Here, we present a modified TGA method for estimation of lignin content in herbaceous plant biomass. This method estimates the lignin content by forming specific thioether bonds with lignin and presents an advantage over the Klason method, as it requires a relatively small sample for lignin content estimation.
Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls and is an aromatic heteropolymer primarily composed of three monolignols with significant industrial importance. Lignin plays an important role in plant growth and development, protects from biotic and abiotic stresses, and in the quality of animal fodder, the wood, and industrial lignin products. Accurate estimation of lignin content is essential for both fundamental understanding of the lignin biosynthesis and industrial applications of biomass. The thioglycolic acid (TGA) method is a highly reliable method of estimating the total lignin content in the plant biomass. This method estimates the lignin content by forming thioethers with the benzyl alcohol groups of lignin, which are soluble in alkaline conditions and insoluble in acidic conditions. The total lignin content is estimated using a standard curve generated from commercial bamboo lignin.
Lignin is one of the vital load-bearing components of plant cell walls and the second most abundant polymer on Earth1. Chemically, lignin is a crosslinked heteropolymer made up of high molecular weight complex phenolic compounds that form a natural renewable source of aromatic polymers and synthesis of biomaterials2,3. This natural polymer plays significant roles in plant growth, development, survival, mechanical support, cell wall rigidity, water transport, mineral transport, lodging resistance, tissue and organ development, deposition of energy, and protection from biotic and abiotic stresses4,5,6,7. Lignin is primarily composed of three different monolignols: coniferyl, sinapyl and p-coumaryl alcohols that are derived from the phenyl propanoid pathway8,9. The amount of lignin and the composition of monomers vary based on the plant species, the tissue/organ type, and different stages of plant development10. Lignin is broadly classified into softwood, hardwood, and grass lignin based on the source and monolignol composition. Softwood is primarily composed of 95% coniferyl alcohol with 4% p-coumaryl and 1% sinapyl alcohols. Hardwood has coniferyl and sinapyl alcohols in equal proportions, while grass lignin is composed of various proportions of coniferyl, sinapyl and p-coumaryl alcohols11,12. The composition of monomers is critical as it determines the lignin strength, decomposition, and degradation of the cell wall as well as determining molecular structure, branching, and crosslinking with other polysaccharides13,14.
Lignin research is gaining importance in foraging, textile industries, paper industries, and for bioethanol, biofuel, and bio-products due to its low cost and high abundance15,16. Various chemical methods (e.g., acetyl bromide, acid detergents, Klason, and permanganate oxidation) along with instrumental methods (e.g., near infrared (NIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and ultraviolet (UV) spectrophotometry) were used for lignin quantification9,17. The analysis methods of lignin are generally classified based on electromagnetic radiation, gravimetry, and solubility. The principle behind lignin estimation by electromagnetic radiation was based on the chemical property of lignin by which it absorbs light at specific wavelengths. These results were estimated based on the principle that lignin has a stronger UV absorbance than carbohydrates. In 1962, Bolker and Somerville used potassium chloride pellets to estimate lignin content in wood18. However, this method has drawbacks in the estimation of lignin content from herbaceous samples due to the presence of non-lignin phenolic compounds and the absence of an appropriate extinction coefficient. In 1970, Fergus and Goring found that the guaiacyl and syringyl compound absorption maxima were at 280 nm and 270 nm, which corrected the extinction coefficient issue of the Bolker and Somerville method19. Later, infrared spectroscopy, a highly sensitive technique for characterizing phenolics, was also used for lignin estimation with a small amount of plant biomass samples. One example of such technology was diffuse-reflectance Fourier transform spectrophotometry. This method, however, lacks a proper standard similar to the UV method20. Later, the lignin content was estimated by NIRS (near infrared spectroscopy) and NMR (nuclear magnetic resonance spectroscopy). Though, there are disadvantages in these methods, they do not alter the chemical structure of lignin, retaining its purity20.
