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

Zhiwen Wang,Xiaotian Zhu,Peter J Deuss

doi:10.1016/j.indcrop.2021.113493

Abstract

Methodologies for the high-yield recovery of lignin with retention of its native CO 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.

Highlights

Lignin is a highly abundant natural aromatic polymer which ac counts for 18–35 % weight of lignocellulosic biomass and of great interest as for the production renewable aromaticpolymers (Rinaldi et al, 2016)
Cellulose and hemi cellulose amounts were significantly reduced in the first cycle but a high percentage of saccharide was still retained in the residues of the four biomass samples: pine (32.4 %) > reed (28.4 %) > walnut shell (25.9 %) > birch (19.7 %), in which hemicellulose content was relatively harder to be removed than cellulose
The enhancement of ball milling and enzyme addition for hemicellulose removal depended on the biomass type, while the relative enzymatic recalcitrance of the four biomass was different with each other and followed a roughly the order of walnut shell > reed ≈ pine > birch, which made the optimized con ditions for residual enzyme lignin (Rel) isolation of the four biomasses different

Summary

Introduction

Lignin is a highly abundant natural aromatic polymer which ac counts for 18–35 % weight of lignocellulosic biomass and of great interest as for the production renewable aromatic (co)polymers (Rinaldi et al, 2016) It is build-up out of p-hydroxyphenyl, guaiacyl, and syringyl moieties connected by different linking motifs that arise from the radical nature of the biosynthesis from the parent monolignols of which the aryl β-aryl ether (β-O-4) motif is dominant (Humphreys and Chapple, 2002; Ragauskas et al, 2014; Ralph et al, 2008; Rinaldi et al, 2016). An extremely high molecular weight (MW, ~100,000 g/mol) compared to lignins isolated by extraction (~2000 g/mol) can be obtained by enzymatic mild acidolysis (Guerra et al, 2006). It is important to develop proper protocols for the isolation of high MW native-like lignins and understand how closely these resemble the native lignin that is part of the parent biomass sample

Objectives

Results

Conclusion