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Stellenbosch University has received national and international recognition in research on the production of liquid bio-fuels from lignocellulose. The Forest and Wood Science department has vast expertise in the characterisation, processing and product performance testing of wood and wood based products. The department therefore plays a significant role in the development of protocols and advanced analytical techniques for characterisation of raw materials, intermediate and final products in all types of processing. Current research in the department as hosted by the bio-fuels research chair involves the isolation and characterisation of lignin-carbohydrate complexes (LCCs). The existence of covalent bonds between lignin and carbohydrates is of considerable interest in connection with a number of issues in wood chemistry, such as the reactions taking place during the formation of wood, the natural molecular weight distribution of lignin and carbohydrates, swelling and accessibility properties and the reactivity of wood during its processing, e.g., during chemical pulping or bio-processing into ethanol. The research is much more fundamental in nature and yet more advanced as among other things, it seeks to understand the changes that take place not only in the lignin carbohydrate bonds but also in lignin-lignin bonds as affected by chemical or bio-processing of biomass.
Recently, the Paper Manufacturers' Association of South Africa (PAMSA) has recognized a critical need for both technology and human resources development to ensure the economic sustainability of this important industry in South Africa, to improve the industry's international competitiveness, and to support developing players in the local industry. In particular, the scarcity of skilled technical staff has become a significant threat to the SA industry, with a shortage of process engineers with research experience in pulp-and-paper processing being the most urgent. Such technical staff will become key process innovators and problem solvers in the industry. At the same time, new technologies are required by the industry to improve current processes and develop new industries around pulp mills to improve economic value added to the feedstock. Key areas of technology and process development required are fiber processing and bio-refining, recycling of fibers, fiber engineering and improvement in the overall energy consumption and associated environmental impact of pulp mills. These objectives can only be met through research capacity building at universities; a vision shared by PAMSA.
The proposed study forms part of the biomass process development program, which considered the application of world-leading technologies for bio-ethanol and pulp production. The work done will contribute critical components of biomass characterisation before, during and after processing. The proposed study identifies critical mechanisms in the development of biomass to ethanol and pulping processes. Aspects of chemical characterisation of the substrate are integrated into research on pre-extraction of hemicelluloses, pulping and or biomass pre-treatment and enzymatic hydrolysis process development, to demonstrate the importance of advanced chemical characterisation of substrates for process development. The proposed work plays a leading role in strengthening the integration of research activities in various sections of the cellulosic-ethanol and pulping value chain.
Scope of the study
This work is aimed in developing protocols and advanced analytical techniques for characterisation of woody biomass raw materials, intermediate and final products in all types of processing, which will focus in particular on the isolation and characterisation of lignin-carbohydrate complexes (LCCs) in order to obtain relevant information on biomass formation and on the delignification process from an LCC perspective.
Investigate changes in lignin carbohydrate complexes when the biomaterials are subjected to pre-treatment and later to pulp samples. This will give knowledge about the ultra-structure of wood for making chemical pulp as the study was developed for softwood biomass materials. The method for the isolation of lignin carbohydrate complexes has been developed for softwoods (Lawoko et al., 2003). In this study the application of the method will be employed together with an inorganic method to have comparable results based on different methods.
Linkages between lignin and carbohydrates have been suggested to be a major obstacle to complete delignification of biomass feedstocks. Covalent lignin-carbohydrate linkages exist in lignocellulose from wood and groups of herbaceous plants although ambiguity in the types, frequency and quantity exist. Xylan-linked lignin is more resistant to oxidative reactions and tends to remain in the biomass, while galactan-linked lignin tends to dissolve during oxygen delignification. The reduction of reactive phenolic and carboxyl groups during delignification is also responsible for the low level of subsequent delignification. In addition, the location and accessibility of residual lignin may also have a significant effect on delignification. An understanding of the structure and composition of lignin carbohydrate complexes (LCCs) of Eucalyptus grandis and sugarcane bagasse prior to and after Kraft pulping could be developed through experimental data collection. In this way, the pulping process conditions can be tailored to the specific chemical and structural composition of the Eucalyptus grandis or bagasse. This in turn can be modelled for other lignocellulosic biomass feedstocks such as bamboo, sorghum etc. In addition, even more importantly, the effect of hemicelluloses pre-extraction on LCCs bonds prior to and after pulping of Eucalyptus grandis can be established. The data collected will be of great significance in the later stages of the pulp processing such as bleaching. The analytical techniques developed for isolation and analysis of LCCs will not only be important for Kraft pulping but for other biomass application processes such as the hydrolysis of biomass for bio-ethanol production and as such, this study can serve as a model for such applications.