The gravimetric Klason method is a direct and the most reliable analytical method for lignin estimation of woody stems. The basis for gravimetric lignin estimation is the hydrolysis/solubilization of non-lignin compounds and the collection of insoluble lignin for gravimetry21. In this method, the carbohydrates are removed by hydrolysis of the biomass with concentrated H2SO4 to extract lignin residue20,22. The lignin content estimated by this method is known as acid insoluble lignin or Klason lignin. Application of the Klason method depends on the plant species, the tissue type and the cell wall type. The presence of variable amounts of non-lignin components such as tannins, polysaccharides and proteins, results in proportional differences in the estimation of acid insoluble/soluble lignin contents. Hence, the Klason method is only recommended for lignin estimation of high-lignin content biomass such as woody stems17,23. Solubility methods such as acetyl bromide (AcBr), acid-insoluble lignin, and thioglycolic acid (TGA) are most commonly used methods for estimation of the lignin content from various plant biomass sources. Kim et al. established two methods for lignin extraction by solubilization. The first method extracts lignin as an insoluble residue by solubilizing cellulose and hemicellulose, while the second method separates lignin in the soluble fraction, leaving cellulose and hemicellulose as the insoluble residue24.
Similar methods employed in lignin estimation based on the solubility are thioglycolic acid (TGA) and acetyl bromide (AcBr) methods25. Both TGA and acetyl bromide methods estimate the lignin content by measuring the absorbance of the solubilized lignin at 280 nm; however, the AcBr method degrades xylans during the process of lignin solubilization and shows a false increase in the lignin content26. The thioglycolate (TGA) method is the more reliable method, as it depends on specific bonding with the thioether groups of benzyl alcohol groups of lignin with TGA. The TGA bound lignin is precipitated under acidic conditions using HCl, and the lignin content is estimated using its absorbance at 280 nm27. The TGA method has additional advantages of less structural modifications, a soluble form of lignin estimation, less interference from non-lignin components, and precise estimation of lignin due to specific bonding with TGA.
This TGA method is modified based on the kind of plant biomass sample used for lignin content estimation. Here, we modified and adapted the rapid TGA method of rice straws27 to cotton tissues to estimate the lignin content. Briefly, the dried powdered plant samples were subjected to protein solubilization buffer and methanol extraction to remove proteins and the alcohol soluble fraction. The alcohol insoluble residue was treated with TGA and precipitated lignin under acidic conditions. A lignin standard curve was generated using commercial bamboo lignin and a regression line (y = mx+c) was obtained. The "x" value uses average absorbance values of lignin at 280 nm, while "m" and "c" values were entered from the regression line to calculate unknown lignin concentration in cotton plant biomass samples. This method is divided into five phases: 1) preparation of plant samples; 2) washing the samples with water and methanol; 3) treatment of the pellet with TGA and acid to precipitate lignin; 4) precipitation of lignin; and 5) the standard curve preparation and lignin content estimation of the sample. The first two phases are primarily focused on the plant material preparation followed by water, PSB (protein solubilization buffer) and methanol extractions to obtain the alcohol insoluble material. Then, it was treated with TGA (thioglycolic acid) and HCl to form a complex with lignin in the third phase. At the end, HCl was used to precipitate lignin, which was dissolved in sodium hydroxide to measure its absorbance at 280 nm28.
1. Preparation of plant samples
2. Washing samples with water, PSB, and methanol
3. Treatment of pellet with TGA and acid to precipitate lignin
4. Precipitation of lignin
5. Standard curve preparation and lignin estimation in the sample
Two different cotton experimental lines were compared for differences in their lignin contents in different tissues. The extracted lignin content of each sample was measured at 280 nm and recorded its respective absorbance values. The average absorbance values of each biological replicate were compared against the regression line of the lignin standard curve (Table 2, Figure 3C). The regression line, y = mx + c, is used to calculate the unknown lignin content of the extracted experimental lines, sample 1 and sample 2. The results of average OD values were substituted in "x" while "m" and "c" values were plugged from the regression line of lignin standard curve to obtain lignin concentration "y" in mg (Table 3, Figure 3B). In the next step, to calculate per 1 mg of lignin content, divide the "y" value by the weight of the sample (15 mg) after methanol extraction. In the following step, to calculate per gram (= 1,000 mg) the y/15 value was multiplied by 1,000. To get % of lignin we divide y/15 value by 1,000 and multiply by 100. The average of lignin % for three biological replicates (of each line, sample 1 and sample 2) was compared between the two experimental lines sample 1 (11.7%) and sample 2 (10.3%). The lignin values were consistent among biological replicates suggesting that the TGA method is a reliable method and highly specific to measure the lignin content. Comparison studies were also made between different tissue types (root, stem and leaves) of two experimental lines of cotton, and both lines showed relatively lower lignin content in leaves (3.4%) compared to stems (9.4% to 9.9%) and roots (9.4% to 9.2%) (Table 4, Figure 4).
