Isolation and Physicochemical Characterization of Lignin from Chromolaena Odorata and Tithonia Diversifolia

: This study dealt with isolation of lignin from soft woods namely; Chromolaena odorata (Siam Weed) and Tithonia diversifolia (Mexican Sunflower) using alkali pretreatment method. The raw samples were characterized by some physico-chemical parameters before extraction. Chromolaena odorata gave higher lignin yield and lesser ash content of 15.12 % and 4.22 % respectively compared to Tithonia diversifolia with 7.40 % and 9.56 % respectively. The isolated lignins were characterized by some physico-chemical parameters and spectroscopy methods like Futran Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscopy (SEM). Lignin from Chromolaena odorata was found to be more pure than the one from Tithonia diversifolia as evident from the values of ash content (4.22%) and klason lignin (33.65 %) for Chromolaena odorata while ash content (9.56 %) and klason lignin (21.96 %) were obtained from Tithonia diversifolia . The FT-IR spectra of both lignins revealed the presence of syringyl and guaiacyl units. © http://dx.doi.org/10.4314/jasem.v19i4.29


Introduction
Lignin is a renewable material obtained in huge quantities as a by-product of the pulp industry.It has mainly been used as a fuel and only a small amount has been isolated and commercialized.Nevertheless, worldwide, amount of lignin has been estimated at 2% and corresponds to 1 million tons per year (Ammar et al., 2014).This macromolecule is complex and it is widely accepted that the biosynthesis of lignin stems from the polymerization of three types of phenylpropane units, normally referred to as monolignols (Lipersguer et al., 2009).These units are coniferyl, sinapyl and p-coumaryl alcohols.The three structures are depicted in Figure 1.
The amount and composition of lignin vary among taxa cell types and individual cell wall layers.Softwood lignin consists almost exclusively of Gtype lignin, hardwood lignin consists of G and S-type units (H units being minor components), while gramineae has all the three (Kang et al., 2012).Additionally, based on its interesting functionalities and properties, lignin offers a perspective for higher value-added applications.The interest in developing lignin-based applications is nowadays driven by three important factors, namely (i) the availability of new lignin sources, such as, sulfur-free, organosolv, steam explosion lignins and so on.(ii) the growing interest in the bio-refinery concept, where lignin valorization is becoming relevant, because it is the only biosourced molecule containing aromatic moieties, and (iii) the approach of sustainable chemistry, where green processes and bio-based products are in focus (Ammar et al., 2014).
However, it has long been recognized for its negative impact on forage quality, paper-making and cellulosic biofuel production.Therefore, elucidating the lignin structure can play an important guiding role on efforts to remove lignin fractions from lignocellulosic materials.Moreover, effectively overcoming the recalcitrance is an important and urgent research priority for the development and implementation of the lignocellulosic biorefinery concept (Meng, et. al., 2012).

Fig 1:
The major building blocks of lignin (Dence and Lin, 1992) Many works on isolation of lignin from different plants and/or agricultural wastes are available in literature (Ammar et al., 2014;Rencoret et al., 2011;Mohammad et al, 2006).et al., 2013).(Baxter (1995) has noted that the plant spreads widely throughout the tropics and has been declared as a noxious weed due to the difficulty in controlling it by both curative and preventive measures.Tithonia diversifolia commonly known as Mexican sunflower was probably introduced into West Africa as an ornamental plant, a member of the family Asteraceae.It is an annual, aggressive weed growing to a height of about 2.5 m and adaptable to most soils.It had been observed to be widely spread in Nigeria where it is found growing on abandoned/waste lands, along major roads and waterways and on cultivated farmlands (Olabode et al., 2007).

Chromolaena odorata commonly known as
This study is aimed at isolating and examination of physico-chemical characterization of lignin from both Chromolaena odorata and Tithonia diversifolia stems.

MATERIALS AND METHOD
Materials: The C. odorata and T. diversifolia used in this work were collected from along road side towards the gate of university of Ilorin, Nigeria.The leaves were detached and the stems were washed, rinsed with distilled water, in order to eliminate sand and contamination, cut into small pieces, then air dried under laboratory conditions of temperature of 30 o C. The obtained materials were milled and sieved to particle sizes in the range of 0.5-1.0mm.The powdered sample was kept in air tight container prior to extraction.