Experimental time plan
The LCC isolation procedure chosen is non-modifying to the fibres and the fractionation components are obtained in high yields unlike previous procedures. In addition, they are nearly free from contamination. Ball milling is an integral part of this procedure. Therefore, optimum ball milling conditions have to be established in order to obtain intact LCC networks from biomaterials and their products. The optimization stage could not be performed previous year as the ball mill was hindering due to its unavailability. The optimization step is to be followed by chemical characterization of LCC structures using advanced analytical procedures such as NMR, HPLC and GC-MS. Previous studies show that glucomannan-rich LCC fraction (GlcMan-L-Xyl) is relatively more stable than the other LCC types toward the end of Kraft cook.
Table 1: Detailed activity time plan
March - May 2011
Redo chemical composition of samples both Eucalyptus grandis and sugarcane baggase
Book FTIR to characterise the chemical bonds before the lignin carbohydrate complexes (LCCs) are isolated.
Submit first year progress report to Process Engineering Department and PAMSA
Submit chapter one based on the chemical composition of the samples
May - June 2011
Establish conditions of ball milling samples from raw materials to pulp samples
Acetylation will been done to determine the molecular mass distribution of the lignin samples
May - August 2011
Start the isolation of LCCs from Eucalyptus grandis
Do thioacidolysis to quantify the β-O-4 bonds
Characterise the samples with FTIR after the LCCs have been isolated
Submit chapter two, based on the literature review
Acetylation will been done to determine the molecular mass distribution of the lignin samples after the LCCs have been isolated
August - September 2011
Start isolation of LCCs from sugarcane baggase samples
Characterise the samples after the LCC isolation stage
Submit chapter three based on the methodology of the study
Submit chapter 4A based on the results and discussions of the study for E. grandis
Write research paper based on the findings for the isolation of LCCs from hardwoods
Submit chapter 4B based on results and discussions for sugarcane bagasse
November 2011 - January 2012
Rework on isolation and characterisation of LCCs for conclusive results
Write exams (WPS144)
February and March 2012
Submission of thesis and corrections
Materials and Preliminary results
The Eucalyptus grandis that is used in this study was supplied by Sappi Ltd, the sugarcane bagasse was from industrial bagasse. The pulp and pre-treated materials were supplied by a PhD student (Ms PF Vena) at the University of Stellenbosch.
The process of converting renewable lignocellulosic biomass to ethanol requires a number of steps, and pre-treatment is one of the most important. Pre-treatment usually involves a hydrolysis of the easily hydrolyzed hemi-cellulosic component of biomass using some form of thermal/chemical/mechanical action that results in a product that can be further hydrolyzed by cellulase enzymes. The sugars produced can then be fermented to ethanol by fermentative microorganisms. If the pre-treatment step is not severe enough, the resultant residue is not as easily hydrolyzed by the cellulase enzyme. More severe pre-treatment conditions result in the production of degradation products that are toxic to the fermentative microorganism.
The materials used in the study are Eucalyptus grandis and sugarcane bagasse with their corresponding chemically treated (alkaline and dilute acid pre-treatment, Kraft and soda AQ) materials.
Chemical analysis of sugarcane bagasse and E. grandis raw materials
The modified Klason lignin method was used (NREL). Samples of 0.3 g were treated with 3 mL of 72 % H2SO4. After an hour of continues stirring at 30 °C in a water bath, 81 mL of water was added to the mixture , which was post hydrolysed under 121 kPa for an hour. The product was filtered and the insoluble lignin (Klason lignin) was quantified by weight. The hydrolysate was analysed by high performance liquid chromatography (HPLC).
Table 2: Chemical composition of E. grandis and sugarcane bagasse.
