A brand-new integrated biological saccharification process using wood-based cellulose

The economic viability and environmental sustainability of lignocellulosic bioconversion have long been the primary barriers to the large-scale utilisation of agricultural and forestry waste, such as crop straw. Currently, the mainstream strategy for lignocellulosic bioconversion is the simultaneous saccharification and fermentation (SSF) process based on free cellulases; however, the core enzyme technology is monopolised by foreign companies, and the cost of enzymes is difficult to reduce further, rendering existing processes uncompetitive in the market. Consolidated bioprocessing (CBP) is a lignocellulosic conversion strategy proposed in recent years. It integrates the production of cellulases, the enzymatic hydrolysis of lignocellulosic substrates and the fermentation of final products within a single reactor, offering advantages such as a simplified process, reduced costs and lower equipment requirements. However, as the CBP strategy involves carrying out multiple steps simultaneously within a single reactor, it requires a compromise in reaction conditions, making it difficult to achieve high levels of enzyme production, hydrolysis and fermentation simultaneously. Furthermore, the end product is a single compound and difficult to modify, which significantly limits its scope of application.

To this end, the Metabolomics Research Group at the Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, took a novel approach and proposed a completely new strategy for the consolidated bio-saccharification (CBS) of lignocellulose based on a whole-cell catalyst comprising fibrillosomes [Liu S, et al, 2019, 12(1):35]. This strategy organically integrates enzyme production with the hydrolysis step, whilst maintaining a degree of separation in the downstream fermentation stage. With fermentable sugars as the target products, it offers advantages such as low enzyme costs, a simple process and flexible downstream output options, and is expected to find widespread application in the bioconversion of lignocellulose to produce various chemicals, functional foods and pharmaceuticals.

Fibrilles are supramolecular complexes secreted by anaerobic microorganisms such as Clostridium thermocellum that can efficiently degrade lignocellulose; they are among the most efficient lignocellulose degradation systems known in nature. The Metabolomics Research Group has long been committed to research into the genetic modification, mechanisms of action and metabolic engineering of cellulolytic bacteria such as Clostridium thermocellum and their fibrils. Utilising genetic manipulation hardware and software tools independently developed by the group, systematic studies have been conducted on the physiology and biochemistry of Clostridium thermocellum, the assembly of fibrils, synthesis regulation and product inhibition, as well as product uptake and metabolism. This has led to an in-depth understanding of the functional mechanisms and regulation of Clostridium thermofibrum and its fibrils. Building on this foundation, in 2017 the group successfully developed the first whole-cell catalyst based on fibrils, achieving the efficient conversion of lignocellulosic substrates into fermentable sugars [Zhang J, et al, 2017, 10(1):124]. A preliminary prototype of the CBS process was established; however, issues such as insufficient resolution of product inhibition, reduced yields of key enzymes and slow saccharification rates remain.

To address these issues, the research team optimised the second-generation whole-cell biocatalyst by re-engineering the fibrous bodies in situ. They also optimised key factors affecting process efficiency—such as culture medium composition, inoculum volume, seed culture and substrate loading—thereby significantly improving saccharification efficiency and reducing saccharification time. Under optimal conditions, using pre-treated wheat straw as the substrate, the total saccharification process time was reduced by 50 per cent, with a sugar yield of 0.795 g/g and a sugar recovery rate of 89.3 per cent (as shown in the figure). When combined with a compatible lignocellulose pre-treatment process developed by this laboratory, this process significantly reduces the cost of lignocellulose saccharification and is now ready for industrial application.

This work provides a new whole-cell biocatalyst and a corresponding optimised process for the implementation of CBS, confirming that CBS is a viable strategy for the low-cost, high-efficiency utilisation of lignocellulose. The Metabolomics Research Group reported this process strategy in a research paper published online on 18 February in *Biotechnology for Biofuels*, and formally proposed the concept of ‘Integrated Biocellulase (CBS)’. The Metabolomics Research Group is currently scaling up the CBS process to pilot scale and establishing an industrial demonstration system based on this process, which is expected to significantly advance the industrialisation of lignocellulose bioconversion.

Liu Shiyue, a PhD student in the research group, and Liu Yajun, an associate researcher, are the co-first authors of the paper, whilst Research Fellow Cui Qiu is the corresponding author. This work was supported by the Strategic Priority Research Programme of the Chinese Academy of Sciences, the National Natural Science Foundation of China, and the Shandong Provincial Natural Science Foundation.该论文的共同第一作者是研究小组的博士生刘诗悦和副研究员刘亚君，通讯作者为研究员崔秋。本研究得到了中国科学院战略性重点研究项目、国家自然科学基金以及山东省自然科学基金的支持。