Klason lignin method

2.3.1. Background

There are a number of methods used to quantify lignin such as non-invasive methods for instance infrared spectroscopy (IR) and nuclear magnetic resonance (NMR) and chemical modification such as lignin solubilisation by thioglycolic acid or acetyl bromide. Nevertheless, the most common used are gravimetric methods such as Klason lignin and detergent insoluble lignin (Hatfield and Fukushima, 2005, Tuomela et al., 2000).

The non-invasive methods do not require an extensive sample preparation, however, the overlapping of peaks due to other compounds and the lack of a proper standard make these methods more appropriate for qualitative than quantitative analyses (Hatfield and Fukushima, 2005). Thioglycolic acid and acetyl bromide are used to solubilise the lignin from cell walls. Thioglycolic acid reacts with benzyl alcohol while acetyl reacts with unsubstituted OH, both making lignin soluble. Solution containing soluble lignin is then quantified by absorbance changes (Hatfield and Fukushima, 2005). Although these methods can have less interference from other compounds present in lignocellulose matrix, complete lignin solubilisation and lack of standard make these method less reliable (Dence, 1992).

Klason lignin method consists in acid hydrolyse biomass carbohydrates (hemicellulose and cellulose) in two steps (72% acid at mild temperature (30oC); then 4% acid at boiling

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temperature) and weigh the acid insoluble lignin (AIL) which remains in solid phase (Sluiter et al., 2008).

Acid detergent lignin (ADL) can be quantified by a detergent method that uses a similar principle as Klason, the difference being the order in which higher temperature and acid concentration are applied (Hatfield et al., 1994). In the procedure to quantify ADL, first reported by Van Soest and Wine (1967), low concentrated acid (4%) is mixed with a detergent solution at high temperatures to hydrolyse mostly hemicellulose. Then, the solid left (cellulose+lignin) is hydrolysed in concentrated acid (72%) detergent solution at mild temperatures (25-30o_C) (Van Soest and Wine, 1967). After both hemicellulose and cellulose are removed in the 1st- and 2nd-step, respectively, remaining solid lignin can be weighed. However, it has been suggested that ADL procedure underestimates lignin quantity significantly, particularly for grasses, due to lignin solubilisation (Kondo et al., 1987, Lowry et al., 1994). Therefore, Klason lignin was chosen as analytical method for quantification of lignin in Miscanthus.

Prior to Klason procedure, biomass needs to be ground and biomass extractives needs to be removed. These procedures are detailed below.

2.3.2. Grinding biomass using liquid nitrogen

MxG was ground using liquid nitrogen in order to obtain a small particle size (<1.4mm) needed for Klason analysis. 5g of MxG was weighed and placed in a blender (Philips, 400W, 1.5L). Liquid nitrogen was added until biomass was completely covered and frozen. Frozen biomass was then ground for 15s.

Ground MxG was passed through a 1.4mm sieve and portion retained in 1.4mm sieves (d>1.4mm) was ground again and placed once more in the sieves. This step was repeated three times in total.

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2.3.3. Removal of extractives

Biomass extractives, composed mostly by non-structural carbohydrates (starch), proteins and waxes (Hatfield and Fukushima, 2005), are important interfering in Klason lignin method, therefore they need to be removed prior the procedure. Solvents used for extraction were water followed by ethanol, according to the National Renewable Energy Laboratory (NREL) procedure for determination of extractives in biomass (Sluiter et al., 2005).

2.3.3.1. Material

Cellulose thimbles (Whatman®, 26×60mm, thickness 1.5mm) were used for the extraction. Extraction solvents were HPLC grade water (Sigma) followed by ethanol absolute (Fisher Scientific). The Soxhlet apparatus was composed by a heating mantle, a glass extractor, a glass condenser and a 250mL glass flask.

2.3.3.2. Method

The thimble was weighed (E1) and used for extraction of about 6g of nitrogen ground MxG. Thimble+MxG were weighed (E2) and thimble was closed by folding its top to avoid MxG to spread once submersed in the solvent.

Thimble was then placed into a Soxhlet apparatus and HPLC grade water was added until the thimble was completely submersed. Soxhlet extraction works by boiling the solvent, which accumulates in the extractor and extracts the soluble components from the biomass; when the extractor is full of solvent, a siphon empties the extractor, returning the solvent to the flask to boil again. The cycle is then repeated. Water extraction was carried for two consecutive days during 8h per day.

