm) PVF (%) Thickness (mm) PVF (%) Thickness (mm) PVF (%)

ML þ P 0.1e0.2 9.3 0.06e0.12 12.3 0.05e0.1 10.2a

S1 0.1e0.3 0.2e0.3 0.15e0.2 9.9

S2 1.8e2.5 83.5 0.5e0.8 80.7 0.7e2.0 75.9

S3 0.15e0.3 0.1e0.2 0.1 4.0

CC e 5.4 e 7.0 e e

a Containing the CC region.

b i om a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1e1 6 3

thickness. In wheat straw these fibres occur as coarse bast

fibres just inside the epidermal layer (Fig. 2A). Unlike wheat,

the cross-section of rice straw reveals a different type of ul-

trastructure, beside the tubular concentric ring structure, the

centre of the rice straw internodes contains a core (Fig. 2B and

C). Moreover, rice is an aquatic plant and has a different type

of protecting layer, including substances composed of lignin,

amorphous silica, and other inorganic substances.

3. Analytical techniques for structuralanalysis of straw lignin

The need of properly characterising and classifying lignin for

the promotion of the new applications is clear. At the product

development level, appropriate analysis will facilitate the

definition of specifications for commercial purposes and also

allow the reliable monitoring of physical and chemical mod-

ifications which eventually will promote the understanding of

Fig. 2 e Micrograph of cross sections of (A) wheat straw under

epidermis, (b) parenchyma, (c) lumen, (d) vascular bundles [24];


the relationship between properties and performance. The

utilisation of lignin for a range of natural and industrial pur-

poses is dependent on the analysis of lignin and the charac-

teristics of this polymer. Also a comprehensive understanding

of lignin is needed when considering processes such as

various pre-treatment techniques, including physicochemical

and biological pre-treatments. The complicated and hetero-

geneous structure of lignin has been traditionally revealed by

a series of chemical analysis, such as thioacidolysis (TA) [26],

nitrobenzene oxidation (NBO) [27] and derivatization followed

by reductive cleavage (DFRC) [28]. Nevertheless recent de-

velopments in nuclear magnetic resonance (NMR) technology

have made this technique the most widely used technique in

the structural characterisation of lignin, the reason being its

versatility in indicating structural features and structural

transformation of lignin.

The studies on lignin are divided into two different sec-

tions: qualitative and quantitative analyses. Some researchers

(e.g. Ref. [3]) divided the analytical methods of lignin

light microscope [23]; (B) wheat straw internode SEM (a)

(C) rice straw internode SEM [25].




Table 3 e Assignments of FT-IR absorption bands (cmL1)[7].

Absorptionbands

Assignment

3429 OH stretching

2945 CH stretching of methyl, methylene

or methane group

1732, 1726 C¼O stretch in unconjugated ketone

and carboxyl group

1660, 1653 C¼O stretch in conjugated ketone

1606 Aromatic skeletal vibration


1460 Aromatic methyl group vibrations


1374 Aliphatic CeH stretch in CH3

1328 Syringyl ring breathing with CeO stretching

1242 Aromatic CeO stretching

1165 CeO stretch in ester groups

1135 Aromatic CeH in-plane deformation

for syringyl type

1043 Aromatic CeH in-plane deformation

for guaiacyl type

855, 844 Aromatic CeH out of plane bending


characterisation into two groups of destructive and non-

destructive methods. Thioacidolysis (TA) [26], nitrobenzene

oxidation (NBO) [27] and also derivatization followed by

reductive cleavage (DFRC) [28] are amongst the most

destructive methods used. The non-destructive methods

include various spectroscopic methods (e.g. UV and Fourier

transform infrared spectroscopy (FT-IR spectra)) and Nuclear

magnetic resonance (NMR) techniques.

The chemical composition of agricultural biomass can also

be analysed with near infrared (NIR) spectroscopy, e.g. Kelley

et al. [29] tested the effectiveness of NIR for measuring the

chemical composition of biomass that has been subjected to a

wide variety of extraction and chemical treatments; Sander-

son et al. used NIR for directly measuring the chemical

composition of biomass [30].

Another technique to analyse lignin in the cell wall is based

onmercurization whichwas firstly used in the early history of

lignin chemistry to characterise its structure [31]. Mercuriza-

tion is a reaction occurring under mild conditions; it seems to

be less sensitive to the substitution pattern of the aromatic

ring. The determination of lignin, thought apparently an easy

matter, is one of the least satisfactory of the analysis

commonly carried out on plant materials and woods. Almost

all current methods contain the solution and hydrolysis of all

other plant constituents and assume that the residue after

such treatment is lignin. Various investigators have pointed

out that this is not the case, but in spite of this vital objection,

considerable reliance has been placed on figures so obtained.

3.1. Quantitative analysis

Quantitative measurement of lignin structures is an impor-

tant aspect when it comes to investigating lignin; the mea-

surement of various structures of lignin is possible when

appropriate standards or pulse sequences are applied [32e34].

Quantitative analysis is based on gravimetry or UV-absorption

[35] either to estimate lignin as an insoluble residue after

strong sulphuric acid treatment (Klason lignin) [36] or to oxi-

dise away the lignin from a holocellulose preparation (acidic

chlorite lignin or permanganate lignin) [37]. The spectroscopic

techniques, such as infrared (IR) and 13C nuclear magnetic

resonance (13C NMR) spectroscopy, are complementary to the

above procedures since they provide information on the

whole structure of the polymer and avoid the possibility of

degradation artefacts [38]. This information is presented with

quantitative values.

3.1.1. FT-IRAmong all the quantitative analysis techniques, FT-IR spec-

troscopy shows interesting characteristics, including high

sensitivity and selectivity, high signal-to-noise ratio, accu-

racy, data handling facility, mechanical simplicity and short

time and small amount of sample required for the analysis

[39]. In addition, the spectrum of a lignin sample gives an

overall view of its chemical structure [40]. FT-IR spectroscopy

is non-destructive method of lignin investigation, which

opens perspective to find structural discrepancies in lignin

isolated by different methods.

