Bioprocessing Techniques

The dominant predicament in cellulase manufacturing is its low enzyme titer. Numerous scholars have endeavored to crack distinctive representations to amplify cellulase enhancement in fermentation technology. It encompasses superior bioprocess performance, employing low-cost material or rudimentary raw materials as a substrate to produce enzymes and genetically obtained microorganisms etc. [47]. There are commonly two kinds of fermentation techniques that focus on enhanced cellulase production: solid-state fermentation (SSF) and submerged fermentation (SMF). These fermentation techniques use microorganisms such as fungus and bacteria. Recently, a new bioprocessing practice developed successively by using bioprocessing techniques in a single reactor. All the bioprocessing practices are discussed in detail in this section, including advancements in new bioprocessing techniques, i.e., CBP and other methods for better enzyme generation.

Currently, scientists mainly focus on the scale-up process of both 2G and 3G biofuel. However, both the sectors are facing problems that are causing interferences in the scaling-up process.

In the 2G biorefinery, in-house enzyme production and enzymatic hydrolysis play a crucial role in reducing the cost and scaling up 2G-ethanol production. From the two types of fermentation, SSF has many advantages but its use is confined at mostly lab scale because of some obstacles in controlling specific parameters and operating variables, which hugely impact microbial growth and metabolite production [25,29]. Moreover, some other drawbacks corresponding to SSF are heat and mass transfer, and high equipment costs [25].

As stated by techno-economic research, the potential to operate biomass at high solid loading during enzymatic hydrolysis will be essential in 2G biorefineries, principally. The equipment cost will come down and can lead to minimizing the requirement process energy and wastewater treatment. However, the enzymatic hydrolysis at high solid loading is somehow not an easy occupation to handle as pretreated solid biomass viscosity increases enormously and starts showing firmly non-Newtonian flow properties. The complications with both mass and heat transfer in the material and mechanical problems are pumping and adequate mixing of the biomass slurry. In SSF, researchers observed ethanol production at high solid loading during simultaneous saccharification and fermentation (SSnF) requires approximately 60% of the energy content while mixing in SSF. All processes are studied at lab scale. Nevertheless, there are several parameters like flow behavior (or even the flow regime) and particular power inputs that change with the reactor scale [48]. According to [49], their study focused on evaluating a prospective fungal strain *Penicillium oxalicum* IODBF5 in the production of cellulase under submerged fermentation. The first shake flask experiment was performed with this strain at 28 ◦C, 180 RPM and incubated for 8 days and initial cellulase activity reported was 0.7 FPU/mL, which later increased by 1.7 fold i.e., 1.2 FPU/mL in 8 days. After optimizing specific parameters, the same experiment was performed at 7 L reactor level in which the temperature maintained was at 28 ◦C and pH at 5.0 by adding 1 M HCl or 1 M NaOH. Fungal spores inoculated around 10<sup>9</sup> spores/L, and initial airflow kept was of 1vvm which after 72 h increased to 1.5 vvm and the incubation time was reduced from eight to four days with the same reproducibility. For effective enzymatic hydrolysis with the crude enzyme, the temperature and pH were maintained at 50 ◦C and 5.0, respectively. At 50 ◦C, the natural enzyme preserved their 50% and 26% of their activity at 48 h and 72 h, respectively. This property of Truffle was reported by [50] in their study where the capability of *Tuber maculatum* to secrete an extracellular cellulase during SMF was explained for the first time in their research. In SMF basal salt media was used with sodium carboxymethyl cellulose (0.5% *w*/*v*) as a carbon source at pH 7 and the cellulase activity reported was 1.70 U/mL after seven days of incubation. The stability test was performed, and it was observed that the enzyme was stable at 50 ◦C and pH 5.0. The enzyme was thermostable as well as maintained its 99% activity at 50 ◦C temperature. Tray Bioreactor, Packed bed Bioreactor (PBB), Rotary Drum Bioreactor (RDB), Fluidized Bed Bioreactor (FBB) and Instrumented Labscale Bioreactor (ILB) are usually used as bioreactors for SSF systems [51]. According to the study performed by [52], cellulase was produced by microorganism *Trichoderma reessei* RUT C-30 under SSF. The substrate used in SSF was wheat bran and cellulose, where cellulose was used as an inducer for further enhanced cellulase production. The combination of wheat bran and cellulose was added in a 250 mL Erlenmeyer flask. The substrate moistened with mineral salt medium and pH was maintained at 4.8 by adding 1 N HCl and 1 N NaOH. This experiment was optimized by involving a two-stage statistical design of experiments, resulting in an increase of CMCase by 3.2 fold i.e., 959.53 IU/gDS. This method was repeated at pilot scale and the reactors used were tray fermenters, and the same lab conditions were used, resulting in 457 IU/gDS yield. Cellulase produced in this experiment was used for enzymatic hydrolysis of alkali pretreated sorghum stover with or without BGL and the BGL used in this experiment was extracted from *Aspergillus niger*. The result from hydrolysis with BGL was reported to be 174% efficient. The hydrolysate produced from hydrolysis of sorghum

stover was used in the fermentation process to generate ethanol, which resulted in being around 80% effective. In another study, crude olive pomace and exhausted olive pomace was used in SSF as substrate. They both were pretreated by ultrasound pretreatment, and their comparative study was also performed. In SSF, both wastes were checked and used as a substrate in SSF to yield cellulase and xylanase by fungi. The use of exhausted olive pomace as substrate by *Aspergillus* resulted in higher titers in the screening. They were therefore used in the experiment of ultrasound pretreatment. The outcome of sonication showed a 3-fold increase of xylanase activity and harmed cellulase activity. Furthermore, the liquid part acquired from ultrasound pretreatment was used to maintain substantial moisture, resulting in a positive effect on enzyme activity, which caused a 3.6-fold increase and 1.2-fold increase, respectively [53].

To date, large-scale algal cultivation still presents various mechanical threats that interfere with the process of commercialization as a promising aspect of algal biomass as a renewable feedstock for biofuels production. These threats or challenges are related to upstream or downstream processing.

In upstream processing, these are the algae and algal cultivation process selection process, the energy input for handling closed-photobioreactors, nutrient sources, water reusability, and footprint and susceptibility of algae towards the surrounding.

In downstream processing, this is attributed to harvesting and drying technology for algal cells, efficient algal lipid extraction methods, biodiesel conversion technologies from algae and biodiesel, and probability to transform biofuels production from the algal residue after extracting the lipid. After the technological hurdles, merchandising algal biofuels production's profitable viability is still dubious because algal cultivation and correlated biofuels production technologies are in shortage. Enhance algal biofuels' commercial potential: it is crucial to understand and recognize technical and economically related problems [54].