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Water containing the extractives was removed from the apparatus and replaced by ethanol. Ethanol extraction was also carried for two consecutive days during 8h per day. The objective of this step was to remove extractives, therefore, volume of water and ethanol were not measured. Instead, solvents were added until the thimble was completely submersed.

At the end of ethanol extraction, the thimble containing the extractives-free Miscanthus was removed from the Soxhlet apparatus and then dried at 65oC for 48h and weighed (E3). Extractives percentage was calculated as shown in Equation 2-1:

𝐸𝑥𝑡𝑟𝑎𝑐𝑡𝑖𝑣𝑒𝑠 (%) = 𝐸3− 𝐸1

𝐸2− 𝐸1 Equation 2-1

2.3.4. Lignin determination by Klason method

2.3.4.1. Method

Klason lignin was quantified in this work using a standard procedure adapted from the NREL for lignin quantification in biomass (Sluiter et al., 2008).

Glass filtering crucibles (Pyrex, Gooch borosilicate, porosity grade 4, 30mL) were placed in a muffle furnace at 575oC for four hours for drying and cleaning organic residues that might be left in the crucibles. Then, they were transferred to a desiccator until room temperature and weighed (K1).

0.3 ± 0.01g of biomass (B) to be analysed was weighed into a glass test tube and 3mL of 72% sulphuric acid (Fluka) was added to the tube. Mixture was homogenised using a glass stir rod and placed into a pre-heated water bath at 30oC for 60min. Mixture was stirred every 10-15min without taking the tube out of the water bath to maximise carbohydrate hydrolysis.

The tube was removed from the bath after 60min and mixture was transferred to a 100mL glass Duran® bottle and diluted to 4% acid (that was done by adding 84mL of distilled

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water). The bottle was closed with plastic lid and placed in an oven for the 2nd-step acid hydrolysis at 121oC for 60min.

After the 2nd_{-step acid hydrolysis, samples were cooled for 30min and vacuum filtered} in the pre-weighed crucibles. Liquid fraction was stored (-20oC) for carbohydrate analysis. 50mL of warm distilled water (~50oC) was used to rinse the solid left in the crucibles. Crucibles were dried at 105oC for 6h and placed into a desiccator until room temperature before being weighed again (K2).

Crucibles were placed into a muffle furnace at 575o_{C for 4h and then placed to cool} down in a desiccator and re-weighed (K3). This stage was used for quantification of ashes present in the samples.

In order to calculate Klason lignin, the oven dry weight (ODW, in grams) was calculated using Equation 2-2:

𝑂𝐷𝑊 =𝐵 ∗ %𝑡𝑜𝑡𝑎𝑙 𝑠𝑜𝑙𝑖𝑑𝑠

100 Equation 2-2

Total solids percentage was obtained by weighing a small amount (~1g) of biomass (T1) that will be analysed by Klason procedure into a pre-weighted Eppendorf tube (T2). The tube was then dried for 48h at 65o_{C and re-weighed (T3). Total solids and moisture contents were} determined by Equation 2-3 and Equation 2-4.

𝑇𝑜𝑡𝑎𝑙 𝑆𝑜𝑙𝑖𝑑𝑠 (%) =𝑇3− 𝑇2

𝑇₁− 𝑇₂∗ 100 Equation 2-3 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 (%) = 100 − 𝑇𝑜𝑡𝑎𝑙 𝑠𝑜𝑙𝑖𝑑𝑠 Equation 2-4

Klason lignin was calculated by Equation 2-5: 𝐾𝑙𝑎𝑠𝑜𝑛 𝑙𝑖𝑔𝑛𝑖𝑛 (%) = (𝐾2− 𝐾1) − (𝐾3− 𝐾1)

37 2.3.4.2. Limitations

Klason procedure might overestimate lignin in high protein content biomasses as part of protein might remain solid after acid hydrolysis (Hatfield and Fukushima, 2005). There is also the possibility of incomplete acid hydrolysis of carbohydrates (Kondo et al., 1987, Sluiter et al., 2008).

Lower molecular weight lignin (known as acid soluble lignin (ASL)) might be solubilised in acid during the process, (Sluiter et al., 2010). According to the NREL protocol for Klason lignin, ASL should be estimated by UV-absorbance of the liquid fraction after 2nd_- step acid hydrolysis (Sluiter et al., 2008). However, the difficulty of this procedure is to choose the correct wavenumber to read absorbance, because of the potential inferences such as furans and carbohydrates (Hatfield and Fukushima, 2005). Moreover, as ASL only represents up to 0.5-1.5% (w/w) of Miscanthus (Brosse et al., 2009, Visser et al., 2001), it was not quantified in this work.