CHARACTERISATION OF LIGNINS ISOLATED FROM BAMBOO ORGANOSOLV AND KRAFT BLACK LIQUOR

Bamboo biomass was pulped using organosolv methods: Acetic acid and formic acid; hydrogen peroxide and formic acid; ethanol and water and kraft process with varying concentrations of the cooking chemicals. The properties of the isolated lignin were influenced by the black liquor pH as well as the pulping method. Highest pH (12,8) was recorded for kraft at 70:30 liquor concentration while lowest pH of 2,5 was recorded for both Aceticformic (70:30) and Ethanol/water (70:30 and 60:40). There were obvious differences in the characteristic colours, shapes and sizes of the lignin samples. Bamboo kraft lignin samples formed large pieces much easier than that of Organosolv. Aceticformic (70:30) had the best filteration properties (FC) while Etha - nol/water had the poorest. Generally, kraft recorded the highest lignin yield while all the organosolv processes recorded reduced yield. Highest yield (119,25 g/L) was gotten from kraft (50:50) while Peroxyformic (60:40) had the lowest yield of 8,20 g/L. The results showed that higher liquor pH favours total dissolved solid as well as lignin precipitation. As the pH increases, total dissolved solid, yield, Klason and low molecular weight (LMW) lignin content increases while FC decreases. The Klason and LMW lignin content increased with incresing lignin yield.


INTRODUCTION
After cellulose, lignin is not only the second most abundant polymeric organic substance on the planet but also the most abundant renewable aromatic bio-polymer resource in nature (Wang et al. 2021, Gellerstedt andHenriksson 2008) with complex molecular structure, containing cross-linked polymers of phenolic monomers mainly consisting of p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol (Inkrod et al. 2018, Soongprasit et al. 2020. Lignin is one of the most potential renewable and sustainable energy resource which is present in a huge amount of waste such as wood, maize, rice straw, corn stover, sugarcane bagasse accounting for 15-30 wt% in the dry lignocellulosic biomass . Lignins have been isolated from different biomass such as olive tree prunnings (Erdocia et al. 2014), oil palm (Faris et al. 2017); rice husk (Singh andDhepe 2016, Kim et al. 2021), baggass (Inkrod et al. 2018); palm oil empty fruit bunch (Hidayati et al. 2018); wood and bamboo . Fractionated lignins can be further used for various applications, including liquid fuel, composite materials, and production of low molecular weight aromatic or phenolic compounds (Inkrod et al. 2018) and other value-added chemicals, and functional materials due to its high-energy density and intrinsic aromatic-based structure (Jardim et al. 2022).
Kraft process is the dominant alkaline pulping process and most used pulping process for wood and nonwood such as bamboo (Kamthai and Puthson 2005). Kraft process is a cheap conventional way of extracting lignin, however with negative health effects (Krief et al. 2008). The residual liquor gotten when wood is digested to pulp is called black liquor. Black liquor disposal consitute a major problem to the environment and a threat to human health. The black liquor consists of almost all the inorganic chemicals used in pulping and organics in the form of dissolved wood constituents (Hidayati et al. 2018). Most importantly, black liquor contains lignin which is about 46 % of its total solid (Hidayati et al. 2018). Lignin extraction from black liquor is an attractive option for pulp mills as it allows for increased production capacity without increasing recovery boiler load (Jardim et al. 2022). It also enables the industry to become a biorefinery by allowing for the manufacture of multiple products from biomass.
Although, Kraft liquor yields are encouraging, but environmental concern on the pulping process are discouraging. The organosolv process on the other hand has the advantage of using substances which are less harmful to human health and the environment compared to conventional processes (Kraft and Sulfite) that cause major problems of pollution (FAO 1996, UNEP 1996, Suhr et al. 2015, Gavrilescu et al. 2008, Ruiz et al. 2011. However, few papers present the utilization of Organosolv methods because they are mostly expensive. Organosolv pulping is a clean chemical process that uses organic solvents to aid the removal of lignin and hemicelluloses from lignocellulosic raw materials. The process involves the cooking of lignocellulosic biomass in a mixture of water and an organic solvent that leads to the deconstruction of both lignin and hemicelluloses and their dissolution in the cooking liquor (Sannigrahi et al. 2011). Lignin is typically retrieved as a precipitate by dilution of the liquor with water. The process provides facility for solvent recovery after pulping (Ruiz et al. 2011). The process has been successfully used as a pretreatment and fractionation process for a variety of lignocellulosic materials, such as agricultural, hardwoods and softwoods (Galbe and Zacchi 2002).
Separation of lignin from various cellulosic materials has been reported using different techniques, e.g., alkaline treatment, solvent extraction, organosolv pretreatment, ionic liquid extraction, and milled wood lignin isolation which resulted in isolation of lignin with variation in purity and chemical characteristics (Raita et al. 2017). Also, many methods have been employed to recover pure lignin from black liquor such as acidification, ultrafiltration and electrolysis (Do et al. 2020, Kihlman 2016. Research has also made progress in two techniques; LignoBoost and Sequential Liquid-lignin Recovery and Purification (SLRP) (Öhman et al. 2007, Tomani 2010, Velez and Thies 2013. However, the use of acid to precipitate lignin has been widely practised (Hubbe et al. 2019, Ma'ruf et al. 2017, Minu et al. 2012.
Lignin structure and physic chemical properties depends on isolation method and raw material source (Hidayati et al. 2018). The biomass source is the primary factor determining the lignin composition, especially the contents of the three structural units. Biomass source as well as extraction methods have been reported to affect the final structures of lignin, in terms of bond linkages, functionalities and structural units (Domínguez et al. 2018). Various methods have been developed to isolate lignin from lignocellulosic biomass. Organosolv lignin (Patwardhan et al. 2011) and milled wood lignin (Jiang et al. 2020) as well as acidolysis of cellulose and hemicellulose to obtain Klason lignin and hydrolyzed lignin have been investigated. Milled wood lignin and alkali lignin have been reported to generate more phenols owing to the abundant ether bonds and side chains because of their lower bond strength. While organosolv lignin produced more CO -O) and CO 2 in the non-condensate gas due to its high carbonyl (C-content (Wang et al. 2015). The organosolv lignin has chemical structure closer to native lignin. More importantly, the delignification process is more environment-friendly (Chen et al. 2019, Qin et al. 2021. However, studies on yield and other properties of lignin have been more on Kraft process. There is need to evaluate the concentration and pulping type that favours the best lignin in terms of yield and functionality from kraft and organosolv pulping processes using Bambusa vulgaris as biomass source. In Nigeria, bamboo is underutilized, lignin can be extracted from these bamboos for high value-added use.

