**8. Microbial immobilisation**

Immobilised microbial systems are becoming increasingly popular in various fermentation processes [144, 145].

The benefits of immobilised microbes over free-cell batch methods come from the ability to use immobilised microbes in continuous operations. Additionally, immobilised microbial cells maintain high cell densities per unit of bioreactor volume long after the nominal washout rate and produce incredibly high fermentation rates [146]. The immobilisation process imitates the phenomenon of microorganisms naturally attaching to various surfaces in nature [147]. Most ethanol production methods are constrained by low ethanol production rates and issues with recycling and separation depending on the microorganism employed [148]. Immobilised microbial cells used in continuous fermentation operations have the potential to boost ethanol output while cutting costs [149].

In contrast to batch processes, continuous systems allow for higher cell densities inside the bioreactor and smaller reaction volumes [3, 146]. Whole-microbial cell immobilisation has received attention from numerous research teams as a potential replacement for traditional microbial fermentation techniques [148]. In particular, microbial cell immobilisation is also simple to apply in sterile circumstances. The production procedure can be performed under minimally stressful circumstances without applying chemical reagents that could seriously harm the environment and hinder cell activity [150].

#### **8.1 Microbial encapsulation**

As the demand for biorefineries increases, it becomes increasingly important to efficiently use all sugars generated from biomass materials [151]. One of the significant obstacles to the viability of the bioprocess is the presence of inhibitory agents in biomass hydrolysates [150]. While First-generation (1G) bioethanol is produced from fermentable sugars directly extracted from food, second-generation (2G) bioethanol is made from lignocellulosic biomass [152]. One of the advantages of 2G is that it is not in competition with food production. However, there are still some challenges in producing 2G ethanol because of the challenge of releasing fermentable sugars completely, the need for effective biomass pretreatments, and low conversion efficiency and yield [153, 154]. Amutha and Gunasekaran [155] found that a higher ethanol yield from liquefied cassava starch was obtained with co-immobilised *Zymomonas mobilis* and *Saccharomyces diastatitus* cultures than with free-state cells. Notably, the immobilised cells caused fermentation processes to end earlier due to the sizeable cellular biomass within the support material, implying reduced processing time.

Additionally, Amutha and Gunasekaran observed that the microbial cells maintain their activity throughout numerous successive batches. Compared to pure-alginate beads, the hybrid alginate-chitosan gel produced improved yeast activity at crude hydrolysate of sugarcane bagasse hemicellulose [150]. Soares *et al.*'s finding showed the possibility of a hybrid gel boosting Second-generation (2G) bioethanol output and prolonging microbial recycling.

Because of its toxic effect and nonbiodegradability, heavy metal pollution poses a significant threat to both human health and the integrity of the ecological system [156]. The use of microorganisms to clean up hazardous metal wastes has already attracted the considerable interest of scientists due to its excellent benefits, which include high efficiency, low cost, and environment friendliness [156]. Utilising Polyvinyl alcohol, sodium alginate, and multiwalled carbon nanotubes, Pang *et al.* immobilise *P. aeruginosa* for hexavalent chromium Cr(VI) detoxification [157]. The beads Pang *et al.* used were immobilised, frozen, and thawed to increase their mechanical strength. The immobilised *P. aeruginosa* bacteria were able to decrease 80 mg/L Cr (VI) in 84 hours, but the free cells were rendered inactive at that concentration of the heavy metal. Also, *P. aeruginosa,* immobilised using alginate and biochar as composite carriers, was used in removing the contaminant acenaphthene from wastewater [158]. According to Lu *et al.*, the immobilised system was promising and thus can be applied to many sewage treatment reactors and the on-site clean-up of contaminated water. Guo *et al.* [159] immobilised *Bacillus subtilis* to remove ammonia nitrogen from swine effluent using chitosan-sodium alginate composite carriers. The immobilised *B. subtilis* was tolerant to high pollutant concentrations, with promising potential application for removing ammonia nitrogen from wastewater.

*Alginate-Based Applications in Biotechnology with a Special Mention to Biosensors DOI: http://dx.doi.org/10.5772/intechopen.110737*


