Consolidated bio-saccharification: Leading lignocellulose bioconversion into the real world

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Research review paper

Consolidated bio-saccharification: Leading lignocellulose bioconversion into the real world ⁎

⁎

Ya-Jun Liua,b,c, , Bin Lia,b,c, Yingang Fenga,b,c, , Qiu Cuia,b,c,

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a

CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, China b Dalian National Laboratory for Clean Energy, Dalian, China c University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing, China

A R T I C LE I N FO

A B S T R A C T :

Keywords: β-glucosidase cellulase cellulosome Clostridium glucose lignocellulose pretreatment saccharification

Lignocellulosic biomass is the most abundant sustainable carbon source on the planet and has enormous potential to substitute fossil resources on the premise of cost-effective conversion. Efforts have been made to develop various lignocellulosic bioconversion strategies to overcome biomass recalcitrance, promote product conversion efficiency and reduce process cost. Consolidated bio-saccharification (CBS), a consolidated bioprocessing (CBP) derived strategy, is herein proposed for lignocellulose bioconversion by integrating enzyme production and hydrolysis steps but separating fermentation from the integrated process. This strategy employs cellulosome-producing microorganisms as a biocatalyst to enhance lignocellulose solubilization and produces lignocellulose-derived fermentable sugars as a platform product for fermentations aiming at various products. The success of CBS depends on robust biocatalysts with high activity, suitable pretreatments for efficient delignification, and downstream fermentations with process compatibility. The review introduces the updated progress on lignocellulose bioconversion following the CBS route, discusses key factors for optimization of the CBS process, and, more importantly, highlights challenges and promising solutions for the CBS strategy in the industrial application of lignocellulose bioconversion.

1. Introduction Plants produce biomass photosynthetically by fixing solar energy and carbon dioxide. Lignocellulose, the major biomass component, comprises about half of the plant dry matter (Sanchez, 2009). The world annual production of lignocellulosic biomass is estimated to be over 200 billion tons, and lignocellulose is considered the most abundant sustainable carbon resource on the planet (Michelin et al., 2013). Lignocellulosic residues, especially the agricultural lignocellulosic wastes, represents one of the best substitutes of fossil resources, because of its low price, high availability and wide distribution (Taha et al., 2016). The products of crop photosynthesis are half in the seed and half in the straw, so enhanced utilization of agricultural lignocellulosic wastes would come from the latter half of the valuable organic agricultural resource. The development of fuels and chemicals from lignocellulosic

wastes has become a worldwide research hotspot (Alonso et al., 2012) and is attracting significant attention from governments worldwide. The US Energy Independence and Security Act requires 36 billion gallons of biofuels by 2022. The European Union also set a target of using up to 18% of the total agricultural land in the EU for biofuel production by 2030. In both cases, about 25% (v/v) of the transportation fuels are required to be replaced by biofuels for energy security and lower greenhouse gas emissions (Biofuels Research Advisory Council, 2006; Public law 110-140, 2007). However, over 90% of bioethanol is currently produced from corn instead of lignocellulose in the US (Renewable Fuels Association, 2019). China is the largest agricultural country in the world, and its annual production of agricultural wastes has thus far exceeded 900 million tons. The country has been promoting effective conversion and application of agricultural wastes through laws, but the scale of cellulose ethanol is only about 0.1 million tons, and approximately 200 million tons of crop straws are still incinerated

Abbreviations: AFEX, ammonia fiber explosion/expansion; BGL, β-glucosidase; BSES, biological simultaneous enzyme production and saccharification; CBP, consolidated bioprocessing; CBS, consolidated bio-saccharification; CELF, co-solvent-enhanced lignocellulosic fractionation; CT, co-treatment; SHF, separate hydrolysis and fermentation; SSCF, simultaneous saccharification and co-fermentation; SSF, simultaneous saccharification and fermentation ⁎ Corresponding authors at: CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, China. E-mail addresses: [email protected] (Y.-J. Liu), [email protected] (B. Li), [email protected] (Y. Feng), [email protected] (Q. Cui). https://doi.org/10.1016/j.biotechadv.2020.107535 Received 11 December 2019; Received in revised form 3 February 2020; Accepted 12 February 2020 0734-9750/ © 2020 Elsevier Inc. All rights reserved.

Please cite this article as: Ya-Jun Liu, et al., Biotechnology Advances, https://doi.org/10.1016/j.biotechadv.2020.107535

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and fermentation simultaneously to reduce capital cost and contamination risk (Galbe and Zacchi, 2002; Öhgren et al., 2007). CBP integrates all bioconversion steps to further reduce the investment cost and simplify the process (Vinuselvi et al., 2014). Both CT and CBS are CBP-derived strategies. CT combines the CBP process with aggressive mechanical milling. Rather than further integration, CBS separates fermentation from the CBP process to avoid the compromise of different reaction conditions, and produces fermentable sugars as the products instead of solvents and other end metabolites (Liu et al., 2019b). Current lignocellulosic bioconversion strategies mainly differ in the integration of the steps by considering technical feasibility and cost efficiency of the whole process. Lignocellulose is mainly considered an alternative carbon source for bioconversion, which means lignocellulose-derived sugars must compete with starch-based sugar in the real world. Therefore, the cost competitiveness of the saccharification process is critical for a promising strategy in practical lignocellulose application, which requires rational technology development rather than straightforward combination of available technology with high cost or low compatibility. In this review, we will first briefly present each of these bioconversion strategies, and then focus on the CBS route to discuss the technical factors that play a key role. The current challenges and promising solutions, which can help improve CBS to lead the lignocellulose bioconversion into the real world, will also be discussed.

in situ every year (Zhang et al., 2016). The low-rate of development of the lignocellulosic industry is mainly due to the low economic and environmental benefits (Liu et al., 2019a). Lignocellulosic biomass is recalcitrant and difficult to deconstruct (Himmel et al., 2007; Zhang et al., 2016). Lignocellulose contains lignin, hemicellulose, and cellulose as the main components. Although, the proportions of the components vary from different biomass sources (Michelin et al., 2013; Singh et al., 2014; Van Dyk and Pletschke, 2012), generally, cellulose is main constituent of plant cell walls and represents up to about 50% of the cell wall matter with D-glucose as the repeating unit. The glucose units, linked by β-1,4 glycosidic bonds, are tightly bound together to form the crystalline structure by extensive intramolecular and intermolecular hydrogen bonding networks (Tian et al., 2018; Van Dyk and Pletschke, 2012). Thus, cellulose degradation is one of the main obstacles for lignocellulose bioconversion (De and Luque, 2015). Hemicellulose is the second most abundant polymer, mainly in the form of xylan (Beg et al., 2001). Unlike cellulose, hemicelluloses have a random and amorphous structure, composed of both C5 and C6 units. Hemicelluloses can interact with cellulose fibers and lignin by hydrogen and chemical bonds, respectively, to form complex linking networks (Tian et al., 2018). Lignin is a three-dimensional polymer of monolignol units and can strengthen the whole lignocellulose structure. The three primary units include syringyl (S), phydroxyphenyl (H), and guaiacyl (G) units, which are linked by aryl ether or C-C bonds (Zeng et al., 2014). Both hemicellulose and lignin, especially lignin, have a negative effect on cellulose accessibility and result in low hydrolysis rates. Compared to cellulose and hemicellulose, lignin is difficult to be degraded and assimilated by organisms, and no evidence has revealed any organism that can grow using lignin as the sole carbon source (Ding et al., 2012). Thus, pretreatment is generally required for delignification to enhance enzymatic hydrolysis of polysaccharides to fermentable sugars. Lignocellulose bio-refinery is mainly composed of two platforms, the syngas platform and the sugar platform. The syngas platform, also called thermochemical platform, transforms biomass to hydrogen, carbon monoxide and carbon dioxide as the substrate for downstream fermentation (Molitor et al., 2017). In terms of the sugar platform, lignocellulosic sugar is considered to be the important intermediate to produce other biofuels and biochemicals (Arevalo-Gallegos et al., 2017). Both chemical and biological methods have been developed for lignocellulose conversion, and bioconversion strategies are preferred taking into account environmentally friendly requirements. Complete deconstruction of lignocellulose to fermentable sugars requires an enzyme cocktail containing cellulases, hemicellulases, and ligninases (Gupta et al., 2016). Cellulases include three primary components: endoglucanase, exoglucanase, and β-glucosidase, which together play a major role in cellulose hydrolysis (Morag et al., 1991). Hemicellulases such as xylanases are indispensable for breaking glycosidic linkages in hemicellulose molecules during saccharification (Beg et al., 2001). Ligninases (peroxidases and laccases) and other accessory enzymes like lytic polysaccharide monooxygenases (Hemsworth et al., 2015) and expansin-like proteins (Artzi et al., 2016; Chen et al., 2016) are also necessary to increase cellulose accessibility for effective saccharification. Two types of enzyme cocktails have mainly been used for lignocellulose saccharification, the free cellulases and the cellulosome system, mainly produced by fungi and anaerobic bacteria, respectively. The feasibility and economic efficiency of industrial production would be crucial issues in terms of bioconversion (Zhang et al., 2016), which generally include enzyme production, enzymatic hydrolysis and fermentation processes besides pretreatment (Taha et al., 2016). Different strategies have been developed for lignocellulose bioconversion, including separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), consolidated bioprocessing (CBP), co-treatment (CT), and consolidated bio-saccharification (CBS) (Fig. 1). SHF is designed to perform enzymatic hydrolysis and fermentation sequentially, whereas SSF performs enzymatic hydrolysis