2.59 ± 1.17
0.18 ± 0.08
10.89 ± 1.90
24.7 ± 1.69
39.3 ± 0.12
13.8 ± 0.29
44.38 ± 0.20
7.0 ± 0.05
1.60 ± 0.03
16.7 ± 0.05
26.4 ± 0.96
46.1 ± 0.22
13.6 ± 0.21
49.16 ± 0.20
The sugarcane bagasse and Eucalyptus grandis analysis is presented in Table 2. The amount of ash is lower than that mentioned by Pate (1982), owing perhaps to the influence of different factors on sugarcane cultivation and processing. It also shows that 13.6 % is composed of xylose and 46.1 % is composed of glucose. This difference in reducing and fermentable sugars could be ascribed to the presence in the hemicelluloses of arabinose, mannose and oligomers like celotriose, originating in incomplete molecule hydrolysis. It could also be ascribed to the presence of compounds like galactose, 4-o-methyglucuronic acid and aldobiuronic acid, which were not detected by High Performance Liquid Chromatography (HPLC). Similar differences were also found by Roberto et al. (1994); Fox et al. 1984; Morjanoff and Gray (1987).
The hydrolysis of the hemicelluloses fraction during acid pre-treatment involves solubilization and partial destruction of the reducing sugar produced. As a consequence, the amount of reducing sugar recovered from the bagasse depends on treatment time, temperature and acid concentration. The time intervals necessary for heating and cooling were not considered here.
Several Kraft and soda AQ pulp samples from Eucalyptus grandis and sugarcane bagasse were used in this study. The samples were alkaline and acid pre- extracted. Eucalyptus grandis and sugarcane bagasse samples were subjected to following chemical treatments:
Table 3: Pulping conditions and resulting Kappa numbers
E. grandisKraft puling
2M NaOH at 40 °C for 240 minutes
E. grandissoda AQ pulping
0.3 (%v/v) at 140 °C for 20 minutes
SCB auto hydrolysis
SCB soda AQ pulping
1.5M NaOH at 65 °C for 240 minutes
Kraft pulping was done on sugarcane bagasse, but the generated pulp was not in a standard of producing papers. Therefore, the method was discarded and soda AQ was one of the pulping conditions for the biomaterial. Soda AQ pulping was done on dilute pre-extracted E. grandis samples while Kraft pulping was done on alkaline pre-extracted biomaterial.
Table 4: Chemical composition of pulp samples
Total Lignin (%)
E. grandisraw untrd pulp
E. grandisRM- Kraft pulp
E. grandisdil acid- Kraft
E. grandisRM- soda AQ
E. grandisdil acid- soda AQ
SCB RM - soda AQ
SCB alkaline - soda AQ
SCB dil acid - soda AQ pulp
(E. grandis = Eucalyptus grandis; RM = raw material; SCB = sugarcane bagasse; dil = dilute; untrd = untreated)
The hemicelluloses are varying in yields. The acid hydrolysis to determine monosaccharide composition was analysed by HPLC. It is evident that the total composition of total carbohydrates is very low as the table indicates that the materials contained more xylose and glucose than arabionose. The analysis has to be re-done as the yields are very low. The low yield may be due to analytical error. According to literature, the glucose yiled should be in the range of 50-70 %. For acid and alkaline treated materials, xylose yields should be in the range of 75-90 and 60-75 % respectively (Hamelinck et al., 2005). The increase in fermentable sugars is due to the high lignin content removed during chemical treatment.
Relative distribution of guaiacyl-type degradation has to be done in order to compare the samples with Kraft pulped samples. This is for the determination of differences in the formation of guaiacol from hemicelluloses. It is important to do the analysis as it will also assist in determining the changes that occur within the lignin-carbohydrate bonds.
FTIR spectroscopy of raw; pre-treated and pulp samples of sugarcane bagasse and E. grandis
FTIR spectra of samples were obtained directly from untreated, pre-treated and pulp samples utilising diffuse reflectance infrared with Fourier transform technique (Perkin Elmer - Spectrum GX). The spectra were normalised by the absorption at 900 - 2000 cm-1, after baseline correction.
Figure 2: Normalised FTIR spectra of raw, pre-treated and pulp samples of sugarcane bagasse and Eucalyptus grandis.
The effects of chemical treatment on samples (raw, pre-treated and pulps) were assessed by FTIR combined with PCA. The FTIR normalised spectra of different samples in the region of 800-2000cm-1 are presented in Figure. It was observed that there was significant difference among samples. The changes were assessed by PCA. The PCA score plot of the matrix containing spectral data showed that the samples were significantly different which were clearly separated by PC1. This PC described 69 % of variation in the data set.