Figure 1: Preparation of plant biomass sample. (A) Collected cotton plant material from green house. (B) Gently flipped pots to separate roots. (C) Thoroughly washed in water to remove all the dirt. (D) Separated root, stem and leaf tissues. (E) Air-dried tissue for 2 days after separating the tissue. (F) Air dried tissue is transferred to the incubator at 49 °C for 10 days. (G) Biomass grinder was used to grind plant biomass samples. (H) Ground plant biomass samples of root, stem and leaf. (I) Ground samples are loaded into the grinding vials, placed in the freezer mill chamber, grounded in the freezer mill at a rate of 10 CPS for 3 cycles. (J) Grinded vials showing finely ground tissue powder after grinding in the freezer mill. (K) Finely ground tissue powder of root, stem and leaf after using freezer mill for grinding. Please click here to view a larger version of this figure.
Figure 2: Critical steps involved in TGA mediated lignin extraction. Flow chart of critical steps involved in lignin extraction from plant biomass to lignin content estimation using TGA method: 1. Preparation of plant samples by sufficient drying and grinding into fine powder using freezer mill; 2. 20 mg of tissue powder was subjected to PSB, methanol and water washes, dried and extracted alcohol insoluble material; 3. Using TGA and acid, lignin was precipitated; 4. Preparation of lignin standard curve using commercial bamboo lignin; 5. Estimation of lignin content. Please click here to view a larger version of this figure.
Figure 3: Standard curve preparation and lignin estimation in the sample. (A) Table showing different concentrations of commercial bamboo lignin used for generating lignin standard curve from absorbance readings at 280 nm. (B) Scattered plot generated with Excel program using the values from table A. (C) Bar graphs representing the estimated root tissue lignin contents of sample 1 and sample 2. Please click here to view a larger version of this figure.
Solution | Stocks needed | Preparation | ||
Protein solubilization buffer (PSB) | 1 M Tris HCl pH 8.8 and 0.5 M EDTA pH 8.0 | To prepare 100 mL of working solution of PSB with final concentration of 50 mM Tris, 0.5 mM EDTA and 10 % SDS, add 5 mL of 1 M Tris, 1 mL of EDTA and 10 g of SDS to 80 mL sterile water, mix, dissolve and make up the final volume to 100 mL with sterile water. Autoclave at 121 °C, 15 psi pressure, for 30 min. | ||
1 M Tris HCl | To prepare 100 mL of 1 M Tris, add 12.1 g of Tris HCl (molecular weight = 121.14 g) in 80 mL of water. Mix Tris HCl by stirring on a magnetic stirrer, adjust the pH with NaOH to 8.8 and make up the volume to 100 mL with sterile water and autoclave at 121 °C, 15 psi pressure, for 30 min. | |||
0.5 M EDTA (Ethylenediamine tetraaceticacid) | To prepare 100 mL of 0.5 M EDTA add 18.6 g of EDTA in 70 mL water. Adjust the pH to 8.0 (EDTA completely dissolves at pH 8.0) using sodium hydroxide pellets and make up the volume to 100 mL. Autoclave the solution at 121 °C, 15 psi pressure, for 30 min. | |||
3 N Hydrochloric acid (HCL) | To prepare 100 mL of 3 N HCl, add 26 mL of concentrated HCL to 74 mL of sterile water. | |||
4 % Sodium hydroxide (NaOH) | Prepare 1 N sodium hydroxide solution, add 4 g of sodium hydroxide in 90 mL of sterile water, dissolve, make up the volume to 100 mL and autoclave at 121 °C, 15 psi pressure, for 30 min. |
Table 1: Preparation of solutions used in the protocol. Table showing the preparation of different solutions used in the protocol.
Table 2: Lignin standard curve prepared from 0.5 mg to 3.5 mg of industrial bamboo lignin. Scattered graph with regression line showing m and c values. Please click here to download this Table.
Table 3: Lignin template used for calculation of unknown lignin content using absorbance readings of samples at 280 nm (as x) and standard curve regression line 'm' and 'c' values from the standard curve. Please click here to download this Table.
Table 4: Lignin content from different tissues (root, stem and leaves) of cotton plant at post flowering stage. Please click here to download this Table.