Biomass collection and preparation
Bamboo biomass wastes were collected during industrial bamboo processing at Forest Product Development Unit of the Forestry Research Institute of Nigeria (FRIN), Ibadan, Oyo State. The bamboo wastes were dried to constant weight and there after screened with 40 mesh sieve to obtain homogenous particle size. All reagents used such as NaOH, Na 2 S, H 2 O 2 , CH 2 O 2 , H 2 SO 4 , C 2 H 5 OH, were analytical grade.

Organosolv pulping of Bamboo wastes
Three organosolv pulping types were employed which were based on the type of reagents used.

Acetic-formic acid (PFA) treatment
In the acetic-formic treatment, the volume ratio of acetic acid to formic acid were varied. The ratios were 50:50, 60:40, and 70:30 respectively. The biomass was delignified at 90 ºC for 90 minutes. Thereafter, the black liquor was filtered from the delignified cellulosic residues.

Ethanol/water (EW) treatment
Delignification procedures followed the procedures suggested by Zhao et al. (2009). In Ethanol/water process, the volume of ethanol to water was varied as 50:50, 60:40, and 70:30 respectively as was done for acetic-formic process. The reaction was catalysed by the addition of 40 ml H 2 SO 4 at 90 ºC for 90 minutes.

Peroxyformic acid (PFA) treatment
Bamboo biomass was delignified by reacting with a mixture of hydrogen peroxide and formic acid of varying concentrations following the procedures of Watkins et al. (2014). The volume of H 2 O 2 to formic acid were 50:50, 60:40, and 70:30 respectively. After delignification, the spent liquor was filtered from the delignified fibre. Lignin precipitation was done as for the two previous processes.

