Development of agarose gel electrophoresis as a novel method to monitor lignin degradation

Lignin represents the sole bulk alternative source of sustainable aromatic platform chemicals that are otherwise produced by the petrochemical industry (Gillet et al., 2017). Currently there is no high-throughput analytical technique that can be used to quantify or assess the degradation of polymeric lignin. This has significantly held back research within this field, as information about polymeric lignin is crucial if a process is to be developed to efficiently valorise it. The aim of this project was to use agarose gel electrophoresis to develop a novel technique to quantify and assess changes in the structure of polymeric lignin. Another aim was to utilise agarose gel electrophoresis to assess the quantity of low molecular weight aromatic products produced by lignin degradation. Agarose gel electrophoresis has, for the first time, been shown in this study to separate effectively polymeric lignin from low molecular weight components. Electrophoretic variables such as pH, voltage, gel-type, buffer, level and edge- effects have been explored and optima determined. Migrating bands have been characterized and confirmed by multiple methods, as lignin. Band visualisation and quantification techniques have been developed to quantify and assess the polymeric fraction of a lignin sample. Purified samples of lignin have been prepared by cutting bands from the gel and extracting into solution. The method has been shown to separate Kraft, protobind and organosolv lignins corresponding to their respective molecular weight to charge (m/z) distribution, and the results were closely comparable to laborious analyses using Gel Permeation Chromatography (GPC). Electrophoresis with 1% agarose gel in sodium borate buffer at pH 8.75 was found to be highly effective in assessing the concentration and m/z distribution of polymeric lignin after biological, chemical and hydrothermal degradation. Trace profiles of the imaged bands show possible differences in the mechanisms of degradation and indicate lignin shape as another mobility factor. High throughput methods were developed with 40 simultaneous lanes. The method was also shown to be successful in separating low molecular weight products from polymeric lignin, and therefore has the potential to be used to assess the production of platform chemicals.