2. Current strategies for lignocellulose bioconversion It is agreed that the major current limitation of the cellulosic bioethanol and other biochemicals at the commercial scale is the degradation of lignocellulosic biopolymers into reducing sugars (Taha et al., 2016). In other words, enzymatic hydrolysis is one of the most crucial and costly steps in lignocellulose bioconversion. Thus, the cost and efficiency of the enzymes, or biocatalysts, used for lignocellulose hydrolysis, is of great concern (Chandel et al., 2019; Passos et al., 2018). According to how and what kind of biocatalysts are produced for enzymatic hydrolysis, we hereby divide current lignocellulose bioconversion strategies into two types: off-site and on-site saccharification approaches (Fig. 1). On-site saccharification means that the lignocellulosic enzymes are produced together with the hydrolysis process, while off-site saccharification indicates that the enzymes are produced earlier, prepared under specified conditions, and supplemented into the saccharification system. 2.1. Off-site saccharification Both SHF and SSF mainly employ free cellulases derived from fungi as the biocatalysts to perform off-site saccharification (Guo et al., 2018a; Liu et al., 2019a). SHF is designed to perform enzymatic hydrolysis and fermentation sequentially (Akhtar et al., 2013), which is considered the most straightforward strategy that combines available techniques according to the needs of multi-step lignocellulose bioconversion. When the two process steps are combined and performed simultaneously, it is referred to as simultaneous saccharification and fermentation. It is reported that SSF enjoys an advantage over SHF in terms of low capital cost and contamination risk (Galbe and Zacchi, 2002; Öhgren et al., 2007). To further stimulate the conversion of fermentable sugars, SSCF was adapted for simultaneous saccharification and co-fermentation of both pentose and hexose sugars on the basis of SSF (Chandrakant and Bisaria, 1998). Trichoderma reesei has been considered one of the best cellulase producers (Bischof et al., 2016). Novozymes, Inc. is the major provider of commercialized enzyme cocktails so far (Chandel et al., 2019). Several facilities, such as Beta Renewables in Italy, GranBio in Brazil, Poet-DSM Advanced Biofuels in Iowa, Abengoa refinery in Kansas, DuPont refinery in Iowa, and Tianguan Biofuel Ethanol in China, have been constructed globally to produce cellulosic ethanol following the off-site saccharification approach, 2

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Lignocellulose bio-conversion strategies ON-SITE saccharification

OFF-SITE saccharification

Biomass Advanced biofuels

Pretreatment

Biochemical

Co-treatment Fungal cellulase

Fungal cellulase

Fungal/bacterial biocatalyst

Cellulosome biocatalyst

Cellulosome biocatalyst Hydrolysis

Hydrolysis

Hydrolysis

Hydrolysis

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Fermentation

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Fermentation

Protein Fatty acids Other products

Fermentable sugars

Bioethanol

Separate Hydrolysis & Fermentation

Simultaneous Saccharification & (Co)Fermentation

Consolidated BioProcessing

CoTreatment

Downstream Fermentation

Consolidated BioSaccharification

Fig. 1. Schematic representation of current lignocellulose bioconversion strategies. According to how the biocatalysts are produced for saccharification, the developed strategies are divided into two types: off-site saccharification and on-site saccharification. The former includes separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF), both of which use free cellulases derived from fungi as the biocatalysts. The latter strategy refers to consolidated bioprocessing (CBP) and its derivates, i.e., co-treatment (CT) and consolidated bio-saccharification (CBS). Among the known approaches, only CBS determines fermentable sugars as the target product, which can be further used as the substrate in downstream fermentations aiming at various bio-products. Red dash frames indicate integrated steps.

fermentation products. More recently, the CBS strategy has been developed by separating the fermentation step from the integrated CBP process (Liu et al., 2019b; Zhang et al., 2017) and focuses on the costeffective production of lignocellulosic fermentable sugars rather than the end metabolites.

using aerobically pre-produced fungal cellulases as the biocatalyst The baseline cost for fungal cellulase production is considered to be about $10 per kg protein (Klein-Marcuschamer et al., 2012). If the sugar yield is 80% and a loading of 10 FPU/g cellulose (about 20 mg cellulase/g cellulose) is used, the cost of cellulase is about $250 per ton sugar. Even more enzymes are required for saccharification of high solid loading substrates (Cara et al., 2007; Hodge et al., 2008), which will further increase the cost. On the other hand, cellulosic ethanol production is not feasible if the sugar production cost is more than $100 per ton (Chandel et al., 2019). With the ultimate goal of reducing enzyme cost for off-site saccharification, many efforts have been made to enhance enzyme production and to isolate new enzymes with high thermostability, product tolerance and activity (Chandel et al., 2019; Maki et al., 2009; Viikari et al., 2012). However, the cost of fungal enzymes is still the main bottleneck that constrains the development of lignocellulose bioconversion (Lynd et al., 2017; Taha et al., 2016). In addition, the long and complex preparation period, transportation, and high consumption of carbon and nitrogen sources for fermentation need to be taken into account for the off-site saccharification strategy, and contamination risk is also not negligible (Chandel et al., 2019). Therefore, further improvement or development of novel technology is still required for its real application.

2.2.1. Consolidated bioprocessing (CBP) Compared to SSF, CBP further integrates enzyme production to perform on-site saccharification, thus relying on efficient biocatalysts which can simultaneously perform lignocellulose degradation and sugar conversion to designed products (Lin et al., 2015; Lynd et al., 2005; Xu et al., 2009). Ideally, CBP microbes should have specific traits, including highly efficient cellulase production and secretion for rapid solubilization of lignocellulose, simultaneous assimilation of C5 and C6 sugars, and tolerance to lignin-derived toxins and the end metabolites (Vinuselvi et al., 2014). Various CBP biocatalysts have been designed and constructed following natural or recombinant strategies (Olson et al., 2012). The native strategy involves the enhancement of the yield and titer of certain products by genetic modification of cellulolytic microorganisms (Argyros et al., 2011; Chung et al., 2014; Passos et al., 2018). The recombinant strategy involves expression of heterologous cellulases and hemicellulases in host organisms, such as Saccharomyces cerevisiae, that can produce target products (Guo et al., 2018b; Matano et al., 2012; Tabañag et al., 2018). Some model microorganisms, like Escherichia coli and Bacillus subtilis that can neither degrade cellulose nor produce certain products, have also been used to construct CBP biocatalysts, because they can co-utilize hexose and pentose, and their genetic manipulation is relatively easy (Wood et al., 1997; Zhang and Zhang, 2010). Considering the complexity of lignocellulosic enzymes and the potential burden for recombinant cells to grow rapidly, approaches based on non-cellulolytic chassis are challenging (Olson et al., 2012). Furthermore, the reported recombinant cellulase-producing strains cannot grow using cellulose as the sole carbon source without additional nutrients such as yeast extract. Thus, the native strategy shows more feasibility towards practical application. Hydrolysis rates of cellulases generally increase with increased temperature, but solventogenic organisms usually cannot tolerate high temperatures (Vinuselvi et al., 2014). Thus, it is difficult for CBP to