### **Table 3.**

*Microbial immobilisation techniques and applications.*

The production of renewable hydrogen from biological means is promising. Governments, researchers, and businesses have all noticed the use of biohydrogen gas as an alternative to traditional fossil fuels since it is seen as a green answer to environmental problems [160]. *Clostridium intestinale* immobilised inside 2% calcium-alginate beads were used to produce hydrogen in strictly anaerobic circumstances [161]. Güngörmüşler *et al.* data indicate that although the bacteria inside hydrogel beads experienced a lag at the start of the fermentation process, the immobilised cells outperformed suspended cultures in terms of volumetric rate of production and molar yields of hydrogen. *Chlamydomonas reinhardtii* and *C. vulgaris*, two different microalgae species, were used to assess the effectiveness of nutrient removal [162]. According to Lee *et al.*, the microalgae species removed the nutrients efficiently. Specifically, the photo-bioreactors with 20% algal bead volume fractions removed 95% of total Nitrogen and completely reduced total *phosphorus* in 3 phases of treatment. In another study conducted using immobilised yeast cells (that expressed Laccase from *Streptomyces cyaneus)*, Popović *et al.* completely decoloured Reactive Black 5, Amido Black 10B, Remazol Brilliant Blue, and Evans Blue [163]. Popović *et al.*'s findings suggest that dye decolourisation could be carried out using laccasecoated yeast cell walls encapsulated within dopamine-alginate beads (**Table 3**).

#### **8.2 Microbial entrapment**

*S. Cerevisiae*, immobilised by entrapment in calcium alginate, was shown to maximise ethanol generation at different alginic acid content, size of the bead, concentration of glucose, temperature, and hardening time [164]. Mishra *et al.* employed lignocellulosic hydrolysate from rice straw in a packed bed reactor. The use of rice straw enzymatic hydrolysate makes Mishra *et al.*'s procedure economical and environmentally beneficial since no antibiotics were used and no detoxification was needed. Matthew *et al.* [165] compared the bioethanol production capacity of free-living or immobilised *Saccharomyces cerevisiae* from oilseed rape straw hydrolysate. The yeast cells were either immobilised as a biofilm on grains, Leca, or reticulated foam or entrapped in alginate

beads or Lentikat® discs. Overall, the research's objectives were to evaluate the bioethanol yields produced by free and immobilised systems and to determine the most effective method of immobilisation in terms of bioethanol production and durability of the immobilised cell system. Compared to the free-living cells and immobilised as a biofilm, cell entrapment in alginate beads and Lentikat® discs produced noticeably greater bioethanol yields. Essentially, yeast immobilised on alginate films generated a larger ethanol yield than free yeast cells under the same conditions [166].

#### **8.3 Microbial electrostatic droplet generation**

Electrostatic extrusion is an innovative and effective method for immobilising microbial cells. The specific need to use tiny beads for many different fermentation processes, such as beer, wine, and cider fermentation, makes electrostatic extrusion attractive. To overcome diffusion constraints of metabolic products and nutrients inside the carrier matrix, small immobilisation beads are needed for fermentation [144–146]. A considerable decrease in droplet size is often achieved using electrostatic extrusion. Nevertheless, the presence of microbial cells often slows network formation and reduces the Ca-alginate hydrogel's strength properties [169]. As opposed to emulsion procedures, electrostatic extrusion yields homogeneous and small beads, as small as 50 μm in diameter [170].

In electrostatic droplet generation, the diameter of the microbeads typically increases when microbial cells are present [146]. The microbial cell concentration may be a crucial element in electrostatic droplet generation, which is determined by the microbe type's growth characteristics or the immobilised system's desired functionality [146, 170]. Microbial electrostatic droplet generation relies on the utilisation of electrostatic forces to disrupt a liquid of a needle tip and generate a charged stream of tiny droplets (**Figure 15**) [170]. Nikolić *et al.* examined how immobilisation affected the conversion of corn meal hydrolyzates into bioethanol [167]. The authors immobilised yeast cells in Ca-alginate using the electrostatic droplet generation technique. According to their findings, diffusion and reduced levels in the bead core caused the yeast cells to have a greater tolerance to an increased substrate and product contents than free cells did.

The electrostatic droplet approach was also used to immobilise *Lactobacillus rhamnosus* in a poly(vinyl alcohol)/calcium alginate (PVA/Ca-alginate) composite for use in lactic acid fermentation [168]. Mechanical characterisation revealed that the PVA/Ca-alginate beads had a significant elastic character. *L. rhamnosus* showed remarkable survival in addition to withstanding a relatively abrupt immobilisation treatment involving "freezing-thawing." Furthermore, the immobilised biocatalyst outperformed the free cell fermentation system by 37.1% because of its excellent operational and mechanical stability and capacity to withstand the potentially stressful "freezing-thawing" approach.

