Biomass preparation
The corncob biomass was procured from the farmer fields, Coimbatore, Tamil Nadu, India and dried at ambient temperature (30 °C) for 12 h to reduce the moisture content from 20 to 14%, then the size was reduced in a sequence through shredder (Chudekar Agro Engg Pvt Ltd., Model: 53 H, India), pin mill (Premium Pulman Pvt Ltd., Model: PPM-12, India) and grinder (Aashapura Enterprises, Model: Stylo 750, India) to ≤ 212 µm, which was sieved via ASTM sieve size No. 70.
Enzyme selection
Generally, two families of ligninolytic enzymes are widely considered for enzymatic delignification, which includes phenol oxidase (laccase) and peroxidases (lignin peroxidase, or LiP and manganese peroxidase, or MnP) [37]. Laccase belongs to the copper oxidase enzyme family, similar to other phenol-oxidizing enzymes and it preferably polymerizes lignin by coupling of the phenoxy radicals produced from oxidation of lignin phenolic groups. Laccase enzyme was selected and used as catalyst for this HCE pretreatment [38].
The enzyme laccase used in the study was from microbial source (Trametes versicolor). The laccase was purchased from Sigma-Aldrich, Bangalore, India and used as such. The laccase activity was determined with the reaction mixture contained appropriately diluted enzyme mixed with 1 mM ABTS in sodium phosphate buffer (50 mM, pH 4.5) at 30 °C for 5 min. using 1 mM ABTS by monitoring changes in absorbance at 420 nm (€ max = 3.6 × 104 M−1 cm−1) spectrophotometrically in a Spectramax 360 (Molecular devices, USA). One unit of enzyme activity refers to the amount of enzyme required to oxidize 1 µM min−1 of the ABTS substrate under standard assay conditions.
System description and operating conditions
The main hurdles associated with commercialization of higher biomass loading pretreatment reactors are complexity of design, reactor geometry and upscale, poor mixing characteristics of reactants, and it is an energy-intensive process. For these reasons, low biomass loading rate (< 20%) is largely preferred in most of the biomass pretreatments [39]. The main criteria to be considered for reactor design is rheological properties of different slurries collected from different unit operations in the fermentation process [40]. Mostly, water with catalyst is used as the working fluid in the recent HC biomass pretreatment studies; whereas, in the present study corncob biomass slurry (buffer + biocatalyst + powdered biomass) was used as the working liquid. The process design of this pretreatment is to supply the biomass slurry via holes in the orifice plate to cavitation zone. The rheological study involving different corncob biomass slurries (2.50, 3.75, 5.00, 6.75, 7.50, 8.75 and 10.00%) showed increased viscosity and yield stress, along with increased biomass solid loading and these slurries exhibit pseudo-plastic or shear-thinning behavior [41]. It is also observed that high biomass loading rate (> 6.75%) of corncob slurry have blocked the holes in orifice plate. Based on this result, low biomass loading rates (2.50, 3.75 and 5.00%) were selected for HCE pretreatment. Hydrodynamic cavitation reactor (HCR) consists of circulation tank (6 L capacity), orifice plate, flanges for orifice plate, centrifugal pump, electrical motor, gate valves for priming and bypass, pressure gauges, and pipe accessories (Fig. 6a). Two pressure gauges (P1 and P2) were fixed on both the downstream and upstream sides of the orifice plate to measure the pressure drop. For same area opening in the orifice plate, higher diameter of the hole is recommended for intensive cavitation applications and vice versa. In this reactor, total hole area openings made in the plate were kept as constant (28.26 mm2) and for constant area openings, two configurations of orifice plates were used, viz., orifice plate 1 (OP1:9 holes and 2 mm Ø) and orifice plate 2 (OP2:4 holes and 3 mm Ø) (Fig. 6c). The suction pipe of the pump was connected at the base of the circulation tank. The main pipeline was divided into three sub-pipelines to serve for three purposes namely priming, bypass line, sub mainline to accommodate flanges and orifice plate, and it was connected to the delivery pipe of the centrifugal pump. To make a closed loop circulation, the working fluid was supplied from the circulation tank to the orifice plate with the help of a centrifugal pump and then sent back to circulation tank. The purpose of the bypass valve was to regulate the flow rate/pressure of the working fluid (biomass slurry) by passing through the orifice plate of the reactor.