Infrared radiation is electromagnetic radiation which

covers the wavenumbers between 13,300 cm�1 and 3.3 cm�1.


The infrared region is usually divided into 3 regions: near

infrared (13,300e4000 cm�1), middle infrared (4000e200 cm�1)

and far infrared (200e3.3 cm�1) regions. Organic compounds

have fundamental vibration bands in the mid infrared region,

which is why the region is widely used in infrared spectros-

copy. An infrared spectrum is unique to each substance and

can therefore, in principle, be used as an impartial charac-

teristic to identify the sample. Every lignin IR spectrum has a

strong wide band between 3500 and 3100 cm�1 assigned to OH

stretching vibrations. This band is caused by the presence of

alcoholic and phenolic hydroxyl groups involved in hydrogen

bonds. The intensity of the band increases during demethy-

lation and decreases during methylation since during deme-

thylation the OeCH3 bonds in methoxyl groups bonded to 3rd

or 5th carbon atom of the aromatic ring are split and CH3 is

replaced by a hydrogen atom producing a new OH group.

During methylation the OeH bonds are split and H is replaced

by CH3 group and the amount of OH group decreases hence

the intensity of the band decreases [41]. Acetylation of the

lignin causes a partial or full band loss since almost all of OH

groups are replaced by CH3COO [42].

The assignments of FT-IR absorption bands for the lignin

normally include the aromatic skeleton vibrations at 1606,

1507 and 1434 cm�1, in which the aromatic semicircle vibra-

tion (a vibration involving both CeC stretching and a change

of the HeCeC bond angle) is assigned at 1507 cm�1 [43], the

carbonyl and unconjugated ketone and carboxyl group

stretching at 1732 cm�1 and the small band of the conjugated

carbonyl stretching such as detected in organosolv lignin at

1653 cm�1. More detailed assignment are summarised in

Table 3 [7,44,45].

3.1.2. Thermal analysisThermal analysis has recently become prominent, especially

for fibres, plastics and other polymeric materials. Thermal





analysis is a set of techniques used to describe the physical or

chemical changes associated with substances as a function of

temperature. The thermal stability of the isolated hemi-

celluloses, cellulose and lignin preparations is determined by

thermogravimetric analysis (TGA) and differential scanning

calorimetry (DSC). TGA can be used tomonitor the weight loss

of the lignin as it is heated, cooled or held isothermally, while

DSC is performed to determine the melting temperature and

enthalpy of the lignin. DSC is the most widely accepted

method for determining glass transition temperatures (Tg) of

lignin or modified lignin samples [46]. Thermoanalytical

techniques, in particular TGA and derivative thermogravi-

metric (DTG), allow the information about chemical compo-

sition of straw to be obtained in a simple and straightforward

manner. Pyrolysisegas chromatography/mass spectrometry

(PyeGC/MS) has also been applied to examine reaction prod-

ucts of thermal degradation [47] and an effective tool to

investigate fast pyrolysis of biomass and on-line analysis of

the pyrolysis vapours [48,49].

PyeGC/MS provides a rapid and easy alternative to tedious

chemical degradation procedures for analysing the mono-

lignol composition of lignin samples [50]. It requires only a

small amount of lignin (<1 mg) and samples do not have to be

isolated from the associated plant polysaccharides to be

effective. Compounds separated on a GC column can easily be

identified from their mass spectra as being derived from p-

hydroxyphenyl (H), syringyl (S), or guaiacyl (G) propane units.

Integration of the chromatograph peaks can determine if the

sample in question comes from a G, S/G, or H/S/G type lignin.

For TGA and DSC the heating rate is usually between 5 �Cand 10 �C. The tests are undertaken in both nitrogen and air

atmosphere between room temperature of 25 �Ce30 �C up to

730 �Ce800 �C [51]. Due to the complexity of lignin and the

difficulty in extraction, the literature related to the pyrolysis

behaviour of lignin is scarce. However Muller-Hagedorn and

Bockhorn [52] obtained kinetic parameters for straw lignin in a

TGA based on the LevenbergeMarquardt and improved Run-

geeKutta laws. The activation energies and frequency factor

for barley straw lignin varied from 92 to 102 kJ mol�1 and from

107.3 to 107.9 min�1 respectively. Ghaly et al. [53] used TGA and

differential thermal analysis (DTA) to study the behaviour of

oat straw in an oxidising atmosphere (15% oxygen and 85%

nitrogen); two distinct reaction zones were observed on the

TGA and DTA curves. Higher thermal degradation rates were

observed in the first reaction zone due to the rapid release of

volatiles as compared to those in the second reaction zone.

The activation energies were in the range of 83e102 and

58e75 kJ mol�1 for the first and the second reaction zones,

Table 4 e NMR techniques for lignin analysis.

NMR techniques Quantitative data

13C NMR Aliphatic/phenolic OH groups and methoxy gro

HSQC Inter-unit bonding patterns31P NMR Aliphatic/phenolic OH groups, guaiacyl, siringyl

condensed OH groups, carboxylic acids1H NMR Hydrocarbon contaminant, aliphatic and aroma

protons in methoxyl groups, benzylic protons in


respectively. The thermogravimetric dynamics parameters of

wheat straw enzymatic acidolysis lignin (EMAL) were calcu-

lated by the methods of Kissinger and Ozawa, respectively;

the activation energy of wheat straw EMAL was 103.92 and

107.69 kJmol�1 respectivelywith themethods of Kissinger and

Ozawa [54].

Carrier et al. [55] have also investigated the suitability of

TGA as a new method to obtain lignin, hemicellulose and

⍺-cellulose content in biomass. The study indicated compa-

rable results to common methods used for ⍺-cellulose con-

tent; however, this method cannot be extended to the lignin

content because of important deviations in the correlation

curves. Through this alternative method, which is faster,

easier to implement and less cost effective than the existing

wet chemical techniques, it is possible to determine the

hemicelluloses and ⍺-cellulose contents of biomass samples.

