*3.3. Mechanical Properties of TA1*/*Q235B Sheets Hot-Rolled at Di*ff*erent Temperatures*

Figure 11 shows the microhardness and test schematic of explosive welding plates and hot-rolled sheets at different temperatures. Since the thickness of the sheet is only 2 mm after rolling, it is difficult to test the hardness gradient along a straight line, and to ensure that the adjacent test points are far enough so the test method as shown in Figure 11a,b is adopted. The microhardness of matrix metals is measured at the distance of 225 μm near the bonding interface, as shown in Figure 11b. Assuming that the interface bonding is the origin, the direction from the interface to Q235B side is positive. TA1, Q235B and the interface are tested five times, and the results are averaged. The interface microhardness distribution is shown in Figure 11c. The microhardness of the explosive welded plate is higher than that of the rolled sheets and the hardness of the matrix, and the interface decreases with the increase of the rolling temperature. The microhardness of the interface is higher than that of the matrix. The maximum interface microhardness is 217.8 HV at the explosive welded plate, with an increase of

rolling temperature, which is gradually decreases to 184.2 HV. When the rolling temperature rises, the change of interface microhardness is related to the amount of TiC (as shown in Figure 8), while the decrease of TA1 and Q235B matrix microhardness is related to the recrystallization grain size (as shown in Figure 10).

Shear strengths, yield strength (YS), ultimate tensile strength (UTS), maximum elongation and Young's modulus of rolled sheets under different temperatures are shown in Figure 12. With the increase of the rolling temperature from 1003 K to 1103 K, the shear strength of the composite sheet increases from 142.1 MPa to 171.4 MPa. The interface shear strength of the TA1/Q235 composite sheet is affected mainly by the interface brittle compounds. It can be seen from Figure 8 that the amount of the C element in the transition layer decreases when rolling temperatures rises, it means the amount of brittle TiC decreases with the increase of rolling temperature. Hence, the formation of brittle carbides in TiC may suppress by the increasing rolling temperature, and the shear strength improved with the disappearance of brittle compounds TiC at a relatively high rolling temperature.

**Figure 9.** Electron probe micro-analysis (EPMA) element distribution in the interface of TA1/Q235B sheets hot rolled at (**a**–**c**) 1003 K, (**d**–**f**) 1053 K and (**g**–**i**) 1103 K, (**a**,**d**,**g**) are microstructures, (**b**,**e**,**h**) are Ti elements and (**c**,**f**,**i**) are Fe elements.

**Figure 10.** EBSD Inverse pole figure (IPF) mapping of TA1/Q235B sheets hot rolled at the temperature of (**a**) 1003 K (**b**) 1053 K (**c**) 1103 K.

**Figure 11.** (**a**) Micrograph of microhardness test (**b**) schematic of microhardness test (**c**) microhardness of explosive welding plate and different temperatures rolled sheets.

**Figure 12.** The tensile stress-strain curves and mechanical properties of TA1/Q235B composite sheets at different rolling temperatures.

The YS and UTS of hot rolled TA1/Q235 sheets decrease with the increase of rolling temperature. After rolling at 1003 K, the maximum YS and UTS of the hot rolled sheet are 517.6 MPa and 558.7 MPa, respectively. The sheet rolled at 1103 K shows lower yield strength (414.0 MPa) and ultimate tensile strength (438.1 MPa) than that rolled at 1003 K and 1053 K. The lower strength of 1103 K rolled sheet is due to the increase of recrystallization in titanium and carbon steel matrix at higher temperature (as shown in Figure 10). The increase of recrystallization also leads to a gradual increase in elongation. The Figure 12 also shows that the rolling temperature has little influence on the Young's modulus of the sheet, and when rolling temperature rises from 1003 K to 1103 K, while the Young's modulus of the sheet does not change significantly and only decreases slightly.