Lignin plays a significant role in plant growth and development and recently has been extensively studied for biofuel, bioenergy and bioproduct applications. Lignin is rich in aromatic compounds that are stored in all vascular plant secondary cell walls. It has several industrial applications such as wood panel products, bio dispersants, flocculants, polyurethane foams and in resins of circuit boards29,30,31. Most of the lignin generated from paper and pulp industries is released as waste or burned for heat production. Thus, if efficiently processed, lignin can be utilized as an alternative to both fossil fuel based products32,33 and bioelectricity production34. Hence, precise estimation of lignin content and composition are critical for industrial applications as the composition varies based on the plant species as well as plant organ type. The major limitation for lignin estimation is the difference arising from the method selected for the estimation of lignin content35. The estimation differences among different methods are primarily due to the contamination with other non-lignin components, variation in the solubility, addition of new groups to lignin, xylan degradation/contamination, native structural changes and loss of some lignin fraction during the elimination of other components. Further, the majority of lignin protocols are originally developed based on wood chemistry27. Hence, there is a critical need for establishing lignin protocols for herbaceous samples as more crop/plant species are targeted for biofuels and bio products. The TGA method estimates pure lignin content based on specific bonding with TGA. Therefore, the lignin estimation by TGA yields lower lignin content when compared to Klason and acetyl bromide methods35,36. This is because of the specific bonding of lignin with TGA as well as loss of some lignin content during lignin precipitation (insoluble part).
The lignin content estimated using TGA method is reproducible and consistent. The results obtained in this study were consistent among the biological replicates and showed a significant difference between two lines, suggesting the reliability of TGA method for lignin estimation. For data reproducibility and precise estimation of lignin content, it is important to follow the steps and take following precautions. Inclusion of positive controls in different concentrations, ranging from 0.5 mg to 5 mg in three replicates, and processing them along with samples from the TGA step will avoid experimental errors and results in precise estimation of the lignin content. The standard curve must be generated for each set of samples and regression line statistic R2 must fall in the range of 97% to 99%. Th exact weight of the empty tube and dried methanol extracted tissue is critical for exact lignin content estimation. Additionally, various factors such as specific stage of plants, growing conditions, genotypes, type of tissue and the age of the plant will affect the lignin content30,37,38. Hence, it is important to grow all the experimental lines in the same environment and harvest the same type of tissues at the same time. Results of the current study showed an expected trend of lower lignin content in the leaves, higher lignin content in stems and roots, and demonstrated the applicability of this method to various plant tissues. Further, less variation among biological replicates suggested that TGA can estimate reproducible lignin content in all plant tissues.
The authors have nothing to disclose.
We thank the Department of Plant & Soil Science and Cotton Inc. for their partial support of this study.
BioSpectrophotometer kinetic | Eppendorf kinetic | 6136000010 | For measuring absorbance at 280 nm |
Centrifuge | Eppendorf | 5424 | For centrifuging samples |
Commercial bamboo lignin | Aldrich | 1002171289 | Used in the preparation of the standard curve |
Distilled water | Fischer Scientific | 16690382 | Used in the protocol |
Falcon tubes | VWR | 734-0448 | Containers for solutions |
Freezer mill | Spex Sample Prep | 68-701-15 | For fine grinding of plant tissue samples |
Heat block/ Thermal mixer | Eppendorf | 13527550 | For temperature controlled steps during lignin extraction |
Hotplate stirrer | Walter | WP1007-HS | Used for preparation of solutions |
Hydrochloric acid (HCL) | Sigma | 221677 | Used in the protocol |
Incubator | Fisherbrand | 150152633 | For thorough drying of plant tissue samples |
Measuring scale | Mettler toledo | 30243386 | For measuring plant tissue weight, standards and microfuge tubes |
Methanol (100 %) | Fischer Scientific | 67-56-1 | Used in the protocol |
Microfuge tubes (2 mL) | Microcentrifuge | Z628034-500EA | Containers for extraction of lignin |
Plant biomass gerinder | Hanchen | Amazon | Used for crushing dried samples |
pH meter | Fisher Scientific | AE150 | Measuring pH for solutions prepared for lignin extraction |
Temperature controlled incubator/oven | Fisher Scientific | 15-015-2633 | Used in the protocol |
Thioglycolic acid (TGA) | Sigma Aldrich | 68-11-1 | Used in the protocol |
Vacuum dryer | Eppendorf | 22820001 | Used for drying samples |
Vortex mixer | Eppendorf | 3340001 | For proper mixing of samples |