Thermal degradation of lignin is reviewed by Brebu and

Vasile [56] where the information on the temperature range,

kinetics and mechanism of thermal degradation is presented.

Samples for the thermal analysis of straw such as TGA are

usually the same as those for FTIR spectroscopy test.

3.1.3. NMRNuclear magnetic resonance spectroscopy (NMR) has been

shown to be reliable and comprehensive among the various

physical and chemical methods for the characterisation of

lignin, because it not only gives quantitative data but also

provides some qualitative information (Table 4). An inclusive

review on NMR application for structural analysis of lignin

was published in 1971 [57].

In the past, proton NMR (1H NMR) was mainly used for

lignin characterisation and the 1H NMR spectrum of acety-

lated lignin is used to determine the quantity of different

hydroxyl groups. Lundquist [58e60] has published works

about NMR characterisation of lignin. With the development

of NMR techniques, 13C NMR became popular in lignin char-

acterisation as it is powerful and capable of revealing a large

amount of lignin structural information including the pres-

ence of aryl ethers, condensed and uncondensed aromatic

and aliphatic carbons. Since the 1980s, many studies on 13C

NMR spectra of lignin have been conducted [61e64]. Many

attempts have been made to increase the sensitivity and

signal-to-noise ratios of the quantitative 13C NMR spectra,

especially for understanding the structural changes of lignin

polymer in pulping processes and other isolation processes

[3]. 13C NMR has been used to aid in the elucidation of pulping

or delignification mechanism (soda pulping, kraft pulping and

oxygen/peracid treatments), as well as pre-treatments, which

Qualitative data

ups Inter-unit bonding patterns

Inter-unit bonding patterns

, Assignments of guaiacyl, siringyl, condensed

OH groups, carboxylic acids

tic acetate,

b-b structures

Improved spectral resolution of key

lignin functionality





are discussed in detail in a recent book by Ralph and Landucci

[65].

Quantitative 13C NMR is commonly used for the estimation

of some specific component [65,66]. However, the most recent

practices in the use of quantitative 13C NMR spectroscopy for

the characterisation of lignin are confined to using the aro-

matic and methoxyl signals as internal standards in

expressing the various functional groups per C9 [65]. Such a

practice is applicable to native lignin but inapplicable for in-

dustrial lignin or modified lignin [66]. To overcome the above

defects, Xia et al. [66] suggested a novel protocol for acquiring

quantitative 13C NMR spectra of lignin by using the internal

reference compounds 1,3,5-trioxane and pentafluorobenzene.

Trioxane offers a convenient internal standard for collecting

inverse gated proton decoupled 13C NMR spectra for lignin.

The internal reference compounds provide single and un-

overlapped sharp signals in the middle of the spectral re-

gion, permitting superficial integration.

Detailed approaches for the quantification of different

lignin structures in milled wood lignin (MWL) have also been

reported by using quantitative 13C NMR techniques [67,68],

including the amount of different linkages, various phenolic/

etherified non-condensed/condensed guaiacyl and syringyl

moieties. This approach is comparable to that reported from

other wet chemistry techniques, but requiring only rather

short experimental times. Much progress has been achieved

on the 13C NMR spectra of lignin, but there still remain some

issues to be explained, such as precise signal assignments and

true quantification based on 13C NMR spectra of lignin, which

are difficult due to signal overlap and other factors. Both solid-

state and solution-state 13CNMRhave been used for structural

characterisation of lignin [69,70], however the solution-state

NMR is more informative because of its better resolution,

although soluble lignin preparations may not be representa-

tive of the whole lignin fraction in plants [71,72].

It is worth mentioning that structural characterisation of

lignin could be directly investigated by NMR techniques via

non-derivatization solvent systems, such as DMSO-d6/NMI-d6[73], DMSO-d6 [74] and DMSO-d6/pyridine-d5 (as gelling sol-

vent) [75]. The rapid NMR characterisation method provides

what appears to be the best tool for the detailed structural

study of the complex cell wall polymers. The DMSO-d6 (gel-

state) method is used for lignin analysis, not polysaccharides

analysis. Nevertheless, based on these non-acetylated solvent

systems, lignin composition (notably, the syringyl: guaiacyl: p-

hydroxyphenyl ratio) could be quantified without the need for

lignin isolation [3].

In summary, with NMR techniques, the true quantification

in lignin is difficult due to signal overlap and other factors. The

best quantification method remains the relatively tedious

inverse-gated technique, along with an internal standard

substance. Fortunately, most of the NMR techniques are

adequate when the researchers want to follow changes in

structure of straw biomass over time during a particular

treatment, provided that the desired precision is maintained

[65].

3.1.4. Two-dimensional HSQC NMRThe interest to study the mixture of increasing complexity of

lignin has created the demand to employ high magnetic field


NMR spectrometers to improve resolution and sensitivity. The

strong self-association of lignin chains and harsh treatment

required for lignin-derived fragments solubilisation are major

factors that make studying lignin primary structures difficult

[76]. Quantification of complex samples with 1H NMR spec-

troscopy suffers from resonance overlap even at high mag-

netic fields, which can prevent an accurate quantification of

sample compounds. Protonecarbon correlated two-

dimensional (2D) NMR can exploit the wide chemical shift

range of carbon, thus offering a significantly improved reso-

lution. Therefore, the application of protonecarbon correlated

2D NMR in the determination of molar concentration of the

sample compounds has gained interest in studies of natural

products, biological samples and processes taking place in

living organisms [77] and 2D NMR of both homo-and hetero-

nuclear became popular and much powerful tools for lignin

characterisation [78e80].