2.2. On-site saccharification In order to meet the requirements of industrial production for simple process and low operating cost, the on-site saccharification approaches, including CBP and CBP derivatives have been proposed (Guo et al., 2018a; Lynd et al., 2017; Lynd et al., 2005; Passos et al., 2018; Philippidis et al., 1993). CBP, which was proposed at the beginning of the 21st century by Lynd’s group (Lynd et al., 2005; Lynd et al., 2002), is distinguished from off-site saccharification strategies. All transformations from solid substrates to target products, including the step for cellulase production, occur in a single step and a single reactor for CBP, which can greatly reduce the cost of enzyme production (Lynd et al., 2005). Subsequently, CT was proposed as an updated CBP technology with enhanced degradation efficiency of the lignocellulosic substrates (Balch et al., 2017). Both CBP and CT mainly emphasize the production of cellulosic ethanol and are also applicable theoretically to other 3

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engineering and rewiring (Cheng et al., 2019; Yadav et al., 2012; Yu et al., 2019). For example, Ichikawa et al. reported that C. thermocellum can produce decanol and dodecanol with the expression of a fatty acyl–acyl carrier protein reductase and an aldehyde-deformylating oxygenase from Synechococcus elongatus PCC 7942 (Ichikawa and Karita, 2015). In addition, C. thermocellum has also been used to produce CBP isobutanol (5.4 g/L) and butanol (0.36 g/L) by enhancing the native pathway and introducing heterologous enzymes into the cell (Holwerda et al., 2014; Lin et al., 2015; Tian et al., 2019).

develop compatible reaction conditions that are optimal for both saccharification and fermentation processes. Hence, although several clostridial strains, such as Clostridium cellulolyticum (Cui et al., 2014; Xu et al., 2015), C. papyrosolvens (Zou et al., 2018), C. cellulovorans (Bao et al., 2019; Yang et al., 2015) and C. josui (Jindou et al., 2002) that can degrade cellulose and produce ethanol are considered CBP candidates, the thermophilic cellulolytic bacterium, Clostridium thermocellum (also named as Hungateiclostridium thermocellum, Ruminiclostridium thermocellum), remains the unrivaled choice as a CBP organism, because of its thermal growth conditions and its capacity for simultaneous cellulose degradation and ethanol production in a “one-pot” process (Holwerda et al., 2019; Maki et al., 2009). It is reported that C. thermocellum can produce 22.4 g/L of ethanol from 60 g/L of microcrystalline l cellulose (Avicel) as the substrate. This represents 75% of the maximum theoretical yield, and the potential sugar conversion ratio was proposed to be up to 85% with further genetic engineering (Tian et al., 2016). To the best of our acknowledge, the highest ethanol titer produced by C. thermocellum is 26.7 g/L which consumes 100 g/L Avicel, but the yield is reduced (Hon et al., 2017). This may due to an increase in the inhibition of cell growth and hydrolysis as the concentration of ethanol increases (Rani et al., 1996). Co-cultivation of cellulolytic and solventogenic microorganisms has also been reported to produce ethanol with higher titer. In this approach, cellulolytic biocatalysts solubilize lignocellulosic components to reducing sugars, which are used as carbon sources by the solventogenic bacteria to produce ethanol at a relatively high titer. For example, 38 g/L of ethanol was produced from 92 g/L of Avicel by an engineered coculture of C. thermocellum and Thermoanaerobacterium. saccharolyticum (Argyros et al., 2011; Park et al., 2012; Zuroff et al., 2013). It should be noted that productivity may be limited by the non-solventogenic microorganism in the coculture due to product tolerance. Taking C. thermocellum as an example, cell growth is reduced significantly with increasing ethanol concentration (Rani et al., 1996), although the ethanol tolerance of the wild-type strain can be increased from less than 10 to 80 g/L after long-term adaption (Williams et al., 2007). The reduced biomass may eventually influence the hydrolysis of the lignocellulosic substrate. Additionally, whether or not the solventogenic partner is involved, the CBP process is dependent on the cellulolytic anaerobes and thus has to be carried out under anaerobic conditions. However, the production and fermentation processes of many valuable products only occur under aerobic conditions or require oxygen to achieve high yields. Therefore, the anaerobic CBP conditions will restrict the application of the technology and the types of potential products. Co-treatment (CT) has been proposed as a CBP-derived strategy by combining the CBP process with aggressive mechanical milling under thermophilic conditions with the cellulosome-producing C. thermocellum as the biocatalyst (Fig. 1). A specific bioreactor was constructed for CT, containing baffles and 4.8-mm stainless steel balls for milling (Balch et al., 2017). Interestingly, C. thermocellum is capable of cell growth and ethanol production in the presence of aggressive ball milling, while the fermentation of yeast (i.e., S. cerevisiae) ceases completely, which may be explained by the fact that yeast cells with bigger size are more vulnerable to mechanical milling (Balch et al., 2017). The CT strategy is effective at enhancing substrate solubilization, and the capital cost and the dependency of the payback period on the capital scale could be significantly reduced (Lynd et al., 2017). Energy consumption may contribute to a large degree to CT operating costs, especially under scale-up conditions. The feasibility of lignocellulosic biofuels like second-generation ethanol is of concern, because the energy consumed when converting biomass to ethanol may greatly exceed the output energy of the produced bioethanol. Under current conditions, CBP production of advanced biofuels, such as long-chain alcohols, fatty acids, alkanes, and alkenes, should be developed (Rabinovitch-Deere et al., 2013). Novel CBP biocatalysts have been constructed through systematic metabolic

2.2.2. Consolidated bio-saccharification (CBS) CBP has shown tremendous advantages in reducing enzyme production costs and streamlining operational processes, but this strategy also suffers from the relatively restricted applications and products produced. Owing largely to the limitation of anaerobic cultivation conditions for cellulolytic microorganisms, current CBP technology is mainly developed to produce biofuels but is difficult to couple with aerobic fermentations. To gain flexible downstream applications for lignocellulose, it is necessary to liberate fermentation from the anaerobic and high-temperature hydrolysis step. The CBS strategy has therefore been proposed whereby fermentation is separated from the integrated process. CBS produces fermentable sugars as the target product rather than end metabolites such as ethanol (Liu et al., 2019b; Zhang et al., 2017). Consequently, fermentation would not be limited by hydrolysis conditions, and the cellulolytic capability can be maximized as well. Thus, CBS provides new insight into lignocellulose bioconversion. CBS employs cellulolytic microorganisms as biocatalysts to perform on-site saccharification like CBP. Among natural cellulolytic microorganisms, cellulosome-producing bacteria are preferred because the cellulase-degrading activity of the cellulosome system is generally higher than free enzymes produced by fungi, especially towards crystalline cellulosic substrates (Johnson et al., 1982; Schwarz, 2001; Thomas et al., 2017b). Clostridium thermocellum is considered one of the best candidates for CBS, due to its highly efficient cellulosomal system (Hong et al., 2014), but targeted genetic engineering is required. Most importantly, it must be ensured that CBS biocatalysts can withstand the high concentrations of reducing sugars produced because high saccharification efficiency and sugar yield are critical for CBS to satisfy various fermentation processes. Previous studies have shown that the application of C. thermocellum and its cellulosome in lignocellulose bioconversion is mainly limited by the strong inhibition of the hydrolysate product cellobiose, and the feedback inhibition can be largely eliminated by addition of β-glucosidase (BGL) (Gefen et al., 2012; Prawitwong et al., 2013; Qu et al., 2017). Thus, the expression of BGL is one of the keys to CBS biocatalyst construction. Because C. thermocellum is difficult to manipulate compared to model microorganisms, it has taken years to develop appropriate methods and instruments for its genetic engineering (Mohr et al., 2013; Olson, 2010; Olson et al., 2015; Tripathi et al., 2010; Zhang et al., 2017; Zhang et al., 2015). By using these genetic tools, two generations of CBS biocatalyst have been constructed by fused expression of BGL with cellulosomal exoglucanases (Liu et al., 2019b; Zhang et al., 2017). In this way, high concentrations of glucose (over 88 g/L) can be accumulated for downstream fermentation (Zhang et al., 2017). The improvement of the CBS process is also critical for the enhancement of sugar yield and cost reduction, which will be introduced in detail later. CBS is of great interest because it can produce fermentable sugars at a low cost, which meets the requirement of the sugar platform of lignocellulose bio-refinery. A biological simultaneous enzyme production and saccharification (BSES) approach has also been proposed for lignocellulose saccharification based on C. thermocellum (Ichikawa et al., 2019). Unlike CBS, BSES requires the addition of BGLs in forms of either free enzyme or bacterial cells (Ichikawa et al., 2019; Prawitwong et al., 2013) and can be considered a hybrid route between on-site and off-site strategies. 4