## **9. Application of alginate composites in the development of biosensors**

The driving force in developing biosensors has remained the need to increase the sensitivity, selectivity, and stability or reduce the production costs of the biosensors [67]. Moreover, such development strategies could range from the biological compound exploration of biological sensing elements such as enzymes, DNA, antibodies, cells, and supporting materials for biological compound immobilisation to detector

### *Alginate-Based Applications in Biotechnology with a Special Mention to Biosensors DOI: http://dx.doi.org/10.5772/intechopen.110737*

improvisation. A biosensor is a self-contained analytical device that uses a specific interaction between analytes and their biological recognition to provide qualitative, quantitative, and semiquantitative information about the analyte(s) being probed. It consists of 3 main components – the sensing element, the transducer, and the detection system (**Figure 16**). The sensing element is made of biomolecules (proteins and nucleic acids), that are immobilised on matrix/support and can interact specifically with the analyte of interest leading to a measurable biochemical response. The biomolecular recognition element (BRE) of a biosensor determines the selectivity and specificity of that biosensor, and it has been a subject of intense research. Specifically, more attention is being paid to the functionalisation – the art of immobilising the biological material onto the support, because it constitutes a critical step in optimising the sensor's performance. Functionalisation must ensure that the structure and activity of the immobilised material are at least preserved or enhanced. Thus, simple and efficient immobilisation techniques are continuously sought.

NiFe2O4 nanoparticles-modified alginate cryogel has been used to develop an electrochemical glucose sensor by entrapping a glucose oxidase within the NPsalginate composite gels [67]. The NPs were added to impart electrical conductivity to the alginate so that the oxidation-reduction events at the working electrode could be efficiently detected and thereby increase the sensitivity of the biosensor. When the oxidation and reduction peaks at the enzymatic electrodes prepared by only alginate and NPs-modified alginate were compared, the latter showed higher oxidation and reduction peaks because of the large surface area of the porous cryogel combined with the nickel-ferrite NPs [67]. This biosensor showed a limit of detection (LOD) of 0.32 mM and a limit of quantification of 1.06 mM, which was a landslide sensitivity over a colourimetric alginate-based glucose biosensor [171], and near-infrared alginate-based glucose biosensor [172]. A separate study developed a sensitive amperometric electrochemical glucose sensor by electro-copolymerisation of covalently coupled biotin-pyrrole and alginate pyrrole to immobilise glucose oxidase [77]. The sensor construction consisted of the conjugation of biotinylated-glucose oxidase (B-GOx) to B-Py through avidin (Av) bridges, followed by copolymerisation with Alginate-Pyrrole. When the set-up did not include the pyrrole-modified alginate but

**Figure 16.** *Components of a typical biosensor.*

unmodified alginate, its performance values were significantly less [77]. Another electrically conductive alginate-polypyrrole composite has been investigated for biosensor development. In 2005, Abu-Rabeah *et al*. synthesised alginate-pyrrole conjugate to develop an amperometric sensor. The electrochemical polymerisation of pyrrole monomers generated alginate-polypyrrole. During the electrochemical synthesis of alginate-polypyrrole, the polyphenol oxidase (PPO) enzyme became physically entrapped within the alginate composite matrix. The entrapped enzyme was used to examine its amperometric determination of catechol, providing a sensor sensitivity of 350 and 80 μA M<sup>1</sup> cm<sup>2</sup> , respectively, for polypyrrolealginate and alginate biosensors [47]. The pyrrole-based electroconductive alginate gel has also been used in the entrapment of algal cells of *C. vulgaris* to develop amperometric sensors [70]. The same research group investigated the enzyme retention capacity of an electropolymerised polypyrrole-alginate matrix used for glucose oxidase-based biosensor construction. Like other reports on alginate-pyrrole enzyme immobilisation, this study showed an improvement in enzyme retention compared to the preparations involving only alginate. Electropolymerised alginate-polypyrrole protected the gel from the destructive effects of phosphate anions that could otherwise have competed for the Ca2+ used for the gelation of the composite [74]. Alginate composites exhibiting electrical conductivity are continuously investigated in developing highly sensitive biosensors. Antibodies immobilised on solid surfaces continue to find wide applications in immunosensors, affinity chromatography and diagnostic immune assays [173]. Alginate is among the solid surfaces used for immobilising antibodies and proteins due to its non-toxicity and gel-forming properties. Moreso, alginate derived composites have found extensive application in optical sensors development. For instance, covalently linked biotin-alginate was used for the encapsulation of genetically modified bioluminescent reporter cells into microspheres for determination of a model toxin, mitomycin [48]. The biosensor was fabricated by carbodiimide mediated covalent conjugation of biotin to the alginate resulting in a composite which was used to encapsulate the bioreporter within the microsphere (**Figures 17** and **18**).