The most used and valuable 2D NMR is heteronuclear-

single-quantum-coherence (HSQC) that provides the correla-

tion between directly bonded protons and carbons in two di-

mensions [67,81]. Overlapping protons that are attached to

carbons with different shifts are separated by their carbon

shift difference; while overlapping carbons may be separated

by their attachment to protons with differing chemical shifts.

Therefore, the apparent resolution of 2D spectra is much

better than anything that can be achieved in 1D spectrum [12].

Thus, quantitative measurement of various structures of

lignin is possible when appropriate standards or pulse se-

quences are applied [33,67]. The assignment for lignin corre-

lations comes from the extensive database of lignin model

compounds along with data from a long history of NMR of

both isolated and synthetic lignin [32,82e87]. The 2D-HSQC

experiments of non-acetylated lignin samples have been

valuable in assigning major structures (b-O-4, b-b, b-5, etc.) in

the lignin samples according to the previous studies and the

previously mentioned database of lignin model compounds

[80].

The HSQC approaches have also been valuable in assigning

major structures of non-acetylated lignin from different ori-

gins in recent years, such as some non-woody plants [32], Jute

fibres [82], bamboo [84,85,87], Triploid poplar [88e90] and

wheat straw [83]. HSQC spectra have been crucial in recog-

nising new and minor lignin structural units. The clear iden-

tification of dibenzodioxocins (5e5 linkages) as major new

structures in lignin has been a significant finding [91,92].

Evidence provided by 2D NMR is far more diagnostic than

1D data purely because of the simultaneous constraints that

are revealed in the data. Consequently, the observation that

there is a proton at 4.9 part per million (ppm) directly attached

to a carbon at 84.4 ppm and a proton at 4.1 ppm attached to a

carbon at 82.5 ppm is more revealing than just observing two

new carbons at 84.4 and 82.5 ppm in the 1D spectrum [92].

Zhang and Gellerstedt [33] presented a new analytical method

based on the 2D-HSQC NMR sequence and quantitative 13C

NMR, which can be applied for quantitative structural deter-

mination of complicated polymers, such as lignin and cellu-

lose derivatives. This method is a combination of HSQC and

quantitative 13C NMR techniques, which gives more reliable

data about the lignin linkages. A recent study showed that 2D-

HSQC NMR spectra of enzymatic hydrolysis residue (EHR)




Table 5 e H/S/G ratios for types of biomass resources.

Type Lignin (%) H S G Reference

Wheat straw 16e21 9 46 45 [109]

Rice straw 6 15 40 45 [109]

Rye straw 18 1 53 43 [110]

Hemp 8e13 9 40 51 [111]

Jute 15e26 2 62 36 [112]

Flax 21e34 4 29 67 [109]


delivered very well resolved spectra that can be compared to

that of MWL [93]. Based on the results obtained, it was found

that in situ characterisation of pre-treated biomass by HSQC

NMR analysis is a beneficial structural analysis methodology

in the emerging biomass research field for the characterisa-

tion of EHR.

Heteronuclear multiple quantum coherence (HMQC) or

HSQCspectraof ligninhavebeenreviewed[80]andwell reported

by previous researchers [4,74,75,78,94e98]. The assignments in

HMQC/HSQC spectra should be made with comprehensive

knowledge of both carbon and proton chemical shifts for each

structural type. 2D HSQC NMR technique is also useful in

determination of the structural characteristics of oxygenated

aliphatic region of lignin polymeric framework [32,99].

3.1.5. DFRC methodThe derivatization followed by reductive cleavage method

(DFRC)was developed by Lu and Ralph in 1997 as an alternative

to thioacidolysis [28,100]. Because of the mild conditions used

and its unique selectivity, theDFRCmethodhas becomewidely

used for characterising lignin from various origins [101e103].

One unique feature of the DFRCmethod is that esters on lignin

remain fully intact during the DFRC procedure [104]; therefore,

p-coumarates on lignin from grass plant materials can be

detected and measured [105]. With a few modifications to the

standard DFRC procedure by replacing the acetyl-containing

reagents (acetic acid and AcBr) with the propionyl analogues,

the modified DFRC method was applied to detect and quantify

the naturally occurring acetates on lignin from some plants

such as kenaf, aspen or other non-woody plants [106].

The DFRC procedure, as a basic component, has been

modified or combined with other techniques to provide more

precise and specific quantitative data about lignin structures

[107]. The combination of DFRCwith quantitative 31P NMRwas

shown to have significant potential for the determination of

arylglycerol-a-aryl ether and other linkage. Coupled with the

spread of advanced NMR techniques, during the past decade,

lignin structural enquiries have been greatly facilitated by the

development of various degradative protocols, such as DFRC

which efficiently cleaves the b-aryl ethers in lignin. DFRC is a

flexible method due to its three distinctly separated steps,

allowing modifications designed to the quantification of

different monomeric units and structures. On the basis of this

flexibility, Tohmura and Argyropoulos [108] proposed the

combination of DFRC with quantitative 31P NMR data.

3.1.6. H/S/G ratioQualitative or semi-qualitative indication of H/S/G ratio units

are not too informative and on the other hand they are very

laborious, lengthy and also prone to errors. Table 5 shows the

p-hydroxyphenylesyringyleguaiacyl (H/S/G) ratio for

different types of biomass which vary quite considerably

amongst each other.

Thioacidolysis is the most used analytical method for

lignin analysis to obtain information about uncondensed

structures (H/S/G ratios) [12]. Thioacidolysis was developed by

Lapierre, as an extension of acidolysis, to cleave b-aryl ethers

in lignin so that the basic units linked by b-aryl ethers are

released as monomers to be quantified by gas chromatog-

raphy [26]. Therefore, the yields of monomers released by


thioacidolysis reflect the proportion of uncondensed units in

lignin. When thioacidolysis is used for grass lignin, esters on

grass ligninmay decrease the efficiency of b-ether cleavage by

thioacidolysis [113,114]; hence this should be taken into

consideration when interpreting thioacidolysis results for

grasses.