For HCE biomass pretreatment, the biomass slurry was prepared by mixing appropriate quantities of biomass powder and a biocatalyst (laccase enzyme) in an acetate buffer pH 4.5. For example, 200 g of biomass and 6.5 U g−1 of enzyme were added to 4000 mL of acetate buffer to make biomass slurry for 5% biomass loading and 6.5 U g−1 of enzyme. The prepared slurry was added to circulation tank and made to circulate via selected orifice plate continuously, which enabled the biomass to get exposed to cavitation action. Cavitation involves production and aggressive collapse of micro bubbles to generate more hotspots, having higher temperature and pressure. This is sufficient to make the chemical and physical transformations in the lignocellulosic biomass. During the process, decomposition of water molecule causes free radical formation, such as hydroxyl radicals leading to turbulence action of working fluid in the cavitation zone, which eventually helps in rupturing the lignin barrier. Laccase enzyme can oxidize a variety of phenolic subunits of lignin and other aromatic compounds via radical-catalyzed mechanism to yield oxygen-centred free radical and quinine for subsequent reduction in the polymerization reaction [42,43,44]. Hence, HCE-based biomass treatment helps in the generation of highly reactive free radicals in HCR, which can improve the mass transport process rates and enhance the lignin degradation. Based on these results, three biomass loadings (2.50, 3.75 and 5.00%) were selected for HCE pretreatment.
Compositional analysis
After each run, the biomass slurry was collected from the circulation tank, and filtered via filter cloth to separate the pretreated biomass from supernatant. The supernatant was stored at 4 °C for further analysis. The pretreated biomass was washed twice with distilled water to obtain a neutral pH of 7.0 and the biomass samples were dried in a hot air oven at 45 °C for compositional analysis. National Renewable Energy Laboratory procedure was adopted for analyzing biomass composition of raw and pretreated samples [45]. The percentage of lignin reduction by the pretreatment process was calculated using the following equation.
$$\text{Percentage of lignin reduction = }\frac{{\text{lignin in raw biomass{-}lignin in pretreated biomass}}}{{\text{lignin in the raw biomass}}} \times 100$$
Experimental design
Response surface methodology (RSM) is a statistical and mathematical technique widely used for optimization of process parameters and their interactions on output response(s). The added advantages of optimization via RSM approach are (i) only a minimum number of trials are sufficient to find optimum, (ii) to find a correlation between independent inputs and output responses and (iii) reduction in time, materials and cost because of the less number of trials are needed [46]. The optimization of biomass pretreatment process involves studying the influence of operational parameters and their interactions on lignin removal from raw biomass. For enzymatic biomass pretreatment, process parameters such as biomass loading, enzyme loading and reaction time are crucial factors in deconstruction of the lignin structure as well as lead to release of reducing sugars due to solubilization of biomass.
Response surface methodology (RSM) was employed to determine the optimal conditions for HCE pretreatment to attain the maximum percentage of lignin reduction in the corncob biomass samples. The response was assumed to be influenced by three independent variables, catalyst concentration (A), biomass to liquid ratio (B) and reaction time (C). The range of three selected independent variables used for pretreatment process are: biomass loading of 2.5–5.0%, enzyme loading of 3–10 U g−1 of dried biomass, and reaction time of 5–60 min. Based on the results of the preliminary trials, the above range of levels of the three independent variables were fixed. A total of 17 experimental trials of the three variables were designed by Box–Behnken design via Design-Expert software 10.0 (Stat-Ease, Inc., USA) [47].
Structural composition of biomass
FT-IR analysis
The FT-IR spectra of the tested biomass samples were obtained using an FT-IR (FT-IR 6800 JASCO, Japan). Absorbance spectra were recorded between 4000 and 400 cm−1 wave numbers with a spectral resolution of 4 cm−1 and 64 scans per sample.
SEM analysis
The morphology of raw and pretreated corncob biomass was analyzed by scanning electron microscope (SEM; Quanta 250, FEI, Hillsboro, OR, USA) using an Everhart–Thornley Detector (ETD) detector. The SEM was operated in a vacuum, 10 kV, with a spot size of 4 and a pressure of 20 Pa. The sample images were taken at ×4000 magnification.
