I have no experience with fungi, but in general you need to remove all non structural cellular material prior to hydrolysies. In original NREL procedure you get rid off pectines, starch and proteins as well as extractives and ash prior to lignins determination. Another issue could be that fungi produce some sort of compounds that absorbed at the same wavelength as ASL or due to the treatment condensation of furanics from carbohydrates is accelerated and you get rise in humins (pseudo lignin) that will increased your AIL.

Thanks Maxim. I’ll try to prolong the extraction time first to make sure complete removal of the extractive components.

Dear Idris Musa Tanimu , interesting question and discussion on pseudo lignin ( Maxim V. Galkin ). Do you have any additional advice for Mohamad Zaihan Zailan , Idris?

Among the factors that may affect the quality /quantity of lignin extracted from biomass are: (1) Extractives present (2) Particle size of biomass (3) Inadequate drying (4) inadequate washing of final lignin residue.

A knowledge of the substrate type is important here as well as your main target product (AIL or ASL?). Was the untreated substrate adequately characterized to determine its chemical components in comparison with the treated before subjecting to lignin extraction?

My assumption is that certain components in the original substrates (e.g. extractives) that could interfere with lignin extraction were utilized by the fungi, resulting in a better yield or "increase". Find out also what other factors may have contributed to the increase: Is the increase due to ash content or inadequate hydrolysis of Holocellulose and or inadequate washing step? If the ash content of your lignin is significant can you carryout a balance of ash content between initial substrate, treated substrate and final lignin residue? This in conjunction with FTIR may reveal the source of ash as well as actual lignin quantity/ quality. All the best