The effect of ball milling on birch, pine, reed, walnut shell enzymatic hydrolysis recalcitrance and the structure of the isolated residual enzyme lignin

Abstract Methodologies for the high-yield recovery of lignin with retention of its native C O bonded structure is an essential prerequisite for many novel high-end lignin applications. Enzymatic residual lignin isolation is such a methodology that leaves the lignin untouched by using enzymatic desaccharification. Thus, a series of representative lignocellulose substrates (birch, pine, walnut shell and reed) were evaluated for effective native lignin isolation, with emphasis on the effect on the lignin structure and purity. The effect of enzyme loading and ball milling severity were studied by tracking residual saccharides and the structural integrity of the isolated lignin. Prolongation of ball milling time could achieve a higher carbohydrate removal and avoid the loading of extra enzyme. However, the application of two or more steps of enzymatic hydrolysis with higher enzyme loading and short ball milling time was shown as an alternative to long ball milling time to achieve similar carbohydrate removal and avoid extensive decrease of the lignin molecular weight (MW). This MW decrease was caused by breaking of some linkages, but not too a large enough extent to cause significant differences in the 2D HSQC NMR spectra. The recalcitrance towards increased enzyme hydrolysis activity by ball milling was different for the four representative biomasses and followed an order of walnut shell > reed ≈ pine > birch by comprehensive analysis the obtained data. Overall, the results showed a clear two-way synergy between enzymatic treatment and ball milling efficiency to isolate lignin with high yield, high native linkage content, purity and minimal MW reduction.