Kraft pulping
The cooking liquor (white liquor) comprises of sodium hydroxide (NaOH) and sodium sulphite (Na 2 S). The concentration of NaOH to Na 2 S were varied as done for organosolv process; 50:50, 60:40, and 70:30 respectively. Delignification was carried out at a temperature of 120 ºC for 2 h. After delignification, black liquor was filtered off the delignified fibre.

Lignin isolation
For the organosolv spent liquor, the black liquors were concentrated at 40 °C, the lignin in the black liquor were then precipitated by addition of water, five times the volume of the black liquor for 1 hour with constant agitation. The filter cake recovered after filtration, were then subsequently washed three times with 200 mL of the corresponding organic solvent-water (50/50, v/v) and finally washed severally with 300 mL of distilled water. Acid (60 % H 2 SO 4 ) was used to precipitate lignin from kraft liquor. The precipitated lignin was filtered, washed with acidified water followed by de-ionised water. The filter cake was thereafter air dried and then further dried in an oven at 30 °C to constant weight.

Characterisation of the lignin
The total dry solids (TDS) content of the black liquor was determined according to the TAPPI T650 om-09 method (2009): 10 mL each of the black liquor samples was put in a ceramic cup and put into an oven with a temperature of 105 °C overnight. After constant weight was obtained, the total dissolved solid was determined gravimetrically, and the pH of the black liquor was measured with a digital pH meter. Ease of filtering (filtration property) which is the quantity of precipitated lignin that can be filtered per unit time. This was done by taking note of the time it took to filters 150 mL of mixture of the black liquor and precipitating agent. Yield was determined gravimetrically by measuring the quantity of lignin that was recovered from a certain quantity of spent liquor and thereafter estimated in g/L. The Klason lignin content was determined following TAPPI T222 om-02 method (2006).
The filtrate was analysed for residual lignin (low molecular weight lignin) using Atomic Absorption Spectrometry (AAS) at 270 nm. Briefly, the filtrate from Klason lignin was diluted to 100 mL in a volumetric flask. A solution that was 50 (for precipitated lignin) or 100 (for black liquor filtrate) times weaker was prepared for UV measurement. The concentration of acid-soluble lignin was measured based on the absorbance value determined by UV at a wavelength of 270 nm in a Specord 205, Analytik Jena.

Analysis of the functional groups
Fourier transform infrared spectroscopy (FTIR) was used to identify and determine the lignin content, the functional group and bond linkage contained in the lignin samples. The spectra were recorded on a Nicolet iS5 FT-IR Spectrometer. Isolated lignin sample was vacuum dried at room temperature for 24 h. The dried lignin sample (2 mg) was powdered and mixed with 300 mg of anhydrous KBr. The mixture was pressed under vacuum to obtain the pellets, and the spectrum between 4000 cm -1 and 500 cm -1 was measured. The background spectrum of air was subtracted from the spectra of the samples. The Organosolv and Kraft lignin samples were investigated using this technique.

Statistical analysis
The experiment was two factors experiment in a Completely Randomised Design (CRD) with five replications. Factor A: Pulping type (4 levels: Ethanol, Peroxyformic, Acetylformic and Kraft); Factor B: Liquor concentration (3 levels: 50:50, 60:40 and 70:30). Data were analysed by one-way analysis of variance (ANOVA). Significant difference among means were determined. Means were separated using Duncan Multiple Range test.

