**6. Microbial cells immobilisation**

Among the commonly immobilised biomolecules are enzymes and microbial cells. Enzymes are biomolecules capable of accelerating the rate of chemical reactions by acting as biological catalysts. The molecules upon which an enzyme might act is called substrate, which is transformed into products. As biocatalysts, enzymes participate in chemical reactions but are not consumed. Therefore, a particular enzyme can be reused repeatedly under optimum assay conditions and a sufficiently high substrate concentration, provided the products are continuously removed. Immobilisation of an enzyme onto solid support appeared to be an effective means of achieving improved recovery of the enzyme for reuse, better operational and storage stability, enhanced pH and thermal resistance, and product separation and purification [109–111].

According to the definition of immobilised enzyme given at the first enzyme engineering conference in 1971, "immobilised enzymes are physically confined or localised in a certain defined region of space with the retention of their catalytic activity, which can be used repeatedly and continuously" [112]. A significant aspect of this definition involves the retention of enzymatic activity, which need not be complete but should be high enough to be of practical interest. Typically, a residual activity of about 50% is typical, whereas a residual activity below 25% may be unacceptable [113]. Enzyme immobilisation has therefore continued to attract research interest from fundamental academic research to various industrial applications, inspiring more innovative immobilisation approaches in terms of simplicity of approach and enhanced stabilisation and performance.

The choice of using immobilised or soluble biomolecules (enzymes and cells) in industrial processes is driven by the cost of the biomolecules and the application. However, immobilised species are mostly preferred because of their reusability and adaptability to different process formats [114]. Basso and Serban summarised the factors that affect enzyme immobilisation which must be considered in the study (**Figure 12**) [115]. In a typical immobilisation process, the selectivity, stability, and kinetics of enzyme are carefully considered alongside the immobilisation matrix's physical, chemical, and mechanical properties to maximise the process's productivity (kg product/unit of the immobilised entities) [115].

The basic idea behind enzyme immobilisation started with the entrapment of enzymes within semipermeable materials that would allow the substrate and cofactors

**Figure 12.** *Factors affecting biomolecules immobilisation.*

to pass through them while the enzyme is retained within the matrix [17]. Thus, the control of the porosity of the matrix became a critical criterion. Depending on the type of enzyme and intended application, the material should be at least non-degradable and compatible with the enzyme's optimum assay condition. Also, the immobilisation process should be simple and mild enough in order not to denature the enzyme in the process, and in the case of *in vivo* application, the material must not be immunogenic. Given these requirements for the immobilised enzymes, alginate hydrogels fulfil these requirements and have thus continued to gain popularity in many enzyme and cell immobilisation studies.