**Figure 17.**

*Biotin-alginate microspheres conjugated to an optical fibre via avidinbiotin affinity interactions:(a) attachment of a lone bead to the end face of the fibre and (b) coating of the fibre with a number of microspheres [48].*

*Alginate-Based Applications in Biotechnology with a Special Mention to Biosensors DOI: http://dx.doi.org/10.5772/intechopen.110737*

#### **Figure 18.**

*Biotin coupling to alginate via carbodiimide chemistry [48]. Where EDC represents 1-Ethyl-3-(3 dimethylaminopropyl) carbodiimide), Sulfo-NHS is (N-hydroxysulfosuccinimide) and R represents the alginate polymer bearing carboxyl functional groups.*

The biotinylated microspheres were conjugated to the surface of a streptavidincoated multimode optical fibre which served as a transducer for the generated light. It was possible to attach both lone and multiple microspheres to the end of the optical fibre via avidin-biotin affinity [48]. The biosensors performance of the composite showed that biotin-alginate microsphere prevented diffusional loss of the encapsulated bioreporter that would have occurred using alginate alone. In 2017, Li et al. prepared an alginate-methacrylate based whole cell biosensor for the detection of quorum sensing molecules [174]. The biosensor development involved the encapsulation of the genetically reporter bacteria within the double crosslinked alginate-methacrylate microbeads. The entrapped bioreporter produces fluorescence by a dose-dependent expression of green fluorescent protein in response to the *P. aeruginosa* secreted autoinducer signalling molecule. The resulting biosensor unit is facile, as the combination of ionic cross-linking and photo-cross-linking affords the formation of stable and robust alginate-based microbeads with decreased swelling ratio, increased stability, and good permeability of dye-labelled autoinducers [174]. The encapsulation efficiency and the viability of the encapsulated reporter bacteria were remarkable, while the increased bead stability reportedly led to 10 times decrease in bacteria leaching from the beads. Alginate and its derived composites have been continuously evaluated for sensors and biosensors applications.

## **10. Conclusion and future perspective**

The exploitation of alginate and its composites as immobilisation support matrices remains a promising research field with limitless potentials of creating innovative and advanced functional materials from the sustainable natural resources on earth. Thanks to their attractive features, including non-toxicity, ease of preparation, excellent biocompatibility, biodegradability, and amenability to chemical functionalisation, alginate and its composites have continued to find widespread biotechnological and biomedical applications. Incorporating other substances (such as natural or synthetic polymers and nanoparticles) into alginate results in alginate composite materials with enhanced or novel physicochemical properties. Thus, the preparation and characterisation of various alginate composites have become increasingly attractive to most biomaterial engineers.

Alginate composite as an immobilisation matrix has witnessed tremendous advancements in the past few decades ranging from the essential encapsulation of molecules to a more stable immobilisation, engaging two or more strategies. The concept of composite formation of alginates derives from the need to overcome the apparent limitation associated with the alginate in terms of physicochemical parameters such as enhanced physical strength, controlled porosity, improved interaction between the alginate support and the biomolecules as well as the impartation of other features such as electrical and magnetic responsiveness among others. So far, better immobilisation performance in terms of porosity and chemical reactivities has been achieved.

Any given immobilisation approach should be simple and able to maintain the integrity and activity of the immobilised entity. The facile nature of immobilisation by encapsulation has drawn much interest in most biotechnological applications, directing enormous research efforts towards improving the encapsulation performance of alginate. The concept of composite formation has led to a tremendous advance in the immobilisation efficiency of alginate hydrogels, one of which is the emergence of the core-shell technology, widely used in targeted delivery and controlled substance release. Depending on the configuration, alginate (or its composite material) could be either the core or the outer shell, as discussed above. The advent of core-shell technology was a breakthrough in immobilisation studies. Furthermore, alginate composites demonstrate different stabilities as well as swelling behaviours in different ionic environments. With a careful choice of dopant in composite alginate formation, one can have the freedom to exert control over alginate.

Hu *et al*. prepared a dual layer of alginate-carboxymethyl cellulose (Alg-CMC) and polyacrylamide (outer layer) for the encapsulation of protein intended for targeted delivery application [13]. In their study, the swelling behaviour of the inner layer (Alg-CMC) was regulated by the outer layer (synthetic polymer) with negligible swelling capacity under the experimental condition. This concept could be expanded for the immobilisation of catalytic biomolecules and cells where the outer layer could serve as a selective permeability barrier with controllable porosity to allow for material exchange and protect the inner alginate layer against degradation. Another aspect of interest is the possibility of immobilising the biomolecule on the dopants and subsequent encapsulation within the alginate matrix. This approach could address the unpredictable diffusional loss of the encapsulated materials.

Chemical (covalent/affinity) immobilisation does not suffer diffusional loss. However, the immobilisation chemistry must be carefully selected to have a negligible effect on the structure and activity of the immobilised species. The chemistry must be simple and interact with a site other than the catalytic site (in the case of enzymes),