Influence of liquor concentration and cooking type on the properties of isolated lignin
The properties of the black liquor and isolated lignin from organosolv and kraft lignin are presented on Table 1 and Table 2 as well as Figure 1, Figure 2 and Figure 3. The properties of the isolated lignin were influenced by the black liquor pH as well as the pulping methods. The ANOVA results (Table 2) showed significant statistical differences in the pH, TDS, FC, yield, Klason content, LMW and HMW lignin content. Liquor concentration and cooking type had significant influence on pH, TDS, FC, yield, Klason content and LMW lignin. However, HMW lignin concentration were not influenced by liquor concentration (Table 2).  The mean value for the properties of lignin from Organosolv and Kraft process are presented in Table  3 and Table 4. Highest pH (12,8) was recorded for kraft at 70:30 liquor concentration which was closely followed by 60:40 with pH value 12,4. Lowest pH of 2,5 was recorded for both AF (70:30) and EW (70:30 and 60:40). This indicates that kraft process is alkaline while all the Organosolv processes were acidic in nature. Also, the result further shows that Kraft (50:50) had the highest TDS of 394 % followed by kraft (60:40)   The pulping processes and liquor concentration with the best filtration property was Aceticformic (70:30) which was filtered within 39 seconds. This was closely followed by Kraft (70:30) with filtration time of 60 seconds. All the Ethanol/water (EW) lignin had the poorest filtration (Figure 2 and Figure 3). Highest lignin yield (119,25 g/L) was gotten from kraft (50:50) closely followed by kraft (70:30) while Peroxyformic (PF) (60:40) had the lowest yield of 8,20 g/L. The result showed that higher liquor pH favours TDS as well as lignin precipitation. This is in line with the report of Hidayati et al. (2018) and Xu et al. (2013) where they reported that yield is influenced by different process condition, i.e, the pH, temperature and ion strength of black liquor. Generally, kraft process recorded the highest lignin yield while all the organosolv processes recorded reduced lignin yield. The reason for the low yield of organosolv lignin might be as a result of the volatility of the organic solvents compared to the inorganic chemicals which resulted in low delignification at low temperature. This result was in agreement with the study of Vazquez-Torres et al. (1992). Also, the higher the black liquor pH, the higher the lignin yield. This indicates that the lignin in the bamboo biomass was more solubilized at low pH than high pH. However, low pH favours lignin precipitation from Bamboo Kraft black liquor. This is because the lignin was more soluble at high pH than low pH, and therefore more low-molecular fragments of lignin were dissolved at higher pH such as 11,8 g/L, 12,4 g/L and 12,8 g/L than at low pH (Gellerstedt et al. 1994).
Klason lignin is the solid residual material that is obtained after a sample of BL has been subjected to hydrolysis treatment with 72 % H 2 SO 4 . Klason lignin content of kraft (50:50) was highest with value of 49 % followed by kraft (70:30) while lowest (11,20 %) was recorded for AF at 50:50 liquor concentration. The content of acid-soluble lignin, the purity and the components of isolated lignin, was determined using UV spectroscopy. The absorbance of lignin was recorded at wavelength of 270 nm. The concentration of the High Molecular Weight (HMW) and Low Molecular Weight (LMW) lignin showed EW (60:40) and kraft (70:30) to be highest with values of 0,164 nm and 0,229 nm respectively while the lowest of 0,003 nm and 0,024 nm were recorded for HMW and LMW lignin respectively (Figure 4).
Higher liquor pH favours LMW lignin. Although, HMW lignin was not influenced by liquor pH but seemed that concentration of HMW lignin were higher for Organosolv than Kraft process. Although there were statistical variations in the properties of the black liquor and the precipitated lignin based on the liquor concentration and pulping type. However, the pH of Aceticformic and Ethanol as well as TDS of Aceticformic and Peroxyformic were statistically similar ( Table 3). The Klason lignin content of Aceticformic and Ethanol/ water were similar as well as LMW lignin concentration of Ethanol/water and Peroxyformic lignin (Table 4).    According to the intrinsic structure of lignin, several absorptions attributed to different purity level were observed, as shown in Table 5. Based on the Lambert-Beer's Law, UV spectroscopy can be used for the semi-quantitative determination of the purity of lignin and its degradation product (Prozil et al. 2012). Because of the cross-linking structures of lignin with carbohydrates, cellulose, and hemicelluloses, the isolation of lignin is extremely difficult. The absorption values for the HMW lignins are arranged in decreasing order, indicating that the lignin purity increases with decreasing absorption value. Acetylformic (50:50) had the highest purity with absorbance of 0,226 nm followed by Ethanol/Water (60:40) while Ethanol/water (50:50) had the least purity with absorbance value of 0,004 nm (Table 5). Table 5: Lignin purity based on the UV absorbance of HMW lignin.

Characteristic colour and forms of the isolated lignin
Due to different pH value, the colour of the isolated lignin varies significantly from cream to light brown and dark brown ( Figure 5 and Figure 6). The colour changes from cream to dark brown with decreasing pH. In addition, there were obvious differences in the shape and size of lignin. Kraft lignin colour also varied from brown to dark brow with decreasing pH. In general, the precipitation yield of organosolv and Kraft lignin depended on several factors, for instance, the pH value of liquor and the cooking method. The comparison of organosolv and Kraft lignins according to different pH values are shown in Figure 5. As can be seen, for a certain type of process e.g. Peroxyformic, due to different pH value, the colour of the lignin varied from light brown to dark brown with pH value decreasing. In addition, there were obvious differences in the shape and size of the lignins. Kraft lignin colour too varied from brown to dark brow with decreasing pH. Kraft lignin sample of bamboo formed large pieces much easier than that of Organosolv, and the process also filtered relatively faster than that of Organosolv during the precipitation process. This was related to the work of (Li et al. 2012) when he compared different Kraft lignins based on hardwood and softwood.