Extraction of lignin:
The extraction of lignin from C. odorata and T. diversifolia was carried out by alkali pretreatment method using 5M NaOH solution in a round bottom flask using a Solid to liquid ratio of 1: 10.The flask was equipped with a condenser and placed on heating mantle set at 100 o C for 7 hours.The mixture was filtered and the filtrate (black liquor) precipitated with 50 % H 2 SO 4 solution and made up to pH 2 with the aid of addition of either 2 M of NaOH or H 2 SO 4 .The lignin was isolated through vacuum filtration and washed with acidified water (pH=2) several times.The lignin cake was then socked dry under vacuum and finally dried in an oven at 50 o C for 8 hours (Khan and Ashraf, 2006).

Characterization of extracted lignin:
The physicochemical parameters such as pH, conductivity, bulk density, moisture content, ash content were determined using standard methods as described in literature respectively (Khan and Ashraf, 2006;Ahmenda et al. 2000;Yoshiguki and Yukata, 2003;Toledano et al.,2012).

Determination of acid insoluble and acid soluble lignin:
Acid insoluble lignin (AIL) which is also known as klason lignin was determined by subjecting lignin to an acid hydrolysis process.The acidic hydrolysis was carried out by adding 3.75 ml of sulphuric acid (72%) to 0.375 g of lignin.The mixture was left for 1 hour at 30 •C.Then it was diluted with 36.25 ml of deionized water and heated at 100 •C for 3 hours.The solution was cooled for 15 minutes and then filtered under vacuum.The remaining solid is the acid insoluble lignin (Toledano et al., 2012).
The acid-insoluble lignin was calculated as follows: Where A is the weight of filter paper (g), B is the weight of filter paper plus dried lignin residue (g) and C initial weight of the lignin sample (g).Acid soluble lignin (ASL) was determined by spectrophotometry (UV absorption at 205 nm).The filtrate above was used.The filtrate was diluted with 1M H 2 SO 4 until the absorbance is between 0.1 to 0.8 cm -1 (Vishtal and Kraslaw, 2011).The acid-soluble lignin was calculated as follows: Where A is the absorbance at 205 nm, B is dilution factor, C is filtrate volume (L), D is extinction coefficient of lignin (110 g.L -1 .cm - ) and E is the initial lignin weight (g).
FTIR and SEM analysis: FT-IR spectroscopy (Shimadzu 8400 FTIR spectrometer) was used to collect the IR spectra of the dried lignin sample from C. odorata and T. diversifolia.Scanning Electron Microscope (FEI NOVA NANOSEM 230), which was equipped with an energy dispersive X-ray microanalysis (EDX) system (FEI, Eindhoven, Holland) was used to study the morphology and chemical analysis of the lignin sample.

RESULTS AND DISCUSSION
Lignin extraction efficiency: The yield of C. odorata and T. diversifolia lignins on dry basis is presented in Table 1.CO has higher lignin yield of 15.12 % than TD with 7.40 %.Lignin content of 6.5 % has been reported for T. diversifolia lignins (Olabode et al., 2007).The wide difference in the yield can be attributed to the difference in intrinsic properties of raw materials since the same extraction method was used.Moreover, the geographical location and environmental effect might affect the amount of lignin contents in each of the plant stems studied.

Physico-chemical properties of isolated lignin:
The physico-chemical properties of isolated lignin from C. odorata and T. diversifolia are also presented in Table 1.The pH of the C. odorata lignin and that of T. diversifolia lignin are acidic with the values of 4.57 and 4.28 respectively, which can be attributed to the The conductivity value for T. diversifolia lignin is higher than that of C. odorata lignin which is 639 and 419 µS/cm respectively.This is in agreement with their ash content.The ash content of T. diversifolia lignin is higher than that of C. odorata lignin which has been found to be 9.56 and 4.22 % respectively.This indicates that C. odorata lignin has higher degree of purity than T. diversifolia lignin.This is a requirement in some applications of lignin such as production of phenolic resins, animal nutrition and dispersants where lignin with low ash contents are utilized (Vishtal and Kraslawski, 2011).
Moisture content of a sample is a measure of the amount of water present in the sample.It implies that sample with low moisture content can be stored for a longer period with lower chances of microbial attack and growth.Moisture content of 16.35 % and 18.67 % were observed for C. odorata lignin and T. diversifolia lignin respectively.These high moisture content values in this study imply that these lignins are more susceptible to bacterial attack.
Bulk density of a sample gives a measure of how dense a sample is.Bulk density of 0.538 g/ml was observed for T. diversifolia lignin which is higher than 0.439 g/ml for C. odorata lignin.The values of various properties of dried C. odorata and T. diversifolia plants are also shown in Table 1.