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3. Current progress in the development of CBS

biocatalyst to eliminate the cellobiose inhibition and obtain high saccharification efficiency. Although the native C. thermocellum bacterium is capable of efficient cellulose degradation, the wild-type strains cannot yet be directly used for industrial production. It has been proven that BGL can efficiently relieve cellobiose inhibition of cellulosome enzymes (Gefen et al., 2012; Qu et al., 2017), but supplementation of free BGL proteins should be avoided according to the concept of CBS/CBP. Instead, BGLs should be produced and secreted by the biocatalyst cells as a free enzyme or a cellulosomal component. Attempts have been made to construct engineered C. thermocellum strains producing extracellular BGL proteins. Plasmidbased expression of free BGL in C. thermocellum was not successful (Zhang et al., 2017). In subsequent work, the BGL protein was fused to the key cellulosomal exoglucanase Cel48S of C. thermocellum, which might have additional benefits by forming a substrate-coupled catalyzing channel and rapidly convert produced cellobiose to glucose (Liu et al., 2019b; Zhang et al., 2017). Without the addition of off-site produced enzymes, the resultant recombinant C. thermocellum strain was used as the first-generation CBS biocatalyst and outperformed the parent strain in terms of sugar yield and saccharification efficiency (Fig. 2). Over 88 g/L reducing sugar (in glucose equivalence) was produced using 100 g/L Avicel as the substrate, which represents about 80% of the maximal theoretical yield (Zhang et al., 2017). Nevertheless, the fused protein Cel48S-BGL showed significantly decreased expression level compared to that of Cel48S in parent strain, which may result in decreased saccharification levels, because Cel48S plays key roles in cellulolytic activity of the cellulosome (Liu et al., 2018b; Olson et al., 2010). Therefore, the second-generation CBS biocatalyst was subsequently developed by fusing BGL with another cellobiohydrolase Cel9K in C. thermocellum. The expression of Cel48S was maintained at a stable level in the newly constructed biocatalyst, and the saccharification process was promoted compared to the previous generation biocatalyst. The sugar yield increased further to 89.3% of the theoretical yield with pretreated wheat straw as the substrate through process optimization (Liu et al., 2019b).

The concept of CBS is derived from the CBP strategy, whereby CBS focuses on the conversion of lignocellulosic polysaccharides into fermentable sugars. The produced fermentable sugars should compete with starch-derived sugar for the fermentation of various products, including biofuels, biochemicals, proteins, oil, etc. (Fig. 1). Therefore, in order to lead the newly proposed lignocellulose bioconversion strategy to practical application, the key factors that may affect saccharification efficiency, sugar yield and cost have to be improved. So far, several signs of progress have been made within the framework of CBS. 3.1. CBS biocatalysts CBS requires biocatalysts that are capable of cellulase production and cellulose solubilization simultaneously. Thus, microorganisms that produce highly efficient cellulosomes could be promising CBS biocatalysts. Compared to fungal cellulases, the cellulosome is a highly-organized multiprotein supramolecular complex, containing both enzymatic subunits and non-catalyzing scaffoldins and is mainly produced by anaerobic bacteria (Artzi et al., 2017; Bayer et al., 2004; Bayer et al., 2008; Fontes and Gilbert, 2010). The catalytic enzymes contain type I dockerin modules to interact with type I cohesin modules of the primary scaffoldins (Xu et al., 2016). The primary scaffoldins may also have type II dockerin modules to interact with the type II cohesin modules of the secondary scaffoldins so that cellulosomes can be further assembled together to form polycellulosomes (Bras et al., 2016). The secondary scaffoldins are known as anchoring scaffoldins if they harbor SLH domains that are embedded in the bacterial cell wall. The polycellulosomes can attach to the cell wall-anchored scaffoldins to form protuberance-like structures on the cell surface (Bayer et al., 1994; Gilbert, 2007; Shoham et al., 1999). Additionally, the cellulosomal components can recognize and interact with corresponding lignocellulosic substrates by using carbohydrate-binding domains (CBM) (Venditto et al., 2016; Walker et al., 2015). The supramolecular structure of the cellulosome allows a high load of enzymes with various functions, which is particularly suitable for the degradation of highly complex lignocellulosic substrates (Hirano et al., 2016; Yoav et al., 2017) (Fig.2). Through the interactions between noncatalytic and catalytic components in the complex system, cellulosomal synergy effects at enzyme-enzyme, enzyme-substrate, and enzyme-cell levels are formed to stimulate the cellulolytic activity (Hong et al., 2014; Lu et al., 2006; Van Dyk and Pletschke, 2012). Not only that, substrate sensing and dynamic regulation mechanisms based on alternative sigma-antisigma (SigI-RsgI) factors and other regulatory factors have been reported for different cellulosome-producing bacteria (KahelRaifer et al., 2010; Munoz-Gutierrez et al., 2016; Ortiz de Ora et al., 2018; Wei et al., 2019). That means the components and structures of the cellulosome can be adjusted in time in response to the changes of extracellular polysaccharides, which allows the cellulosome to be more flexible and effective (Li et al., 2018). Among known cellulosome-producing microorganisms, C. thermocellum is of great interest because it produces the most efficient cellulosome hitherto known (Liu et al., 2019b). Besides its efficient cellulosome system, the thermal and anaerobic growth conditions of C. thermocellum favor the cellulolytic enzymatic activity, decreases the probability of contamination, and reduces the cost of aeration and agitation (Taylor et al., 2009). However, the cellulosome of C. thermocellum contains no β-glucosidase (BGL) component and suffers from severe feedback inhibition of cellobiose (Lamed et al., 1991; Lamed et al., 1985) (Fig. 2). Although two different intracellular BGLs have been detected in C. thermocellum (Ahmed et al., 2019; Katayeva et al., 1992), they were not secreted extracellularly to participate in the cellulose degradation. The presence of extracellular BGL activity, which converts the cellobiose product to glucose, is critical for the CBS

3.2. CBS process The newly developed CBS biocatalysts are essentially living cells that produce cellulosomes, and their functional conditions are quite different from fungal cellulases used in off-site saccharification. Thus, the development and optimization of the CBS process are compatible with biocatalysts and indispensable for the reduction of cost and time, which is critical to the industrial realization of this strategy. 3.2.1. Process optimization with high hydrolysis efficiency The whole CBS saccharification process can be roughly divided into two stages, a biocatalyst-producing stage and a hydrolysis stage (Zhang et al., 2017). In the biocatalyst-producing stage, the cells grow rapidly to produce cellulases (e.g., the cellulosome) with sufficiently high cellulolytic activity to initiate and maintain the hydrolysis process. In this stage, carbon source and other nutrients are consumed for cell growth and enzyme production (Freier et al., 1988), and less reducing sugars are accumulated. It seems that the biocatalyst-producing stage can also be considered a lag phase for the second stage, the hydrolysis stage. This represents the contrast between sugar accumulation and assimilation, which may be challenging for CBS to reach a high sugar yield. Moreover, due to the “non-productive” biocatalyst-producing stage, the duration of the CBS process is prolonged, and the saccharification efficiency will be affected. Thus, the most preferential optimization of CBS is the reduction of the biocatalyst-producing stage. It turns out that the use of a relatively large inoculum (10%, v/v) can shorten the lag phase and thus promote the whole saccharification process, and the inoculum can be cultivated with pretreated natural biomass rather than expensive pure cellulose, such as microcrystalline cellulose 101 (~US $35 per kg) (Liu et al., 2019b). Glucose may also be used as the carbon 5

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Fig. 2. Schematic representation of the present- and next-generation CBS biocatalysts, based on how the β-glucosidase (BGL) protein is expressed and introduced into the cellulosome. A. The wild-type cellulosome without engineering. The cellulosome contains both enzymatic subunits and scaffoldins. The enzymes can be assembled to the scaffoldin through type I and II cohesin-dockerin interactions (Xu et al., 2016). The cellulosomal components may have carbohydrate-binding modules (CBMs) to recognize and interact with cellulosic substrates. The whole cellulosome complex can attach to the cell surface through SLH domains that interact with bacterial cell-wall components. However, the wild-type cellulosome has no BGL component and suffers from feedback inhibition of cellobiose. B. Present CBS biocatalysts. The 1st- and 2nd-generation biocatalysts have been constructed by fusing BGL with C. thermocellum exoglucanases (EG), Cel48S and Cel9K, respectively. The expression level of fused EG-BGLs decreases compared to that of EGs in the parent strain and may result in reduced saccharification efficiency of the cellulosomes. C. Next-generation biocatalysts. To avoid the reduced expression of key cellulosomal components, the next generation biocatalysts should be constructed to not only enhance BGL expression but also to maintain the expression of cellulosomal components. BGLs could be expressed as a free secreted protein with or without tag pairs that can interact with each other specifically. For example, the cellulosomal cohesin-dockerin modules can be used for BGL assembly into the cellulosome. Other interaction tags (TagI and TagII), such as the SpyTag/SpyCatcher pair that can form a stable complex through isopeptide bond formation (Zakeri et al., 2012), can also be used to apply free BGL with cellulosomal components like Cel48S.