Permanganate oxidation, nitrobenzene oxidation, GCeMS

pyrolysis, thioacidolysis and DFRC are degradative methods

that reveal theH/S/G composition of the lignin polymer. Itmust

be mentioned that degradative analytical techniques are

lengthy and complicated. All these methods release only a

fractionof the lignin for analysis, theyarebasedon the cleavage

of lignin backbone and analyses of the fragments obtained, and

provide partial data due to the specificity of the treatment.

The quantification of lignin H/S/G ratio has been reported

by 13C NMR and HSQC NMR analysis [115,116].

3.2. Qualitative analysis

Qualitative lignin analysis relies rather much on studies of

lignin degradation products; these methods only provide

preliminary information which could then be useful for the

quantification of lignin within straw.

3.2.1. SEM-EDXScanning electron microscopy (SEM) is used to examine the

microstructure, morphology, crystalline structure and size of

the straw. The microscope could be equipped with energy-

dispersive X-ray (EDX) analyser, which provides useful

element compositions and also information about the chem-

ical composition of the straw.

Despite its importance as imaging technique, SEM is not

capable of quantitatively evaluating the purity of typical lignin

sample. Moreover, there is no published algorithm to convert

such images into numerical data. SEM analysis also provides

information on the particle size and the surface properties of

straw. When the straw is being treated by means of chemical

ormechanical treatments, the particle sizesmight change and

the surface of straw might become smoother or rougher

depending on each step of the treatment.

Field emission scanning electronmicroscopy (FE-SEM) was

used by Koo et al. [117] in order to observe the change in lignin

distribution during pre-treatment; (Fig. 3), which is one of the

key applications of SEM technique used for qualitative anal-

ysis of biomass.

3.2.2. TEM and optical microscopyTransmission electron microscopy (TEM) has been proved to

be an effective tool for determining cell wall ultrastructure.




Fig. 3 e Droplets on surface of pre-treated biomass due to the isolation and migration of lignin (a: untreated biomass; b: pre-

treated at 120 �C; c: pre-treated at 130 �C; d: pre-treated at 140 �C) [117].


TEM observations deliver further details of lignification and

the distribution of lignin. Based on the intensity of the stain-

ing, the concentration of lignin in different morphological

regions of straw could also be qualitatively examined by TEM

micrograph, for example, Fig. 4 shows the distribution and

concentration of lignin under TEM. The dark staining of the

Fig. 4 e TEM micrograph of an ultrathin transverse section

of the cell wall of Forsythia suspensa fibre: S1 [ outer

secondary wall, S2 [ middle secondary wall and

S3 [ inner secondary wall [118].


cell corner middle lamella (CCML) and compound middle

lamella (CML) indicates that both of them are strongly ligni-

fied. Like SEM, TEM analysis will also be helpful in finding

particle size and surface properties of straw.

Optical microscopy is unable to provide the detailed fea-

tures of the materials under examination but is a good tool to

observe the lignin distribution within the straw. It is also a

quick and easy method to observe the overall structure of

straw and hence to observe the changes that may occur after

any treatments used on straw. The evaluation of straw

morphology for preliminary research can usually be carried

out using optical microscopy or light microscopy. Basic parts,

including epidermis, cortex, vascular bundle and pith of the

straw, can be observed, with the epidermis cells arranging in

parallel rows and being closely packed to protect the internal

parts of the plant, the cortex being the zone between

epidermis and the vascular bundles and containing collen-

chyma and parenchyma cells, the pith being the central part

of the stem which is composed of thin walled parenchyma

cells, and the vascular bundles in the stem of the grass plants

varying in number and size but having the same basic struc-

ture composed of xylem and phloem (Fig. 5).

4. Structure of straw lignin

Lignin monolignols are polymerised by a radical coupling

process that links them by carbonecarbon or ether bonds. A

linkagemay occur at any of several different locations on each

phenolic unit, causing many different linkage types to be




Fig. 5 e Light micrograph of cross section of grass stem,

showing four basic parts [23].


possible. The most common linkage types found in a lignin

molecule are b-O-4, a-O-4, b-5, 5e5, 4-O-5, b-1 and b-b

[119,120]. The ether type linkages dominate in native lignin,

estimated to make up one half to two thirds of the total

number of native plant lignin linkages. Monolignols form

branch points within the polymer giving a network-like

structure. Given the variety of linkages that occur, lignin

molecules cannot be depicted as a series of regular, defined

repeating units, as traditional polymers are. In contrast, lignin

is a highly irregular complex polymer [119,120].

Lignin is one of major cell wall component of herbaceous

crops. The composition and structure of lignin vary consid-

erably within and among plants; Lignin has been classified

into three groups: Gymnosperm (softwood), Angiosperm

(hardwood) and Grass lignin. However, it was recently found

that these categories are inadequate because of ignoringmost

of the herbaceous angiosperm lignin and some conifer fam-

ilies containing guaiacyl and syringyl types of lignin. It was

suggested by Gibbes that lignin should be categorised into two

groups: guaiacyl lignin and guaiacylesyringyl lignin according

to their overall chemical constitutions [12]. Each type also

generally contains a small proportion of p-coumaryl units,

though grass lignin generally contains a higher amount of p-

coumaryl units than woody lignins [121]. Other phenolic

monolignols have been identified, but they generally make up

a much smaller portion of the lignin molecule [122]. Other

phenolic compounds (alcohols, aldehydes, acids, esters and

amides) have been reported to act as lignin precursors and

illustrate the structural ‘‘plasticity” of the polymer and the

adaptability of the lignification mechanisms in plants

[123,124], but several of these compounds (such as p-hydrox-

ycinnamaldehydes, ferulic acid or 5-hydroxyconiferyl alcohol)

only provide a significant contribution to lignin in transgenic

plants with modified monolignol biosynthesis [124] and are

minor lignin precursors in normal plants [125]. On the other

hand, some phenolic compounds with saturated or no side-

chains, e.g. dihydrocinnamyl alcohol, sporadically incorpo-

rate to the lignin polymer as terminal units as confirmed by 2D

NMR [126,127].