Principal component analysis of the FTIR spectra
Spectra of the samples were converted to excel using OMNIC software. The normalised absorbances in the range of 900 - 2000 cm-1 were submitted to principal component analysis (PCA) calculations using STATISTICA 10.
Principal component analysis (PCA) groups samples according to their similarities and differences. It provides information on the mass spectral features that are basis of chemical similarities and differences. It allows for easy distinction of samples that have been subjected to a chemical treatment from raw, pre-treated and pulp samples with high concentration of one component from samples with low concentration of a particular component.
According to PCA, component 1 differs significantly from component 2. Component 1 and 2 are 69 and 14 % different respectively. They have a sum square of 83 % (sum square - A statistical technique used in regression analysis. The sum of squares is a mathematical approach to determining the dispersion of data points. In a regression analysis, the goal is to determine how well a data series can be fitted to a function which might help to explain how the data series was generated. The sum of squares is used as a mathematical way to find the function which best fits (varies least) from the data. In order to determine the sum of squares the distance between each data point and the line of best fit is squared and then all of the squares are summed up. The line of best fit will minimize this value). The other components have very little difference which is 17 %. The score plot demonstrates that samples 1,4,5,11,15 and16 are very different from other samples. Samples 2, 3, 6,7,8,9, 10, 12,13,14,17, 18 and 19, have very little difference.
Figure 3: Score plot for raw, pre-treated and pulp samples from different biomass materials. The spectra were previously normalised.
The samples were grouped into 3 groups A, B and C. The grouping was based on the lignin content that each sample contains. Samples in group A had low response when they were subjected to chemical treatment in losing and degrading lignin as they fall in the negative region of the axis. Group C consists of samples that had high loss and degradation of lignin whilst in group B there was no to little change in lignin content.
Figure 4: Scatter plot of multiple variables against variable number.
The loads plot using PC versus variable number (wave number converted to variable number) gave a spectrum of data set in Figure. This gave information on the wave numbers that contributed to the clear separation of samples as in accordance to PC1 values. Characteristic peaks of lignin (C=C of aromatic at 1595 cm-1,1550 cm-1 and 1500 cm-1; C-O-C of aryl ether at 1225 cm-1 and 1263 cm-1, C-O-C of allyl ether at 1137 cm-1 and 1188 cm-1; C-H of aromatic ring at 820 cm-1, 850 cm-1 and 930 cm-1) dominated the spectrum. The peaks of cellulose and hemicelluloses (C-O of 1st alcohol at the range of 1038 cm-1, C-O of 2nd alcohol at the range of 1088 cm-1 seemed to be unaffected by the chemical treatments in all the samples. Components 1 and 2 are very different in the region of lignin this is due to the fact that lignin was degraded when the samples were subjected to different chemical treatment processing methods (pre-treatment and pulping).
Alternative method for the isolation of LCC
Figure 5: Schematic representation of an alternative method for the isolation of LCCs from E. grandis (Li et al., 2011).
There is a possibility of investigating the changes in lignin carbohydrate complexes when a material is subjected to pre-treatment. The hydrolysate can be analysed as the xylan will be isolated by dilute acid or alkaline pre-treatment method. The main aim is to have only lignin and carbohydrates on the material. The purpose of isolating cellulose is to minimize the number of carbon atoms when they material is analysed by NMR and FTIR. The expected peaks are of xylose backbone.
The method that was developed by Lawoko et al., 2003 for the isolation of LCCs was for softwood species. The method is said not to be suitable for hardwood biomass material. According to literature, there have been many arguments for the isolation of LCCs from hardwoods with enzymes. The isolation of lignin by enzymatic hydrolysis is accompanied by minimal structural changes. The literature also says that, the presence of carbohydrates in preparations makes the analysis of lignin more complicated, but on the other hand it allows investigation of lignin-carbohydrate (LC) linkages in pulps (Minor 1986; Tamminen and Hortling 1999). However, enzymatic residual lignin usually contains some protein contaminants, which can affect the characterization of lignin particularly by spectroscopic techniques. An approach using a combination of enzymatic hydrolysis with mild acidolysis has recently been suggested (Argyropoulos et al. 2002). This method inherited both advantages and shortcomings of enzymatic hydrolysis and acidolysis (JÓ“Ó“skelÓ“inen et al. 2003).