influences the beginning of the saccharification process rather than in the later stages of the process (Liu et al., 2019b).

source for inoculum cultivation, although strain domestication is generally required to reduce the long adaptation time (Li et al., 2018). The sugar yield of CBS is closely related to the amount of substrate supplemented in the saccharification system. Thus, the effect of substrate loading on saccharification efficiency should be investigated, and the supplementing method should be optimized for CBS. Previous studies on high-load SSF processes using free cellulases as the biocatalyst indicated that the initial stage of the hydrolysis process is the key factor in determining the efficiency of saccharification, thus suggesting the importance of mass transfer and enzyme-substrate homogenization, especially with high solid loading (Hodge et al., 2008, 2009; Lavenson et al., 2012; Roberts et al., 2011). In the CBS process, by using the horizontal shaking mode, we found that relatively high substrate load (8% w/v, i.e. 80 g/L) may result in a low sugar yield due to inefficient mass transfer in the system (Liu et al., 2019b). Thus, the optimal initial substrate load was determined to be 40 g/L of sulfite-pretreated wheat straw to enhance saccharification efficiency under the laboratory conditions. Fed-batch approaches can further increase sugar yield by repeatedly supplementing 40 or 20 g/L pretreated substrate during the saccharification process to reach a total substrate load of 80 g/L (Liu et al., 2019b). It has also been observed that substrate load mainly

3.2.2. Optimization of the growth medium to reduce cost It is notable that CBS requires the on-site production of cellulases and simultaneous hydrolysis, and the cost of the medium accounts for a major part of the total CBS cost. With 4% (w/v) sulfite-pretreated wheat straw as the substrate for CBS under laboratory conditions (Liu et al., 2019b), one ton of medium is required to hydrolyze 40 kg substrate. Based on a cellulose/hemicellulose content of 75% of the substrate and a sugar conversion ratio of 90%, about 27 kg of reducing sugars would be obtained. If the GS-2 medium were used, the cost of medium per ton sugar would exceed US$1750 while the market price of glucose is about US$400. Thus, an appropriate low-cost medium is indispensable for biocatalyst cultivation, and the growth medium needs optimization to reduce the cost of the CBS process. Because sodium sulfide is listed as a typical reducing form of sulfur in anaerobic fermentation media (Hendriks et al., 2018) and is lower in price than other sulfur compounds, it was used as an alternative sulfur source. Corn steep liquor has been commonly used in the fermentation industry as a universal cheap source of nitrogen, vitamins, and other trace elements, so it was 6

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and monomers, which can be further used to produce furfural, 5-hydroxymethylfurfural and other valuable products (Hendriks and Zeeman, 2009) but usually generates a large amount of acid-containing wastewater (six to ten times the mass of solids). Zhang et al. have recently improved full-solid-filling dilute acid pretreatment to reduce the amount of wastewater (Hsu et al., 2010; Zhang et al., 2011). Other challenges for dilute acid pretreatment include the need of expensive reactors made of corrosion-resistant materials, the neutralization of the acid-containing sugars and the removal of toxic by-products (e.g., furan aldehydes) prior to downstream fermentation (Jönsson et al., 2013; Nguyen et al., 2015). Low-pH pretreatments contribute to the efficient hemicellulose degradation to monosaccharides but have less effect on lignin removal (Kim et al., 2016). Additionally, solubilized lignin components may redeposit onto the solid fractions under acidic conditions, resulting in a decrease of cellulose accessibility (Pedersen and Meyer, 2010). On the other hand, cellulosome-based CBS generally requires higher levels of delignification and cellulose accessibility, because the high-molecularweight cellulosome complexes can only penetrate the larger voids and gaps compared to the small and individual fungal cellulases that can readily penetrate into the pore structure of the microfiber network (Ding et al., 2012). Thus, the typical low-pH pretreatments showed low compatibility with the CBS process (Kothari et al., 2018). A co-solventenhanced lignocellulosic fractionation (CELF) pretreatment has been developed based on dilute acid treatment using tetrahydrofuran as a miscible co-solvent to water (Nguyen et al., 2015), which can greatly enhance lignin removal and biomass depolymerization. With CELFpretreated switchgrass as the substrate, the cellulosome-producing C. thermocellum showed higher lignocellulosic degradation effectiveness than that of fungal enzymes (Kothari et al., 2018; Thomas et al., 2017a). Thus, despite the potential safety issues and increased chemical cost that should be considered, CELF may be used as a CBS-compatible pretreatment method.

used to substitute yeast extract as the alternative nitrogen source. Not only the cost of culturing cells was greatly reduced, but also a high level of cell biomass was obtained using the modified medium (Liu et al., 2019b). By replacing the sulfur and nitrogen source, the cost of the medium was reduced by about 80% (from US$1750 to 340 per ton reducing sugars). Continued optimization of the growth medium is possible and in progress in our lab, in order to further reduce the cost and promote the desired metabolic process by adjusting the amounts of the other macronutrients besides carbon and nitrogen, various metal ions on which enzymes may depend, and proper chelators that can stabilize free ion elements (Hendriks et al., 2018). 3.3. Pretreatment approaches compatible with CBS Due to the recalcitrance and complexity of lignocellulosic biomass, pretreatment is indispensable for either off-site or on-site saccharification (Taha et al., 2016). According to the effects of the structural features of the biomass on enzymatic digestibility and the requirement of industrial application (Chang and Holtzapple, 2000), a suitable pretreatment method should exhibit high recovery of polysaccharides, high cellulose accessibility, effective delignification, low energy requirements, reduced wastewater, production of negligible inhibitors (Hendriks and Zeeman, 2009; Michelin et al., 2013; Singh et al., 2014), and most importantly, the compatibility of the pretreatment with subsequent enzymatic hydrolysis in practical applications. Both mechanical pretreatment and chemical pretreatments (low-pH or high-pH methods) and their compatibility with CBS process are discussed below. 3.3.1. Mechanical treatment Mechanical treatments are often the first step in lignocellulose pretreatment to increase surface area and cellulose accessibility and to decrease the degree of polymerization and cellulose crystallinity of the substrate to benefit subsequent saccharification (Bitra et al., 2009; Miao et al., 2011; Zhang et al., 2012). However, milling and pulverization usually result in high energy costs which are not economically sustainable on an industrial scale (Hendriks and Zeeman, 2009). In addition, the reduced size of the material particles may not always correlate with enhanced digestibility, and particles with larger diameter can be more conducive to the mass transfer (Moniruzzaman et al., 1997; Rivers and Emert, 1988). The effect of pulp refining (PFI) treatment on saccharification efficiency has been assessed for either free cellulase-based saccharification or CBS. The results showed that PFI treatment can enhance the saccharification ratio of free cellulase-based saccharification from 70 to 83% (w/w), but in terms of the cellulosome-based CBS process, the PFI treatment showed inhibitory rather than stimulatory effects on the saccharification ratio, which decreased from 75% to 57% (w/w). Thus, the mechanical milling step is not as necessary for CBS as for off-site saccharification.