The solubility of straw lignin in alkali has been attributed

mainly to the presence of significant amounts of p-hydrox-

yphenyl residues, which are bound to lignin as p-coumarate

units [128]. Ferulic and p-coumaric acids in grasses are known


to be implicated in the cross-linking of cell wall carbohydrates

with lignin [129]. Obviously such cross-linking can have a

dramatic influence on the mechanical properties and biode-

gradability of straw [4].

All grasses have lignin that is acylated by p-coumaric acid.

Early attempts at establishing the acylation regiochemistry

(the site on lignin where the acylation occurs) utilised UV and

concluded that p-coumarates were mainly at the g-position of

lignin side chains [130]. NMR work on corn lignin, subse-

quently on wheat [4] and other grasses showed that the

acylation was exclusively at the g-position, implicating

enzymatic processes in the formation of the ester. Thio-

acidolysis confirmed the presence of g-acylation, although

esters are partially cleaved [113]. The DFRC method, which

leaves such g-esters intact, further established the g-acylation

and, as for acetates, indicated that p-coumarates were pre-

dominantly on syringyl units [105].

4.1. LCC linkages

Lignin is always associated with carbohydrates (in particular

with hemicelluloses) via covalent bonds at two sites: ⍺-carbon

and C-4 in the benzene ring, and this association is called

lignin carbohydrate complexes (LCC). The major inter-unit

linkages within the lignin monomer (H, S, and G) are b-O-4,

b-b, b-5, b-1 arrangements. The chemical linkages limit the

efficient separation of lignin from plant cell wall. Thus, it is

important to understand the LCC linkages of lignin samples.

Four types of linkages between lignin and carbohydrates

have been suggested: glycosidic linkages, ester linkages,

benzyl ether linkages and hemiacetal or acetal linkages [12].

Among these linkages, the possibilities of glycosidic, benzyl

ether and ester linkages have been demonstrated with model

compounds [13]. The association of lignin and carbohydrates

in grass cell walls is largely through free radical coupling of

ferulates or diferulates with monolignols or growing lignin

oligomers. Due to the complexity of LCCs, the isolation of

homogeneous preparation is the most important step for the

following compositional analysis and structural characteri-

sation. Many isolation methods for LCC have been tried, but

the finely divided plant meal [131,132] and MWL [133] were

preferred sources for LCC isolation because any chemical or

biochemical treatment before LCC isolation would break

possible linkages between lignin and carbohydrates.

The cross-linking of lignin and carbohydrates via ferulates/

diferulates in grass cell walls has been demonstrated [12]; it is

generally accepted that the cross-linking of carbohydrates

and lignin by ferulates presents the greatest barrier to efficient

utilisation of grass cell wall [113].

4.2. Physical properties

The physicochemical properties of straw lignin are known to

be different from those of softwoods or hardwoods, with

straw lignin possessing characteristic alkali solubility. The

physicochemical state of lignin dictates how and where it can

be utilised in the production of various products. The source

from which lignin is obtained and the method of extraction

has a strong bearing on its properties [16]. As lignin from

different crops or treatment can be extremely diverse in





structure and hence physical properties, it is necessary to

determine these differences through analytical methods. The

reactivity and physicochemical properties of lignin are

dependent to certain extent on their molecular weight distri-

bution (Table 6). The reactivity of lignin will impact on the

attributes of the end products. For example, Muller and

Glasser [134] found that kraft lignin-based phenol formalde-

hyde resins have superior properties to steam exploded

lignin-based phenol formaldehyde resins. Methods for lignin

molecular weight determinations are developed and

described by previous researchers [135,136]; gel permeation

chromatography (GPC) involves the chromatographic frac-

tionation of macromolecules according to molecular size.

The technique of fractionating macromolecules on Sephadex

gel has been applied to lignin materials since early 1960s

[137,138].

Another important parameter is the glass transition tem-

perature, Tg, which is an indirect measure of crystallinity and

degree of crosslinking and directly indicates the rubbery re-

gion of the material [139]. Lignin cross-linking degree will

depend on the quantity of water and polysaccharides, as well

as molecular weight and chemical functionalisation. Lignin

cross-linking degree generally increases with increasing mo-

lecular weight.

4.3. Lignin distribution and concentration

Lignin’s properties are significantly different with carbohy-

drates such as activity and solubility, according to which the

distribution of lignin in plant cell wall can be analysed. Many

techniques have been applied to the study of distribution of

lignin and organisation of cellulose components. One of the

oldest procedures is selective staining which follows by the

study under light microscope and another method to study

lignin distribution is electron microscopy of lignin skeleton

[140]. Although thementionedmethods are useful in studying

lignin distribution, only qualitative or semi-quantitative data

of lignin in various morphological regions can be provided.

The studies that focused on quantitative data of lignin dis-

tribution in the secondary wall and compoundmiddle lamella

(CML) have been carried out by using UVmicroscopy [141]. UV

absorption is a tool used for lignin identification because

Table 6 e Molecular weight and functional groups oflignin [1].

Lignin type Mn (g mol-1) COOH(%)

OHphenolic

(%)

Methoxy(%)

Soda

(bagasse)

2160 13.6 5.1 10.0

Organosolv

(bagasse)

2000 7.7 3.4 15.1

Soda (wheat

straw)

1700 7.2 2.6 16

Organosolv

(hardwood)

800 3.6 3.7 19

Kraft

(softwood)

3000 4.1 2.6 14


lignin absorption occurs at 280 nm. The weight concentration

of lignin was estimated to be 16% and 73% for the secondary

wall and CML of Norway spruce tracheids respectively [142].