Correlation analysis
The correlation between the liquor concentration and lignin parameters were presented in Table 6. There was strong positive correlation between pH and TDS, yield, Klason and low molecular weight lignin content. Only FC had strong negative correlation with pH. TDS had strong positive correlation with yield, Klason and Low molecular weight lignin. Yield had significant positive correlation with Klason and Low molecular weight lignin content. Likewise, Klason lignin content showed significant positive and negative correlations with Low molecular and High molecular weight lignin content respectively (Table 6). This indicates that as the pH increases, the TDS, yield, Klason and low molecular weight lignin content increases while FC decreases and vice versa. Also, Klason and Low molecular weight lignin content increased with increasing lignin yield. In addition, the Klason lignin content had a considerable influence on the absorbance values of the HMW lignin. This agreed with the work of (Kallavus et al. 2015), who reported that the type and structure of lignin, solvent, and pH of the solution have a considerable influence on the UV spectra values. Table 6: Correlation matrix of the liquor concentration and lignin properties. ** = Highly significant correlation; * = Significant correlation.

Fourier transform infrared spectroscopy (FTIR) analysis of the lignin
The FTIR spectra of the lignin isolated from the B. vulgaris contained most of the characteristic absorption bands for the different chemical structures. The lignin samples were analyzed based on the chemical structures and the peaks were identified by comparing their wavenumbers with literature data. The FTIR spectra obtained for both processes are shown in Figure 7. The characteristic bands between 3500 cm -1 -3000 cm -1 indicate the existence of alcohol and carboxylic group in the samples. It was observed that Ethanol/Water, Peroxyformic, and Kraft lignin resonates at this wavelength. Which means Acetylformic did not provide information about the secondary structure of this functional group. The appearance of the band in the range of 1120 cm -1 -1270 cm -1 and 1330 cm -1 -1375 cm -1 shows the existence of P-hydroxyl (H), Guaiacyl (G) and Syringyl (S) Unit in the bamboo lignin which are the basic precursor for lignins (Table 7). Apparently, this characteristics band were found to be present in all the lignin type. C-H stretching in Alkanes and Alkyls group is reflected in the band of 2901 cm -1 . All the lignin types exhibited this functional groups. Band at 1600 cm -1 -2500 cm -1 were attributed to Alkenes and Carbonyls group which are the characteristic bands found in the infrared spectra of Peroxyformic lignin (Table 7). The absorption band associated with all the lignin type at 1600 cm -1 -1680 cm -1 are attributed to stretching vibrations of the C=H bond of the Alkenes. Absorption associated with the Amides R-C(O)-NH-R band at wave number between 1500 cm -1 -1550 cm -1 leads primarily to bending vibrations of the N-H bond. Furthermore, C-Br stretching in Alkyl halides groups is reflected in the band of 1019 cm -1 -400 cm -1 for Ethanol/Water, Peroxyformic and Kraft lignin.

CONCLUSIONS
The highest lignin precipitated (119,25 g/L) was recorded for Kraft at 50:50 liquor concentration while the lowest (8,20 g/L) was recorded for Peroxyformic at 60:40 liquor concentration. Acetylformic (70:30) was the easiest to filter which was filtered within the shortest period of 39 seconds compared to Ethanol/water (60:40) that took 822 seconds before the filter cakes could be filtered off the residual filtrate. Generally, the precipitation yields of lignin decreased with increasing pH value. Ethanol/Water at 60:40 liquor concentration was found to have the highest purity judging from the UV spectrometric analysis. The cooking type and liquor concentrations have considerable influence on the concentration of the lignin. Functionality of organosolv and Kraft lignin showed that alcohol and carboxylic acids, alkanes and alkyls, carbonyls, alkenes, amide, and alkyl halides are functional groups present in the isolated lignin.