3.3.3. High-pH treatment In contrast to acidic treatments, pretreatment methods using alkaline chemicals and ammonium sulfite (AS) as reagents comprise highpH treatments. High-pH conditions can better promote the delignification of biomass without great loss of polysaccharides compared to acidic methods (Kim et al., 2016). Since the cellulosome-based CBS process benefits more from lignin removal than hemicellulose removal (Kothari et al., 2018), high-pH pretreatments are considered more compatible with the CBS process than low-pH methods. The alkaline-based pretreatments are generally operated under low temperature or moderate conditions compared to low-pH conditions but usually require longer reaction time (Kim et al., 2016). The gross crystallinity index of the biomass material may increase after alkaline treatment, because lignin and part of the hemicellulose fraction can be solubilized by saponification of intermolecular ester bonds of lignin and other polymers, but the structure of cellulose fibers are less affected (Stamatelatou et al., 2014), which will reduce the loss of cellulose and enhance the sugar yield of the hydrolysis process. Additionally, alkaline pretreatments are more effective for lignin removal of grass biomass than wood material and are thus more compatible with the bioconversion of agricultural wastes (Kim et al., 2006; Kumar et al., 2009). The current alkaline-based pretreatment techniques using sodium hydroxide, sodium carbonate, calcium hydroxide (lime), and ammonium (in forms of ammonium hydroxide, liquid or gaseous ammonia) as catalysts have been well-reviewed previously (Kim et al., 2016). NaOHor KOH-mediated treatments are efficient in delignification but suffer from high cost and low reagent recovery. Lime pretreatment is simple, low-cost, and safer, but with less effectiveness (Eggeman and Elander, 2005). Nevertheless, current alkaline pretreatment technologies typically consume large amounts of water for washing and detoxification of pretreated substrates, and the post-treatment of the generated black liquor is also a major concern. Compared to alkali pretreatment,

3.3.2. Low-pH treatment Both hydrothermal pretreatment and acid treatment are considered low-pH treatments and have been widely used in the lignocelluloserelevant industry (Chandel et al., 2019). Hydrothermal pretreatments (steam explosion, liquid hot water, etc.) increase the temperatures to above 150–220oC under high pressures (Mosier et al., 2005; Rodríguez et al., 2017) and the high-pressure hot water exhibits weak acidity and can provide hydronium ions aiding the breakdown of hemicellulose and cellulose. Thus, the hydrothermal process has the advantages of no chemical addition, no need for a special reactor, and little requirement for biomass particles before pretreatment (Coronella et al., 2014). Dilute acid treatment is a favorable pretreatment for industrial application so far (Rocha et al., 2012; Shekiro III et al., 2014; Sipos et al., 2010) using relatively inexpensive chemicals (0.5–1.5% of e.g., sulfuric acid, hydrochloric acid, and phosphoric acid) as the reagents (Singh et al., 2014). Dilute acid treatment can efficiently separate hemicellulose in the liquid fraction from solid-form by converting it to soluble oligomers 7

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Fig. 3. Schematic representation of future improvements for the CBS strategy. The newly developed CBS strategy has demonstrated success on the laboratory scale, while further improvements in terms of biocatalyst construction, saccharification process improvement, and development of suitable pretreatment and downstream fermentation approaches are still needed to lead lignocellulose bioconversion to the real world. First, CBS biocatalysts must express secretive BGL protein to relieve cellobiose inhibition of the cellulosome, and the enzymatic ratio of the cellulosome and BGL is also crucial. In addition, the adaptability of CBS biocatalysts should also be considered for effective saccharification of various lignocellulosic substrates having different components. Secondly, the mass-transfer efficiency of CBS should be improved for high solid loading saccharification by current development of novel equipment and anaerobic fermentation processes. Last but not least, to develop a complete process from biomass to target products, the coupling of the CBS process with both upstream pretreatment and downstream fermentation is essential and challenging. For pretreatment, the first consideration is delignification efficiency, and the reduction of cost and wastewater is also critical. For downstream fermentation, the selection of fermenting microorganisms and target products are both important.

complex composition of lignocellulosic biomass. On the other hand, CBS sugars can be less expensive and are more suitable for the production of biofuels and chemicals that have large market demand and relatively low price. CBS separates fermentation from the integrated CBP process. Thus, the provided sugar-rich hydrolysates can be used for both aerobic or anaerobic fermentation. Noted CBS hydrolysates usually contain both C5 and C6 sugars. In most fermentation cases, the presence of glucose inhibits the utilization of pentose (i.e., xylose) due to carbon catabolite repression (Deutscher, 2008), which may result in low sugar conversion rates and product yield. Thus, microorganisms that can simultaneously utilize pentose and glucose are preferred for fermentation using lignocellulose-derived sugars as the carbon sources. SSCF is considered an updated SSF technique because it employs suitable microorganisms to ferment C5 and C6 sugars at the same time for the production of target products such as ethanol and lactate (Zhang and Lynd, 2010). The xylose-fermenting microbes, such as Zymomonas mobilis, Lactobacillus pentosus, and Saccharomyces cerevisiae (Öhgren et al., 2006; Patel et al., 2005; Zhang and Lynd, 2010; Zhu et al., 2007), can also be applied in fermentations coupling with the CBS process. Besides, considering the existing and potential wide application of engineered model microorganisms such as S. cerevisiae, Escherichia coli and Bacillus subtilis, the growth of model microorganisms should be detected directly using CBS hydrolysates as the carbon sources after supplementation of necessary nutrients. Our preliminary assessment using these model microorganisms suggests that eukaryotes are more resistant to the potential stress in CBS hydrolysates and are preferred for fermentation coupling with the CBS process. Therefore, oil-rich yeast or microalgae species (i.e., Chlorella pyrenoidosa, Candida utilis, and Aurantiochytrium sp.) could be ideal microorganisms for downstream fermentation using the CBS strategy. Our preliminary fermentations using Aureobasidium pullulans, C. utilis, and Aurantiochytrium for production of pullulan, protein-rich cell biomass, and oil, respectively, indicated that the product yields, using CBS hydrolysates as the carbon source without optimization of the media composition, are similar to those using starch glucose as the carbon source. Thus, CBS hydrolysates derived from lignocellulose can be directly used for downstream fermentation, and further media and process optimization improvements would be promising to make CBS

ammonium pretreatments enjoy advantages of high reagent recoverability, moderate reaction conditions, low corrosiveness, and less production of toxins (Pandey et al., 2019). For example, the ammonia fiber explosion/expansion (AFEX) is considered an efficient pretreatment approach for cellulose decrystallization and lignin removal since the 1980s. During the AFEX process, no significant loss of biomass polysaccharides occurs, and ammonia can be recovered to a great extent (Alizadeh et al., 2005; Holtzapple et al., 1991). Ammonium sulfite treatment is a well-applied pulping method for the papermaking industry due to its capability of delignification through the sulfonating reaction between lignin and sulfite (Qi et al., 2018; Tavares et al., 2018). The high chemical dose of ammonium sulfite pretreatment (15–30%, w/w) may greatly increase costs, while the spent ammonium sulfite liquor containing sulfonated lignin and the remaining ammonium sulfite can be further used to produce nitrogenrich bio-fertilizer, making the whole process clean and sustainable (Chi et al., 2019). Therefore, ammonium sulfite-derived pretreatment is a promising option to couple with the CBS process. 3.4. Biological fermentation using CBS sugars as the carbon source Despite the cost-effectiveness of the biocatalyst and saccharification process and the compatibility of the pretreatment method, the feasibility of the CBS strategy also greatly depends on the availability of lignocellulosic sugars in downstream applications. The CBS system contains various nutrient components to grow biocatalyst cells and produce cellulosomal components. Thus, compared to SSF hydrolysates, the CBS hydrolysates have not only sugars but also the remaining nutrients, such as nitrogen, phosphate, and metal ion elements, which are usually indispensable nutrients for downstream fermentation. We have determined that, after a 7-day C. thermocellum saccharification of sulfite-pretreated wheat straw, no significant reduction of total nitrogen was detected in the obtained CBS hydrolysate compared to the original GS-2 medium (Johnson et al., 1981). Negligible amounts of metal ions, including Mg, K, Na, and Fe, were consumed by C. thermocellum cells, except for Ca, which is essential for cellulosome assembly (Leibovitz et al., 1997; Yaron et al., 1995). Compared to starch, which is basically glucose, CBS hydrolysates exhibit relatively low sugar purity, due to the 8