Figure 13 shows the macroscopic morphology of sheets rolled at different temperatures after tensile fractured. It can be seen that titanium and carbon steel are separated near the fracture surface. During the tensile test, the brittle compounds TiC and FeTi in the bonding interface fractured firstly. Subsequently, the fracture occurred in the matrix metals. Figure 14 shows the SEM fracture morphology after tensile fracture of samples rolled at different temperatures. It is obvious that interfacial fractures of the composite sheet rolled at 1003 K is small, and the extent of interfacial fractures increases with the increasing rolling temperature. It illustrates that the interfacial compounds layer causes the cracks to initiate in the composite sheets, and crack propagation is more serious with the thicker interfacial compound layer. It can be seen from Figure 14d–f that the fracture profiles of titanium at different rolling temperatures are similar in fracture characteristics. The fracture profile of the TA1 plate includes cleavage fractures, while the fracture profile of carbon steel is a dimple fracture. As shown in Figure 14g–i, the depth, size, and proportion of the dimple increased with the increase in rolling temperature, and the plasticity of the composite sheets increased simultaneously [32,33]. The more in-depth, more extensive, as well as larger proportion of dimple fractures exist on the carbon steel side of the composite sheet at 1103 K, which is related to the increase in plasticity after proper heating [34]. Because of the appearance of both dimples and cleavage planes in the rolled composite sheet, it infers that the fractures of TA1/Q235B composite sheets are a mixed-mode of ductile and brittle fractures.

**Figure 13.** (**a**) The area of SEM of fracture profiles, (**b**) Macroscopic morphology of sheets rolled at different temperatures after tensile fracture.

**Figure 14.** SEM fracture morphology after tensile fracture of samples rolled at different temperatures: (**a**) 1003 K, (**b**) 1053 K, (**c**) 1103 K, (**d**,**g**) Ti and steel matrices in (a), (**e**,**h**) Ti and steel matrices in (**b**), (**f**,**i**) Ti and Steel matrices in (**c**).


**3. Experimental**

### **3.1. Material**

Commercial powder lipase from *Thermomyces lanuginosus* (fungal lipase, lyophilized, specific activity of 1400 U/mg solid) and *Candida antarctica* lipase A (specific activity of 2500 U/mg solid) were purchased from Codexis Inc. (Pasadane, CA). Sunflower oil and canola oil were supplied by a local company; waste cooking oil (WCO) was used following filtration to remove particles remaining in the oil after domestic use. Polyethyleneimine [PEI; (C2H5N)n] as 50% (w/v) (average molecular weight 750.000) and glutaraldehyde (GA, acidic aqueous solution as 25% (w/v)) were obtained from Sigma (USA) and AppliChem (Darmstadt, Germany), respectively. All other chemicals were of analytical reagent grade and used without further purification.

### **3.2. Experimental procedure**

### *3.2.1. Lipase immobilization*

stable than plant and animal lipases and are available in bulk at lower cost compared to lipases of other origin. Yeasts lipases are easy to handle and grow compared to bacterial lipases. Among the yeast lipases, *Candida rugosa* has gained good commercial importance. The most commonly used biocatalyst for biodiesel production are the microbial lipases that are pro‐ duced by a number of fungal, bacterial, and yeast species [40]. Free enzymes are far cheaper than immobilized lipases. They can be purchased in an aqueous solution composed of the enzyme solution plus nothing more than a stabilizer to prevent enzyme denaturation (e.g. glycerol or sorbitol) and a preservative to inhibit microbial growth (e.g. benzoate) [49].

Immobilization of lipases was carried out using entrapment, physical adsorption, ion ex‐ change, and crosslinking. Carriers for lipase immobilization include polyurethane foam, silica, sepabeads, cellulosic nanofibers. Based on the criteria for selecting the immobilization technique and carrier dependings on the source of lipase, the type of reaction system (aqueous, organic solvent or two-phase system), and the bioreactor type (batch, stirred tank, membrane reactor, column and plug-flow) can be designed. The literature is replete with various lipase producing microorganisms, enzyme immobilization methods, and physical carriers. The challenge will be to select a carrier and immobilization technique that will allow maximum lipase activity, retention, and stability on the oil substrate. Among the immobilization method, adsorption technique is the simplest and most widely used technique for lipase immobiliza‐ tion. Adsorption method consists of bonding the lipase to the immobilization support surface through weak forces such as van der Waals or hydrophobic interactions. However, the main disadvantage of this technique is enzyme desorption from the support due to low bond

