Can Lignin Be Accurately Measured?

too simplistic (Sarkanen and Ludwig, 1971), and there are now many examples showing that other phenolics Forages serve an important role in providing nutrients to ruminants besides the cinnamyl alcohols can be incorporated into while providing positive benefits to the environment. Forage cell wall digestibility is incomplete because of several structural features within lignins (as reviewed by Sederoff et al., 1999). From a the wall, but digestion is mostly inversely correlated with the amount functional point of view, lignins impart strength to cell of lignification that has occurred during cell wall development. Lignin walls, facilitate water transport, and impede the degrais a hydrophobic polymer formed through enzyme-mediated radical dation of wall polysaccharides, thus acting as a major coupling of monolignols, mainly coniferyl and sinapyl alcohols. The line of defense against pathogens, insects, and other herpolymer is highly resistant to degradation and generally passes through bivores. the ruminant unmodified. Though lignin is resistant to degradation, It is important, no matter which definition you prefer, it is not easily quantified within various types of forages. Numerous to be able to determine the concentration of lignin methods have been developed over the years to measure lignin levels within a wide range of cell wall types. Since lignin is in different plant species. Most frequently used among workers infairly resistant to both chemical and biological degradavolved with forage development or utilization are the acid detergent, Klason, and permanganate lignin methods. More recently, acetyl brotion, one would think that it would be relatively easy mide has received attention as a possible lignin determination method. to measure (Sarkanen and Ludwig, 1971). Lignin has The acetyl bromide method is dependent on determining the abbeen defined, at least in general chemical terms, for sorbance of the extract in which all the lignin of a sample has been 50 yr and several well-defined procedures to quantify dissolved. Each of these methods gives different lignin values for the lignin in plant tissues have been developed and apsame type of forage sample. For example, acid detergent, Klason, proved as AOAC (Association of Analytical Communipermanganate, and acetyl bromide lignin methods give quite different ties International formerly Association of Official Anavalues for alfalfa stems: 93, 145, 158, and 135 g lignin kg 1 cell wall, lytical Chemists) or standard wood chemistry methods. respectively. These differences can be even greater for grasses: 25, Of the several types of methods available to determine 77, 45, and 92 g kg 1 cell wall from corn (Zea mays L.) stalks analyzed the lignin in plant samples, not one would be considered by acid detergent, Klason, permanganate, and acetyl bromide lignin methods, respectively. This paper will discuss the different lignin dea standard unambiguous method for all samples. With termination methods and highlight the advantages and disadvantages the increased interest in altering lignin quantity and of each as they relate to forage sample analysis. composition for a variety of reasons (increased ease of pulping, digestibility, etc.), plus availability of molecular techniques to accomplish this task, come the following L is the biochemical process of forming questions. What method should be used to measure the collective of phenylpropanoid macromolecules lignin? Does changing the lignin composition by adding termed lignin (Sarkanen and Ludwig, 1971). Lignin can unique phenolics alter the properties sufficiently to warbe defined from a chemical point of view (i.e., its chemirant concern with the reliability of a favorite method? cal composition and structure) or from a functional view We will review the different types of lignin methods that stresses what lignin does within the plant. It has that have been used, focusing on the main procedures been recognized for over 50 yr that lignin is a polymeric that are used for herbaceous plants. material composed of phenylpropanoid units derived primarily from three cinnamyl alcohols (monolignols): Methods for Determining Lignin p-coumaryl, coniferyl, and sinapyl alcohols (Sarkanen There have been numerous methods developed and and Ludwig, 1971). With the development of molecular modified through the years to quantitatively determine techniques, it has been possible to manipulate the lignifithe amount of lignin that is in a given type of plant cation process by altering enzymes within the monoligtissue. Methods for determining lignin in samples can nol biosynthesis pathway resulting in the incorporation be divided into two basic categories, those that rely on of nontraditional residues into lignin polymers (Sederoff a gravimetric determination and those that are based et al., 1999). The general chemical definition might be on a noninvasive method. Many procedures that have been developed (modified) through the years have their Ronald Hatfield, USDA-ARS US Dairy Forage Research Center, roots in the wood chemistry and paper and pulp indus1925 Linden Drive West, Madison, WI 53706; Romualdo S. Fukushima, Faculdade de Medicina Veterinária e Zootecnia da Universitries. In some cases, they have been adapted, or attempts dade de São Paulo, Av. Duque de Caxias-Norte, 225, 13635-900, have been made to adapt them, to be used with herbaPirassununga, SP, Brazil. This paper was originally presented at the ceous samples. Some of the methods discussed here Lignin and Forage Digestibility Symposium, 2003 CSSA Annual require that the plant sample be subjected to some form Meeting, Denver, CO. Received 15 Apr. 2004. Forage & Grazing Lands. *Corresponding author ([email protected]). of extraction to remove potentially interfering compounds. This probably is the one step that has been Published in Crop Sci. 45:832–839 (2005). modified the most in terms of adapting procedures dedoi:10.2135/cropsci2004.0238 veloped for woody species (or wood derived samples) © Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA for use with herbaceous samples. As always, there are 832 Published online March 28, 2005