3.2.3. Consolidated Bioprocessing (CBP) and Some Case Studies

In recent years, the consolidated bioprocess CBP, initially known as direct microbial conversion (D.M.C.), has been investigated. CBP's critical difference with other biomass bioprocessing strategies lies in the use of a single organism or their consortium for enzyme production, hydrolysis, and fermentation. These are generally carried out at room temperature, which results in the reduction of production costs [64,65,69,70]. Furthermore, the hydrolytic and fermentative processes' compatibility means that a single reactor is required, simplifying its operation. Although CBP has great advantages compared to other production processes, there are issues such as long fermentation periods and low biofuel yields due to the formation of various by-products (organic acids), the sensitivity of microorganisms to alcoholic solvents, and growth limited in the supernatant of hydrolysis, which requires the continuous search for more efficient strategies [64,69].

However, the industrial-scale application of CBP remains challenging so far because of the low efficiency. Derived from CBP: Consolidated Bio-Saccharification (CBS) strategy proposed fermentation separated from the integrated process. CBS produces fermentable sugars as the target product rather than end metabolites such as ethanol. Consequently, fermentation would not be limited by hydrolysis condition. The cellulolytic capability is maximized as well. Thus, CBS provides new insight into lignocellulose bioconversion [11,71]. Figure 2 shows the schematic representation of the consolidated bioprocessing process in terms of biorefinery. The advantages of SSF are linked to the utilization of agro-industrial residues as solid substrate substituting for carbon and energy sources, however enzyme production for industrial purposes still surfaces into certain industrial restrictions [72]. On the contrary, SMF is a universally acknowledged fermentation process for industrial enzyme production as it is more convenient to control all the variables such as pH, temperature, and operational techniques. The CBP strategy proposed in 1996 and increasing evidence supports that CBP may be feasible. CBP's research has focused on developing new and even more effective CBP microorganisms, a critical challenge [73,74]. Several studies were performed for sustainable ethanol production using advanced consolidated bioprocessing. In one research study, the subsequent pairing used in CBP was *Clostridium thermocellum* ATCG 27,405 with mesophilic microaerobe, *Pichia stiptis* NCIM-3498. The biogenic municipal solid waste was pretreated with 0.5% NaOH for reducing the recalcitrance property. It was observed that subsequent CBP (23.99 g/L) was better than CBP alone (18.10 g/L).

In subsequent CBP, exogenous xylanase was added to enhance xylan hydrolysis. Subsequent CBP II biosystem was observed to give maximum ethanol production (36.90 g/L) at pH 5 in a single reactor [75]. In another investigation, the author used yeast engineered with five functional cellulase genes (BGL, XYNII, EGII, CBHI, and CBHII) where ionic pretreated bagasse and laubholz unbleached kraft pulp was used as targeted biomass. The screening was performed to observe that it is essential to breed CBP yeast having the optimized cellulase expressing the ratio for the proposed biomass. In CBP, yeast development is considered a promising and cheap way in consolidated bioprocessing for ethanol production [76]. In one such study, the author used a new approach that is partially consolidated bioprocessing (PCBP). In this process, a mixture of lignocellulosic material was used (mentioned in Table 2). PCBP was used to prepare mixed lignocellulosic substrate by utilizing non-isothermal simultaneous pretreatment. Saccharification methods were used for the hydrolysis of the pretreated substrate. In the saccharification process (*Trichoderma Reesei* RUT C 30) was exploited the combination of laccase (*Pleurotus djamor*) and holocellulase. Then, it was later pursued by the co-fermentation process in a single reactor. The artificial neural network (Feedforward ANN) model was used to optimize all parameters in PCBP that resulted in increased ethanol productivity [77]. In one of the studies, the authors blend two characteristics, i.e., the saccharolytic and fermentation integrated into one microorganism. It diverted a huge interest towards this process for production of ethanol in terms of CBP causing lignocellulosic biorefineries to decrease environmental pollution and economic cost. Therefore, in this study, industrial *S. cerevisiae* strains are used, expressing strong characteristics such as thermotolerance and enhanced resistance to inhibitors. The strain was estimated to be great as it expresses hemicellulolytic enzyme activity on its cell surface. It resulted in increased ethanol productivity with the addition of commercial cellulase [78]. In one of the investigations, a thermophilic anaerobic bacterium was isolated from Himalayan hot spring *Clostridium* sp. DBT-IOC-C19. This strain was considered suitable for consolidated bioprocessing. It expressed a wide range of substrate conversion into ethanol, acetate, and lactate in a single step, where ethanol is considered the main product. DBT-IOC-C19 displayed 94.6% degradation at 5 g/L and 82.74% degradation at 10 g/L of avicel concentration or loading in 96 h of incubation time during fermentation. Rice straw was used as a lignocellulosic substrate for comparative analysis with different strains but total product yield and ethanol production increased in *Clostridium* DBT-IOC-C19 [79]. The same author studied different strains for CBP, e.g., *Clostridium thermocellum* ATCC31924, where crystalline cellulose was used as the sole carbon source. It was reported that cellulosome extracted from the bacterial strain when purified resulted in concentrated cellulase and xylanase enzyme [80]. Ionic liquid pretreated pine needle biomass was used as a substrate in CBP. First, saccharification was performed by using cellulase and xylanase in a single pot. Then, fermentation of hydrolysate was performed by using yeast to yield ethanol [81]. CBP cannot only be used for the production of ethanol, but there are other yields as primary productivity. In one study, the author worked with CBP along with co-cultivation. The author designed a microbial consortium composed of hemicellulose producing bacteria *Thermoanerobacterium, Thermosacchrolyticum* M5, and succinic acid production specialist *Actinobacillus succinogens.* This co-cultivation in CBP resulted in increased production of succinic acid under optimized conditions [82]. The three lignocellulolytic bacteria (*Clostridium thermocellum, C. stercorarium,* and *Thermoanaerobacter thermohydrosulfuricus*) were used as collegial co-culture strains in consolidated bioprocessing. This was performed in terms of a comparative analysis with the monoculture by employing a wide range of lignocellulosic material. The pretreated and unpretreated substrate was used to study the productivity (yield) produced from monoculture and triculture, and co-culture [83]. In another investigation, CBP was used to produce H2. The cellulose-degrading microorganism was retrieved from the bovine ruminal fluid (BRF). *Clostridium acetobutylium* had great potential in increasing H<sup>2</sup> yield [84]. Figure 3 shows the schematic representation of the consolidated bioprocessing process using strategy hydrothermal pretreatment processing in the production of biofuels.

**Figure 2.** Schematic representation of consolidated bioprocessing processing in terms of biorefinery from lignocellulosic biomass.

**Table 2.** Consolidated bioprocessing (CBP) for a mixture of lignocellulosic material in the enzyme production.



### **Table 2.** *Cont*.

**Figure 3.** Schematic representation of consolidated bioprocessing processing using as hydrothermal strategy pretreatment. Adapted and modified from [15].