In recent years, whole-cell immobilized lipases have been studied for biodiesel production. This method is cheaper as it does not require the enzyme purification and isolation steps from fermentation broth. The efficiency of the transesterification process could be increased by using microbial cells that produce intra-cellular lipase as whole-cell biocatalysts [40, 46]. Filamentous fungi have been identified as robust whole-cell biocatalysts for biodiesel production: among these *Rhizopus* and *Aspergillus* have been most widely used [42]. There are several recent works reporting the utilization of bacteria, yeast and fungi as whole-cell biocatalysts in biodiesel

Commercial powder lipase from *Thermomyces lanuginosus* (fungal lipase, lyophilized, specific activity of 1400 U/mg solid) and *Candida antarctica* lipase A (specific activity of 2500 U/mg solid) were purchased from Codexis Inc. (Pasadane, CA). Sunflower oil and canola oil were supplied by a local company; waste cooking oil (WCO) was used following filtration to remove particles

*2.4.2. Immobilized biocatalysis*

26 Biofuels - Status and Perspective

*2.4.3. Whole-cell biocatalysis*

process [27].

**3.1. Material**

**3. Experimental**

strength between the enzyme and the support [40].

Figure 1 shows multi-layer immobilization of *T. lanuginosus* lipase on cotton cloth by aggre‐ gation with polyethyleneimine (PEI). 1 mL of PEI solution (pH=11), containing 2 mg of PEI, was added to each 0.1 g piece of cotton cloth. The PEI solution volume was at a sufficient level to completely wet the cloth. After adsorption of PEI, 50 mg of enzyme (5 mL of 10 mg/mL enzyme solution) was added. Upon the addition of enzyme to PEI-adsorbed cotton, a "milky" turbid solution was formed. The flasks were put into a shaker-incubator (Heidolf Unimax 1010, Germany) at 150 rpm at room temperature (25±1 o C) for 5 min. The white turbidity disappeared within 5 min and the coupling solution was completely clarified. The clarified coupling solution was slowly decanted and PEI-enzyme coated cottons were dipped in a cold GA solution (2.5% (w/v), pH= 3.5) for cross-linking at 5 min. The cross-linked cottons were washed with distilled water and potassium phosphate buffer (1 M, pH= 7). It is important to note that there was no washing step until the completion of GA cross-linking [25, 39]. Based on the degree of immobilization tests, about 80-90% of the enzyme was immobilized in this proce‐ dure. The actual enzyme loading was determined as 180 mg of *T. lanuginosus* lipase per 1 g of cotton cloth.

**Figure 1.** The procedure for PEI-multilayer lipase immobilization on cotton cloth fibrils

### *3.2.2. Biodiesel production with immobilized lipase-catalyzed transesterification*

Production of biodiesel by enzymatic catalyzed transesterification from various vegetable oils was studied in a packed bed reactor (Figure 2). A small piece of immobilized cotton cloth (1 g) was placed in the glass column reactor (1 cm diameter x 12 cm height) with a water jacket maintained at a constant temperature (30 o C). Substrate mixture (oil and alcohol) was contin‐ uously recirculated throughout the immobilized enzyme reactor with a peristaltic pump at a flow rate of 50 mL/min by adding of alcohol in three-steps. Immobilized cotton cloths were washed by *tert*-butanol before adding of alcohol to each reaction medium. Reaction was continued for 10 h. Samples were taken from the flask at appropriate time intervals and analyzed for fatty acid methyl esters (FAMEs) and glyceride contents by high performance liquid chromatography (HPLC) [50].

**Figure 2.** Schematic diagram of the enzymatic column reactor used in the transesterification reaction