Acid insoluble and acid soluble lignin:
Acid insoluble lignin (AIL) also known as klason lignin is a measure of lignin purity since lignin isolation process is based on lignin insolubility in acid media.So, it can be assumed that the measurement of the acid insoluble lignin is related to the lignin purity.The C. odorata lignin has the higher AIL value of 33.65 % than T. diversifolia lignin which has 21.96 %.These results are in agreement with their ash content in which C. odorata lignin with the lower ash content of 4.22 % exhibited the higher AIL.
Acid soluble lignin (ASL) is probably composed of two components; lignin degradation products and secondarily formed hydrophilic materials such as lignin-carbohydrate (Yasuda et al., 2001).The T. diversifolia lignin has higher acid soluble lignin value of 4.42 % than 2.10 % obtained for C. odorata lignin.This also supports the acid insoluble lignin results which in turn reflect on their purity.These results are presented in Table 1.Each measurement is replicated 3 times; the deviation between the experimental values does not exceed 5% andindicates not applicable FT-IR analysis: The infrared spectra of C. odorata and T. diversifolia lignin samples obtained by the alkali pretreatment process are presented in Figure 2 and their corresponding band assignments are given in Table 2. Strong and broad band at 3416 and 3441 cm -1 observed in C. odorata lignin and T. diversifolia lignin spectra respectively are attributed to OH stretching.The bands at 2922 and 2920 cm -1 correspond to the vibration of C-H bond in methyl and methylene groups.The asymmetric deformation of this bond also produced a band at around 1464 and 1462 cm -1 .Three typical vibrations that normally appeared in aromatic compounds such as lignin, are exhibited around 1597, 1510 and 1423 cm -1 .The vibration at around 1728 and 1710 cm -1 are associated with C=O bond stretching in unconjugated ketones, carbonyl and ester groups while the vibration at around 1639 cm -1 is related to the C=C bond stretching of aromatic ring.
The most significant bands in lignin spectra were those that correspond to its main substructures, guaiacylpropane (G), syringylpropane (S) and phydroxyphenylpropane (H) -such as the peak around 1329 cm -1 that was related to the breathing of the syringyl ring with C-O stretching and the bands at around 1265 cm -1 (shoulder) and 1226 cm -1 that were associated to the breathing of the guaiacyl ring with C-O stretching.Around 1159 and 1157 cm -1 is a vibration caused by the deformation of the bond C-H in guaiacyl substructures while the same linkage but in syringyl substructures appeared at around 1116 and 1112 cm -1 .The vibration at around 1026 cm -1 was due to the deformation of the aromatic C-H linkages in guaiacyl substructures.It can also be related to the deformation of the bond C-O in primary alcohols.Similar bands have been reported for lignin (Mohamad et al., 2006;Khan and Ashraf, 2006).SEM analysis: This analytical technique was used for investigating the surface morphology of the isolated lignins in order to have an insight into their applicability in industries in terms of adsorption capacity.Figures 3 and 4 depict SEM images for C. odorata and T. diversifolia lignins and are observed to exhibit porous surface at higher magnification.The pores observed from SEM images are having diameter in micrometer (μm) range.These pores are considered as channels to the microporous network.This suggests that C. odorata and T. diversifolia lignins might be used as biosorbents for removal of organics even without modification.
Siam weed is a tropical species of flowering shrub in the Isolation and Physicochemical Characterization of Lignin 788 *1 FRIDAY, O. NWOSU, 2 MUHAMMAD, M. MUZAKIR, sunflower family, Asteraceae.It is an invasive deeprooted shrub recorded as part of the 100 worst invasive species in the world (Iondoh . NWOSU, 2 MUHAMMAD, M. MUZAKIR, fact that lignin is a polyphenolic compound which contains acidic OH groups.

Table 1 :
Physico-chemical properties of raw and isolated Lignins of two plants

sample Absorption bands (cm -1 )
in unconjugated ketones, carbonyl and ester groups 1639 1639 C=C bond stretching of aromatic ring 1508,1421 1597,1510 Characteristics of aromatic rings due to aromatic skeletal vibrations 1464 1462 CH bond deformation 1329 1329 Breathing of the syringyl ring with C-O stretching 1209 1265 Breathing of the guaiacyl ring with C-O stretching.