Lange and Kjaer [143] proposed interferencemicroscopy based

on the pathways of rays which is another method for quan-

titative analyses of lignin distribution within the cell wall

layers. With the development of electron dispersive X-ray

analysis (EDXA) combined with SEM and TEM, the interfer-

ence microscopy in conjunction with EDXA was applied to

determine lignin distribution in wood and straw, in differen-

tiating xylem and in chips during pulping [144,145]. The

technique is fully quantitative and precise. Donaldson [146]

used confocal laser scanning microscopy (CLSM) to study

lignin distribution in agricultural fibres.

Although many chemical studies of grass lignin have been

carried out, there is still little quantitative data on lignin dis-

tribution in grass species. Zhai and Lee [22] used SEM-EDXA

technique to determine the Br-L X-ray counts of the

different brominated wheat straw cell which are presented in

Table 7. In fibre and non-fibre cells of wheat straw, the lignin

concentration is the highest in the cell corners (CC) and the

lowest in the secondary wall. In the morphological regions of

all cells, the lignin concentration is the highest in parenchyma

cells, followed by fibres, and is the lowest in epidermis cell

[22]. Interference microscopy [144] and CLSM [147] were also

used by researchers to examine the lignin concentration of

wheat straw in the ML and in the secondary wall.

4.4. Lignin functional groups and their characteristics

The key chemical functional groups in lignin are the hydroxyl,

methoxyl, carbonyl and carboxylic groups. The quantity of

these groups depends on the genetic origin and isolation

processes adapted. Functional group characterisation can be

used to determine the lignin structure. Various analytical

methods for determining functional groups in technical lignin

have been studied, while the statistical comparison of the

various analytical methods for hydroxyl groups have been

found not fully comparable [148], the aminolysis and non-

aqueous potentiometry are assumed to be the most reliable

for phenolic hydroxyl and for determining the total carbonyl,

and the oximating method was found the most reliable

method which was first described by Faix et al. [149]. The

Table 7 e Lignin concentration and distribution in wheatstraw [22].

Cells S CML CC

Br-L X-ray counts

Thick wall fibre 1620 2156 2992

Thin wall fibre 1340 2004 3336

Parenchyma cell 2005 2599 e

Epidermis cell 1022 1690 1743

Lignin concentration (g g�1)

Thick wall fibre 0.168 0.412 0.571

Thin wall fibre 0.154 0.339 0.664

Lignin distribution (%)

Thick wall fibre 67.5 18.0 14.5

Thin wall fibre 57.5 20.3 22.22





phenolic hydroxyl group is a significant functionality affecting

the physical and chemical properties of lignin polymer.

Moreover, the chemical reactivity of lignin in various modifi-

cation processes is influenced by its phenolic hydroxyl con-

tent which is significant in the reaction with formaldehyde for

the production of lignin adhesive [150]. Lai described a pro-

cedure to determine free phenolic hydroxyl groups in lignin

[151]. Commonphysicalmethod used to estimate the phenolic

hydroxyl content of lignin is UV spectroscopy, which is based

on difference in absorption of phenolic units in neutral and

alkaline solutions [152].

Methoxyl groups (eOCH3) are found in lignin from all

plants. The amount of the groups depends on plants species

and on the method of isolation. The amount of the methoxyl

groups is often used as a measure of the purity of lignin

preparation, since the isolated lignin sometimes is contami-

nated with hydrocarbons. The classical method for methoxyl

determination of lignin uses hydroiodic acid to promote

demethylation and gas chromatography to determine the

percentage methoxyl. Alternative method, which is less

tedious, involves the use of proton nuclear magnetic reso-

nance (1H NMR) [153].

There are the primary aliphatic hydroxyl groups bonded to

the g-C-atom, secondary aliphatic hydroxyl groups bonded to

the ⍺-C-atom and phenolic hydroxyl groups bonded to the 4-

C-atom of the aromatic ring in lignin. The accurate determi-

nation is difficult since they have very similar chemical

properties and reactivity. The amount of total hydroxyl group

in lignin was determined by potentiometry [154].

Natural lignin contains a low concentration of COOH-

groups. However, when native lignin is subjected to chemi-

cal or biological treatments, carboxyl groups are frequently

detected in significant quantities, hence quantitative mea-

surements of carboxyl groups may provide information

regarding the degree to which the lignin has been degraded or

modified as a result of treatment. Further carboxyl groups are

produced during delignification as a result of oxidation of

hydroxyl and carbonyl groups. Oxidation of the lignin struc-

ture will result in an increase of carboxyl absorption in the FT-

IR spectra as it was observed by Xu et al. [7]. In work on lignin

Fig. 6 e 31P NMR of wheat straw lignin phosphitylated with 2


characterisation by Sahoo et al. [15], FT-IR analysis pointed

out the differences in the bonding pattern and the functional

groups. These different chemical functionalities are very

important components when it comes to utilising lignin in

biomass for composite applications, the final properties of the

composites.

5. Wheat and rice straw lignincharacteristics

Researchers have focused on the structural analysis of wheat

and rice straw by using a combination of qualitative and

quantitative techniques in order to either, optimise these

renewable raw materials for industrial applications or to

investigate the changes within the composition of straw

biomass after a certain treatments. Most of the researches in

this area are comparative studies between different types of

biomass to establish the structural differences between them.

Xu et al. [7] in a comparative study examined the chemical

characteristics of lignin in order to get more information on

chemical structures and relationship between physical prop-

erties and chemical structures. The analytical quantitative

techniques such as FT-IR and 1H and 13C NMR spectroscopy

were used to investigate the changes occurring in lignin

structure during the organosolv treatment processes. 1H and13C NMR spectra revealed that the lignin comprised guaiacyl,

syringyl and a small amount of p-hydroxyphenyl units. The

various covalent linkages and physicochemical interactions

between lignin, cellulose, hemicelluloses, phenolic acids and

other polyphenolic or proteinaceous or mineral extractives all

contribute structural integrity to the wheat straw cell wall

composite [155].