Isolation of enzymatic residual lignin from softwood Kraft pulps is a well-established procedure producing lignin preparations with high yields and relatively low enzyme impurities (Tamminen and Hortling 1999). In contrast, the isolation of enzymatic residual lignin from hardwood pulps is not well developed. There are very few publications on the isolation of residual lignin from hardwood pulps, and all of them report relatively low yields of residual lignin and the presence of rather high amounts of protein contaminations (Tamminen et al. 1999 and Choi and Faix 1999). When the enzyme isolation method is used on hardwoods, it fails to yield quantitative recovery of lignin either present in LCCs or as lignin-free carbohydrates.
An alternative method for the isolation of LCCs from hardwoods has recently been developed by Li et al., 2011. The method permits complete dissolution of wood and pulp samples in the course of which subsequent fractionation into individual LCCs is possible. The samples are subjected to ball milling to destroy the crystalline structure of cellulose without affecting the structure of lignin. Once the amorphous structure is obtained, the material is treated with various inorganic solvents.
Table 5: Summary table for methodology of the study
Chemical composition analysis of E. grandisand sugarcane bagasse
The amount of ash is lower than thatmentioned in literature
FTIR analysis of biomaterials
The materials show significant differences when analysed by PCA.
Isolation of lignin carbohydrate complexes from raw biomaterials
This is important to understand the composition of LCCs before a material is subjected to chemical treatment
Isolation of LCC from processed (pre-treatment and pulping) biomaterial
This serves for the understanding of biomaterials how they change when they have been treated
FTIR and NMR analysis of processed biomaterial
This is to determine the chemical changes within the biomaterial structure
Characterization of biomaterial before and after processing by thioacidolysis
The method is for quantifying β-O-4 linkages
According to FTIR-PCA analysis of the samples, they showed that lignin was removed when the biomass materials were subjected to different chemical processing. The lignin that remained will be further analysed to determine its association with hemicelluloses as the literature elaborates.
Argyropoulus, D.S., Sun, Y. and Paluš , E. (2002). Isolation of residual lignin in high yield and purity. J. Pulp Pap. Sci. 28:50-54. Browning, B.L. Methods of wood chemistry 2:785.
Choi, J.-W. and Faix, O. (1999). Investigation on residual lignins and residual carbohydrates and the covalent bonds between them. In: 10th International symposium on wood and pulping chemistry, 1:368-373.
Hamelinck, C.N., van Hooijdonk, G. and Faaij, A.P.C. (2005). Ethanol from lignocellulosic biomass: techno-economic performance in short-, middle- and long-term, Biomass and Energy 28, 384-410.
JÓ“Ó“skelÓ“inen, A.-S., Sun, Y., Argyropoulos, D.S., Tamminen, T. and Hortling, B. (2003). The effect of isolation method on the chemical structure of residual lignin. Wood Sci. Technol. 37:91-102.
Lawoko, M. and Henrikson, G. (2003). New method for quantitative preparation of lignin-carbohydrate complex from unbleached softwood Kraft pulp: lignin-polysaccharide netwoks J. Holzoforschung 57, 69-74.
Li, J., Martin-Sampedro, R., Pedrazzi, C. and Gellerstedt, G. (2011). Fractionation and characterisation of lignin-carbohydrate complexes (LCCs) from eucalyptus fibres. Holzforschung 65, 43-50.
Minor, J.L. (1986). Chemical linkage of polysaccharides to residual lignin in loblolly pine Kraft pulps. J. Wood Chem. Technol. 6:185-201.
Pate, F.M. (1982). Value of Treating Bagasse with Steam under Pressure for Cattle Feed. Trop. Agric., 59, 293.
Tamminen, T. and Hortling, B. (1999). Isolation and characterization of residual lignin. In: Progress in Lignocellulosics Characterization. Ed. Argyropoulos, D.S. Tappi Press, Atlanta: 1-42.
Tamminen, T., Hortling, B., Ranua, M., Luonteri, E., SuurnÓ“kki, A., Tenkanen M. and Buchert, J. (1999). Enhanced bleachability of spruce kraft pulp by mechanical and enzymatic treatments. In: 10th International symposium on wood and pulping chemistry, 1:584-588.