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for C. thermocellum that RsgIs release the corresponding SigIs with the presence of specific extracellular polysaccharides, and SigIs can recognize specific promoters to initiate cellulosomal gene transcription (Munoz-Gutierrez et al., 2016; Nataf et al., 2010; Ortiz de Ora et al., 2018). Such SigI-RsgI regulating systems, and related promoters can be potentially used to control the expression of BGL and cellulosomal components. According to our results, the BGL-integrated cellulosome hydrolyzes about 80% of the cellulose to glucose rather than cellobiose and other cellooligosaccharides (Zhang et al., 2017). Since glucose is not a preferred carbon source for C. thermocellum (Li et al., 2018), the BGL expression in the early stage for biocatalyst production is not necessary and may even be disadvantageous. Thus, besides the expression intensity, the expression timing of BGL should also be considered. That means the expression of BGL should be turned “OFF” during the biocatalyst-producing stage but turned “ON” afterwards for efficient saccharification. Such induced BGL expression may shorten the whole CBS process and avoid the large inoculum, which is not preferred in consideration of the operational feasibility and cost. Additionally, recent data showed that supplementation of a high dose of hemicellulase can greatly shorten the saccharification process by eliminating the lag phase, indicating that the hemicellulase activity of the present biocatalyst can be further improved for quick initiation of the hydrolysis process (Liu et al., 2019b). Thus, endogenous or heterologous xylanases can be introduced into the cellulosome of the current biocatalyst by further genetic engineering to enhance the capability of hemicellulose degradation. Other cellulolytic microorganisms besides C. thermocellum may also have the potential to be used as CBS biocatalysts. For example, Caldicellulosiruptor bescii is an extremely thermophilic cellulolytic bacterium that grows optimally at ~80oC. It can directly degrade unprocessed biomass and has been used for CBP ethanol production (Chung et al., 2014; Chung et al., 2015; Williams-Rhaesa et al., 2018). Instead of the cellulosome, C. bescii produces various extracellular cellulases including those with multifunctional modules (Brunecky et al., 2013; Yi et al., 2013). Such cellulases have been used to construct hyperthermostable cellulosomes (Kahn et al., 2019), which can be used for the development of novel CBS biocatalysts that can work at elevated temperatures.

hydrolysates an alternative carbon source. 4. Future improvements for the CBS strategy The current progress of CBS strategy development has demonstrated success on the laboratory scale and has provided a promising direction for industrial applications. However, as a newly developed technology, CBS still needs improvement and innovation of existing processes and instruments to make breakthroughs in the real world (Fig. 3). A pilotscale demonstration is currently under construction using the CBS strategy, and the following key factors are particularly considered in future developments. 4.1. Construction of CBS biocatalysts with high robustness and adaptability CBS mainly employs natural cellulolytic clostridia strains, such as C. thermocellum, as biocatalysts, because they produce a highly organized multiprotein supramolecular complex, the cellulosome, to degrade highly complex lignocellulosic substrates (Hirano et al., 2016; Yoav et al., 2017). To release the problem of cellobiose-mediated inhibition of the cellulosome, the expression of a cellulosomal BGL in the biocatalyst is essential. Thus, two generations of C. thermocellum biocatalysts have been developed by fused expression of BGL with cellulosomal cellulases Cel48S and Cel9K, in order to form substrate coupling channels (Fig. 2). However, the fused expression of BGL with either Cel48S or Cel9K resulted in decreased expression intensity of exoglucanases that play key roles in cellulose degradation and sugar production, which may reduce the overall efficiency of cellulosomes in the saccharification (Leis et al., 2017; Liu et al., 2019b; Liu et al., 2018b; Olson et al., 2010; Zhang et al., 2017). The reduced expression may be caused by protein instability, insufficient secretion or incorrect folding of the fusion protein with multiple modules. Thus, other approaches rather than fusion with cellulosomal components should be considered for the introduction of BGL. For example, BGL could be expressed as a stand-alone protein fused with interaction tags (e.g., cohesin-dockerin, SpyTag-SpyCatcher, RIAD-RIDD, etc.) (Hatlem et al., 2019; Kang et al., 2019; Pages et al., 1997), whereby the BGL can be assembled into the cellulosome (Fig. 2). Regardless of the approach used, supplementation of enzymes that are produced off-site should be avoided, and either chromosome- or plasmid-based expression of BGLs should be considered (Mearls et al., 2015; Olson, 2010; Tripathi et al., 2010). The developed CBS biocatalysts have relieved cellobiose inhibition to some extent, but further improvement is still required. Our studies showed that the addition of free BGL protein in the saccharification system with 2nd-generation CBS catalyst can further stimulate the saccharification level (Liu et al., 2019b), indicating that the BGL expression or activity of present biocatalysts could be further improved. It is possible to enhance the expression and secretion of BGL by using an engineered promoter, ribosome-binding sites, and signal peptides. In terms of the BGL activity, although the BGL used for construction of CBS biocatalysts (CaBglA) showed relatively high resistance to glucose and thermal stability (Zhang et al., 2017), other reported BGL candidates (Akram et al., 2016; Sinha and Datta, 2016; Zhao et al., 2013) or engineered CaBglA that may have higher catalytic efficiency and product tolerance can be considered, especially if high solid loading would be used and a high concentration of sugars would be produced in the saccharification system. It has been reported that the enzymatic ratio of cellulase and BGL is crucial for efficient cellulose saccharification by the cellulosome complex (Hirano et al., 2019). Thus, the importance of the activity balance between BGL and exoglucanases should also be addressed by modifying parameters such as promoters, ribosome binding sites, and untranslated regions (Yadav et al., 2012; Yu et al., 2019; Zou et al., 2018). For example, the expression intensity of BGL and exoglucanases can be adjusted by using promoters with different activities and inducing capabilities (Olson et al., 2015; Zhang et al., 2015). It has been proposed

4.2. Improvement of the CBS process for saccharification at high solid loadings CBS aims to convert lignocellulosic polysaccharides, i.e., cellulose and hemicellulose, to fermentable sugars as a platform product for downstream fermentation or chemical catalytic conversion processes. Thus, both sugar yield and concentration are the core criteria for evaluating process feasibility. Under the premise that biocatalysts have high saccharification levels and sugar yield, the sugar concentration is mainly determined by substrate load in the saccharification system and the proportion of polysaccharides in pretreated lignocellulosic biomass, which varies, depending on the biomass types and various pretreatment methods (Chandel et al., 2019; Taha et al., 2016). If the proportion of polysaccharides in pretreated lignocellulosic biomass is at an average level of 60% and no less than 80% of which can be converted to sugar, then about 10% (w/v) pretreated substrate load is required to produce 50 g/L reducing sugar. If 3/4 of the produced reducing sugar is glucose, then 37.5 g/L glucose can be produced from 100 g/L dry weight pretreated biomass, while certain fungal fermentations may require even higher glucose concentrations. Hence, it is essential to develop a highload CBS saccharification method to increase the sugar concentration in CBS hydrolysates, and the increased solid loading may also lead to reduced process cost. A loading of solids over 15% (w/v) is generally considered high-solid loading in lignocellulose pretreatment and saccharification (Kim et al., 2019; Modenbach and Nokes, 2012). However, the CBS hydrolysis efficiency may decrease greatly with the 9

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increase of solid concentration in the saccharification system with solid loadings of more than 8% and 10%, using pretreated wheat straw and microcrystalline cellulose as the substrate, respectively (Liu et al., 2019b; Zhang et al., 2017). The reduction in efficiency is mainly due to mass transfer limitation (Du et al., 2017). Pretreatment and enzymatic saccharification at high solid loadings have been achieved using fungal cellulases as the biocatalysts by process optimization. Fed-batch operations have also been proposed for lignocellulosic saccharification. By using free fungal cellulases as the biocatalyst and pretreated oak as the substrate, Kim et al. performed fed-batch saccharification at a final solid loading of 30% and obtained high-titer glucose (120.2 g/L) and xylose (37.3 g/L) at 75.9% and 58.6% of theoretical maximum yields, respectively (Kim et al., 2019). Additionally, horizontal tank-rotating reactors, rather than conventional stirred-tank reactors, have been applied to promote the mixing quality of enzyme and substrate and the mass transfer efficiency (Du et al., 2017; Du et al., 2014; Pino et al., 2019). Drawing on such approaches, the current CBS process can be further optimized to overcome the reduction in mass transfer efficiency under high solid loading saccharification conditions.

bagasse hydrolysate as a carbon source to produce single cell oil composed of oleic acid and palmitic acid by co-culture of Chlorella. pyrenoidosa and Rhodotorula glutinis (Liu et al., 2018a). Bajpai et al. reviewed the recent research work on the production of single-cell protein from lignocellulosic wastes (Bajpai, 2017). By coupling microbial fermentation with the CBS strategy, the cost of the high-value additives in animal feeds could be reduced, which would be of great interest to the animal feed industry. CBS hydrolysates have been successfully used in different downstream fermentation using various microorganisms, but the fermentation media have not been thoroughly optimized according to the change of carbon source from pure sugar to mixed CBS sugars. The CBS hydrolysates contain both mixed sugars and other nutrients, and the low purity may become an advantage in case of downstream fermentations that are sensitive to the price of raw materials. Further optimization of the cultivation media should be performed to reduce the addition of nutrients already present in the CBS hydrolysate, making the CBS strategy more economically feasible.