Recently, an in-situ quantitative 2D-HSQC NMR technique

(the ball-milled plant cell wall was dissolved in DMSO-d6) was

used for characterising the changes in the cell wall during the

hydrothermal pre-treatment (195 �C for 6 min) process of

wheat straw for second-generation bioethanol production

[156]. This study provides an effective quantitative method to

reveal the structural changes of cell wall components based

-chloro-4, 4, 5, 5-tetramethyl-1, 3, 2 dioxaphospholane.





on in situ 2D-HSQC NMR techniques. The results revealed

substantial lignin b-aryl ether cleavage, deacetylation via

cleavage of the natural acetates at the 2-O- and 3-O-positions

of xylan, and uronic acid depletion via cleavage of the (1/2)-

linked 4-O-methyl-a-D-glucuronic acid of xylan after hydro-

thermal treatment.

By combining mild alkaline hydrolysis with quantitative31P NMR, Crestini and Argyropoulos [4] have been able to

arrive at a protocol for determining the various ester linkages

and their relative contributions to the overall structure of

wheat straw lignin; their technique has been applied on

samples of milled wheat straw (ML), dioxane acidolysis wheat

straw lignin (AL), and a milled wood lignin from black spruce

(BSL). A typical spectrum together with the detailed signal

assignment can be obtained (based on earlier efforts of

Granata and Argyropoulos [17] and Jiang et al. [18]) (Fig. 6).

Rice straw has high lignin and low nitrogen contents which

in combination are responsible for its low nutritive value.

Researchers in recent years have attempted to improve its

nutritive value by chemical treatments [157]. Rice straw is

highly heterogeneous and complex, and its fractionation and

utilisation should be conducted on different levels according

to structural properties. Dohnani et al. [158] evaluated the

variability in chemical and morphological variables of Euro-

pean rice straw in their work with the aim to relate these

variations to changes in “in sacco” degradation. In their work

fifteen rice straw varieties are analysed for their chemical and

morphological compositions, the results indicated that both

chemical and morphological characteristics have great vari-

ability. For instance the ash content of rice straw samples

ranged from 9.6 to 14.1% which are very low compared to the

reported ash content (averaged 14.1%) in Philippine varieties

[159], (16.6%) in Asian varieties [160], and (18.6%) in Californian

varieties [161]. NIR spectroscopy was used for determination

of rice straw analytical parameters such as total ash, mois-

ture, cellulose, hemicellulose and lignin; where the chemical

composition content varied among different rice straw sam-

ples (Table 8). This resulted from the different rice straw va-

rieties, the various tissue parts and the different origins [162].

Thermal gravimetric analysis and differential thermal

behaviour of rice straw were investigated [163], where the

degradation was found to be of first order reaction and hemi-

cellulose was found to be less stable than cellulose. The study

of structural features and physicochemical and thermal prop-

erties of the lignin, isolated with 1 M NaOH at 30 �C for 18 h

from de-waxed rice straw, maize stems and rye straw using a

combination of several destructive and non-destructive

Table 8 e Chemical composition of rice straw [162].

Component Range (%) Average (%)a

Moisture 4.2e9.8 6.9

Hemicellulose 19.8e31.6 25.6

Cellulose 30.3e38.2 33.9

Klason lignin 7.2e12.8 10.2

Acid insoluble ash 3.8e13.2 7.8

Total ash 7.8e15.6 11.8

a Average of 34 samples.


techniques, showed the treatment with 1 M NaOH at 30 �C for

18 h can significantly cleave the ether linkages between lignin

and hemicelluloses from the cell walls of the straws and stems

[6]. The hemicelluloses of rice straw can substantially be

decomposed between 200 and 300 �C and above 245 �C the

decomposition of cellulose starts to take place reaching to a

maximum peak around 300 �C and yielding mainly volatile

products [6]. The decomposition process of lignin covered a

larger temperature range, between 250 and 600 �C, however at

temperatures lower than 400 �C only 40% was decomposed,

rendering charcoal as the residue product. This result implied

that hemicelluloses degraded in first place, while lignin

showed less degradation, and therefore, its structurewasmore

stable. Cellulose, on the other hand, showed an important

degradation process, mainly between 250 and 330 �C.

6. Conclusions

The analytical techniques up to date for the structural analysis

of straw lignin were reviewed and the resultant un-

derstandings of the structure of straw ligninwere summarised.

The studies on lignin could be divided into the qualitative

and quantitative analyses, the former includes SEM-EDX,

TEM, optical microscopy and the latter includes FT-IR, TGA/

DSC, NMR and DFRC. Different analytical methods applied to

same lignin samples could provide significantly different re-

sults that are not directly comparable. This problem, coupled

with the extreme heterogeneity of lignin and its chemical

structure among different crop species, morphology and

maturation degree has made to date the quantitative struc-

tural characterisation of lignin an open debate. More sensitive

and reliable quantitative HSQC techniques are the most

diagnostic and accurate analytical tools to investigate lignin

structure, among them the magnetic resonance techniques

when applied to lignin have proved to be efficient analytical

tools for the structural elucidation of these complex bio-

polymers. These NMR techniques are capable of detecting and

quantitatively determining all functional groups in lignin

possessing labile hydroxyl groups, i.e. aliphatic OH, the

various forms of phenolic OH and carboxylic acids.

The composition and structure of straw lignin varies

considerably within and among plants. The nature of lignin

carbohydrate linkages and occurrence is not completely clear

and the isolation processes could affect the lignin structure.

Because of the complexity of the chemical composition of

lignin in straw, it is difficult to find a single technique to

analyse its structure, hence themost accurateway to study the

straw lignin could be the combination of several techniques,

each providing partial but complimentary information.

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Structural analysis for lignin characteristics in biomass ...€¦ · Hot-water soluble 14.6 7.2 18.9 13.2 Lignin 15.3 14.2 14.1 16.7 Hemicellulose 32.4 33.8 34.2 32.7 Cellulose 37.7