XRD analysis
The cellulose crystallinity of the biomass samples was measured using an Ultima IV diffractometer (Rigaku, Japan). Copper Kα radiation, 30.0 kV of voltage, 15 mA of current, and a rate of 2.0°min−1 for a 2θ continuous scan from 4.0° to 70.0° were applied. The crystallinity index was obtained from the ratio of the maximum peak intensity 002 (I002, 2θ = 22.0) and minimal depression (Iam 2θ = 16.5) between peaks 001 and 002 [48, 49].
$${\text{Crystallinity index}} = \frac{{I_{002} - I_{\text{am}} }}{{I_{002} }} \times 100$$
where I002 is the diffraction intensity at 2θ = 22.5°, which represents both the crystalline and amorphous regions, and Iam is the diffraction intensity at 2θ = 18.5°, which represents the amorphous regions.
Thermogravimetric analysis (TGA)
The sample size of the corncob used in the experiment was 10 ± 2 mg in a thermogravimetric analyzer (TA instruments, Model: TGA Q50, USA). The test sample was heated at a heating rate of 10 °C min−1 for the temperature range from 50 to 800 °C and nitrogen gas was purged at a flow rate of 30 mL min−1 to create pyrolysis conditions. TGA curve was plotted using TA software (TA Universal Analysis 2000) for both raw and pretreated samples and the results were compared.
Effect of cavitation on temperature of working liquid and residual enzyme activity
During the experimental trials, the temperature of the working liquid was measured at an interval of 5 min by digital thermometer (Multi-thermometer, India). Similarly, the residual enzyme activity during the reaction period was also determined [50].
Working liquid temperature
The acetate buffer was used as working liquid in the HCE biomass pretreatment and there was a rise in the temperature of the working liquid from 30 to 50 °C in 60 min, which could be attributed to heat energy dissipation by sudden collapses of bubbles and cavities. Since laccase enzyme was used, the temperature of working liquid was maintained at 30 °C by circulating cold water to circulation tank during the experiment.
Residual enzyme activity
Enzyme’s protein conformation changes in temperature, pH, ion concentration, and mechanical stress, and microenvironment of a solution. In this study it was observed that, the laccase enzyme was still active (OP1:23.5%, OP2:32.05%) after HCE treatment, this implies that the residual enzyme can be reused for another batch of pretreatment. For OP1, operated with inlet pressure of 50 kPa, the residual enzyme activities at 5, 10, 20 and 30 min were 34.2, 28.6, 23.5, and 23.5%, respectively. The residual enzyme activity was initially reduced and stabilized after 30 min. While OP2 operating with inlet pressure of 100 kPa showed that the residual enzyme activity was gradually reduced over time (34.2, 33.1, 32.1 and 32.05% in 5, 10, 20 and 30 min, respectively). Typically, the residual enzyme activity obtained for OP1 was less than that of OP2, possibly due to variation in higher inlet pressure, coupled with hole numbers and diameter. The frequency of turbulence decreases with an increase in the diameter of the orifice hole [51]. Laccase, being a green catalyst, is widely used for delignification. Although it contributes for the cost factor, in this study, we have shown that laccase after HCE treatment did not lose its activity much. This indicates that the enzyme can be reused for further pretreatment.
Cavitational yield
The cavitational yield is the result of several design parameters optimized for cavitating reactor [52, 53]. This yield can be enhanced by changing flow conditions and reactor geometry. The orifice plates with higher holes results higher cavitational yield due to increased cavitational effects. Cavitational yield can be defined as number of molecules degraded per unit energy dissipated.
$${\text{Cavitational yield}}\, = \,8.834\, \times \,10^{ - 11} \left( {P_{\text{collapse}} } \right)^{1.1633}$$
Collapse pressure can be predicted by
$$P_{\text{collapse}} \, = \,7527 \, \left( F \right)^{ - 2.55} \, \times \,\left( {P_{I} } \right)^{2.46} \left( {R_{0} } \right)^{ - 0.80} \left( {d_{o} } \right)^{2.37}$$
where R0 is the initial cavity size, mm; PI is the inlet pressure, atm; Do is the diameter of the hole in the orifice plate, mm; F is the percentage free area of holes in the total cross-sectional area of the pipe
The energy required per kg of biomass in an HCE pretreatment process
The energy consumed for treating one kg of biomass by the HCE pretreatment process was calculated by the following equation.
$${\text{Energy consumption, MJ/kg}} = \frac{{{\text{Inlet pressure}} \left( {\frac{N}{{m^{2} }}} \right) \times \;{\text{flowrate }}\left( {m^{3} } \right) \times {\text{reaction time}}\left( s \right)}}{{{\text{Weight of biomass used }}\left( {\text{kg}} \right)}}$$