4.3. Development of a complete process from biomass to target products

Although many facilities have been constructed globally to produce cellulosic ethanol, such as Beta Renewables in Italy, GranBio in Brazil, Poet-DSM Advanced Biofuels in Iowa, Abengoa refinery in Kansas, DuPont refinery in Iowa, Tianguan Biofuel Ethanol in China, etc., these facilities mainly use fungal cellulases following off-site saccharification strategies, which are distinguished from the CBS technology. CBS requires specific pretreatment methods, anaerobic cultivation of biocatalyst cells, and high-solid-loading fermentation. In addition, CBS may employ fed-batch saccharification to further increase the solid loading and sugar concentration. Therefore, no ready-made fermentation equipment can be used for CBS, and it is necessary to design and construct novel pretreatment equipment and saccharification reactors for the scale-up of the CBS process. Even though moderate pretreatments are preferred for CBS, which may reduce the production of wastewater, the post water treatment is indispensable. Scientists and engineers involved in the construction of water treatment methods and instruments for CBS can learn from present biofuel facilities and paper industry.

4.4. Scale-up and development of novel instruments for CBS

To convert lignocellulosic biomass to target products, the coupling of CBS process with both upstream pretreatment and downstream fermentation is essential and challenging. In terms of pretreatments, acidcatalyzed thermochemical reactions often result in the formation of phenolic compounds, aliphatic acids, furan aldehydes, and oligosaccharides derived from lignin and polysaccharides that may have inhibitory or toxic effect on the enzymes or fermenting organism (Kont et al., 2013; Liu and Wyman, 2003; Wierckx et al., 2010; Zhang et al., 2010). Taking into account reaction severity and delignification efficiency (Kim et al., 2016), high-pH pretreatments are more suitable to couple with the CBS process than acidic methods. Since CBS employs viable cells, e.g., cellulosome-producing anaerobes, as the biocatalysts, the sensitivity to inhibitors produced during harsh pretreatment processes should be different from fungal enzymes used in off-site saccharification. However, no matter what kind of pretreatment is used, detoxification of pretreated substrate through chemical or biological treatment, liquid-liquid extraction, liquid-solid extraction, and evaporation is usually not negligible (Jönsson et al., 2013; Kamimura et al., 2017). The separated detoxification step may prolong the whole process, increase water consumption and cost, and thus reduce the economic viability of lignocellulose bioconversion. Thus, the effects of potential inhibitors should be considered in the development of pretreatment methods coupling with the CBS process. In order to reduce the production of inhibitors, moderate pretreatment methods at low temperatures are preferred, while the deceased reaction temperature may result in an extended processing cycle, which should be considered as well. On the other hand, it is possible to increase the inhibitor resistance of CBS biocatalysts by adaptation, mutagenesis, or genetic engineering of the bacterium to reduce the stringent requirements for pretreatment. With well-developed conversion technology, lignocellulosic sugars can be less expensive but with low sugar purity compared to starch glucose. Thus, based on the critical features, CBS sugars can be used as promising raw materials in the production of not only biofuels such as the 2nd-generation bioethanol and advanced biofuels but also other target products of large demand and sensitivity to cost, such as animal feeds. Besides starch-based crops, the animal feed also requires key nutrition supplements such as amino acids, fatty acids, prebiotics, vitamins and other nutrients to enhance growth and avoid diseases of animals, which will result in an additional cost (Villas-Bôas et al., 2002). The necessary feed additives can be produced by microbial fermentation of oil-, protein-, vitamin-, and polysaccharide-rich microalgae and fungi using lignocellulosic hydrolysates as the carbon source substitutes. For example, Liu et al. used non-detoxified cassava

4.5. CBS feasibility depends on the cost competitiveness towards starch sugar CBS produces fermentable sugars as an alternative carbon source for industrial fermentation. Thus, the feasibility of CBS depends on whether the cost of the lignocellulosic sugars is competitive to sugars produced from corn. The current price of starch-derived glucose is about US$400 per ton, while the price of wheat straw or corn stover in China is 200 to 500 RMB (about 30–70 US$) per ton. Calculated on an average solid recovery of 60% during pretreatment and a sugar conversion ratio of 80% during biosaccharification, about two tons of raw biomass would be required to produce one ton of sugar. In this case, the raw material cost would increase to US$140, and the process cost, including both the pretreatment and CBS, must be kept below 290 US$ per ton sugar. In terms of pretreatment, the process optimization should focus on the reduction of chemical usage and energy reuse. For the CBS process, cultivation media and energy consumption account for the main part of the total cost, which would be significantly reduced by further optimizing the cultivation conditions of the biocatalysts and the reuse of the biocatalysts. Furthermore, the optimization should be carried out by considering pretreatment and biosaccharification as a whole process, rather than separated processes. For example, by coupling the pretreatment and CBS processes, it would be possible to use the waste heat of the pretreatment for CBS so that little extra energy would be required, since energy-intensive stirring is not indispensable for CBS. Additionally, by developing novel instruments, the CBS biocatalyst and cultivation medium, the chemicals and heat, can all be recycled, which 10

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the National Natural Science Foundation of China [grant numbers 31570029, 31670735, 31870568 and 31661143023]; the Key Technology Research and Development Program of Shandong [grant number 2018GSF116016]; and the Major Program of Shandong Provincial Natural Science Foundation [grant number ZR2018ZB0208]; QIBEBT and Dalian National Laboratory For Clean Energy (DNL), CAS (grant number QIBEBT I201905); and the Director Innovation Foundation of Qingdao Institute of Bioenergy and Bioprocess Technology, CAS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

would significantly increase the solid loading of CBS to produce CBS sugars at a cost competitive with starch sugar. 5. Conclusion and perspectives Lignocellulosic biomass has high potential to be used as a substitute for fossil resources, thereby effectively alleviating global dependence on crude oil. With the support of the governments involved, several commercial facilities have been operating to produce lignocellulosic products. However, despite the current fluctuations in oil prices, lignocellulosic products are thus far not competitive in the market compared to fossil derivatives. Three conventional stages are mainly required for the production of lignocellulosic products: the breakdown of biomass by pretreatment, the conversion of pretreated biomass to fermenting sugars by saccharification involving various cellulolytic biocatalysts, and the conversion of sugars to target products by fermentation. Throughout the whole lignocellulose bioconversion process, saccharification may contribute the most to cost, especially for the offsite saccharification that depends on aerobically pre-produced fungal cellulases, and thus is considered the decisive factor in the feasibility of lignocellulose bioconversion (Lynd et al., 2017; Lynd et al., 2005). Compared to off-site saccharifications, CBP and its derivatives, including CBS and CT, have advantages in reducing cost because of the on-site production of enzymes, thus having the potential to lead lignocellulose bioconversion into real industrial production (Balch et al., 2017; Liu et al., 2019b). CBS focuses on the production of fermentable sugars, which can be used as the platform chemical in downstream fermentation. In this way, the CBS strategy can have broad application prospects, as long as the production cost of fermentable sugars competes with starch sugar, the common carbon source in the fermentation industry of bulk and cost-sensitive products. Following the CBS strategy, novel whole-cell biocatalysts have been successfully constructed and the saccharification process has been improved to reduce the production cost and shorten the process. Moreover, compatible pretreatment methods and downstream applications are in development with promising initial results. Thus, the entire lignocellulose bioconversion route based on CBS has been established in a preliminary fashion. With continuous optimizations of the CBS biocatalysts and process, the saccharification efficiency and sugar yield will be further increased, as well as the economic feasibility of CBS. First, the cell tolerance of CBS biocatalyst to pretreatment-derived inhibitors should be considered, and the enzymatic activities of various cellulosomal components should be further balanced to release the product feedback inhibition. Secondly, novel instruments should be developed to increase the mass transfer efficiency for saccharification with high sold loading. Thirdly, the compatibility with CBS should be kept in mind for the development of pretreatment methods and downstream applications. In terms of pretreatment, high-pH based treatment methods that can promote the delignification of biomass are considered more suitable for CBS. In terms of downstream applications, CBS hydrolysates should be considered a sugar-rich mixture rather than a pure carbon source and can provide additional nutrients for fermentation. Microorganisms that can ferment both C5 and C6 sugars are preferred, and products of large demand and cost sensitivity should be chosen. Last but not least, the construction of a pilot-scale CBS demonstration is ongoing, on which the cost of produced fermentable sugars could be estimated upon large-scale production. Through unremitting improvement, we believe CBS will finally lead lignocellulose bioconversion into the real world with economic practicality and sustainability.

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Funding This work was supported by the “Transformational Technologies for Clean Energy and Demonstration”, Strategic Priority Research Program of the Chinese Academy of Sciences [grant number XDA 21060201]; 11

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