**3. Adoption of organic absorbent materials for purification procedures**

The emergence of organic materials such as activated charcoal, *Calostropis procera* latex and imarsil has made significant contributions for industrial applications. Some of these organic absorbents are discussed below:

#### **3.1 Activated charcoal and other carbon particles as purifying agents**

Activated charcoal is an adsorbent extensively utilized in the treatment of wastewater and industrial contaminants by reason of its high shifting ability and adaptability for a wide range of pollutants. It is produced from any crucially carbonaceous materials. Coal, cotton waste, tree barks, palm kernel shell, and many agricultural by- products can be made to produce activated carbon and their capacity to remove colors has been investigated. Ferreira and coworkers [26] demonstrated the production, characterization of activated charcoal from castor seed cake through activation with phosphoric acid. Treatment of fino sherry wine with activated charcoal, in combination with other clarifying agents, produces a wine with lower polyphenolic content, good organoleptic characteristics, but its receptive to browning is indistinguishable to that recognized in untreated wine, despite starting from lower levels of color potency. Activated charcoal is used to remove compounds that cause objectionable color, odor and taste in water treatment while its industrial applications require elimination of harmful gases and pesticides and including purification of organic compounds [27]. It is established that 80% of activated charcoal globally produced is used in aqueous-phase adsorption of both organic and inorganic compounds [28]. However, the application of activated charcoal in the decolorization of enzyme-converted glucose syrup had been described; though its application for the purification of microbial biocatalysts has been sparse.

#### *Prospects of Biocatalyst Purification Enroute Fermentation Processes DOI: http://dx.doi.org/10.5772/intechopen.97382*

One profound advantage of activated charcoal over conventional purification systems is this swift enzyme purification from composite fermentation broth mixture at a very high purification fold. These conventional procedures of purification of enzyme among others include solvent precipitation; gel filtration and salting out technique. From an industrial application stand point of view, they are quite expensive base on the fact that they are associated with some difficulty of scaling up and plugging leading to viscous and particulate materials when treated with crude enzyme extracts. Additionally, it is not economical for developing nations as the materials disposal or enzyme recovery techniques employed in the separation method might escalate the expenses. As a result, the usage of activated charcoal has been considered as preferred option for enzyme purification method.

In addition to the inexpensiveness of activated charcoal, their efficient surface absorption attributes can be exploited for depolarization of fermented medium for efficacious and efficient recovery and purification of industrial enzymes making the downstream processing in large-scale industrial bioprocesses less economical [29]. López et al. [30] in their investigation on the use of activated charcoal in combination with other fining agents as clarifying agents reported that these carbonized materials acted upon the phenolic compounds thus encouraging their precipitation. In the field of enology, many different substances have been employed as fining agents over time such addition of antioxidants (ascorbic acid, sulfur dioxide and bottling under inert atmosphere. The use of bentonite has been well-proven and reported to have a remarkable effect on the protein content of wine and also hastens the precipitation of the thermolabile protein [31] but has also minimized the polyphenolic content of the wine during production [32].

The structure of activated carbon which is based on the graphite lattice corresponds to a non-graphitizable carbon and macromolecular structure of the precursor residues during heat treatment, and losses small molecules by developing and degradation some cross-linking, so that joining cannot occur. Therefore, crosslinking bring about a fixed design with small vigor, thus producing a permeable system and intercepting the ordering expected during graphitization.

### **3.2** *Calotropis procera* **as purifying agent and its industrial applications**

*Calotropis procera* belongs to the family Asclepidaceae being a native of tropical and subtropical region of Africa, the Middle East, and South and South-East Asia [33]. It is a shrub that produces latex with wide pharmacological profile which is a rich source of biologically active compounds [34]. *C. procera* latex contain several chemical compounds which include calotropagenin glycosides/derivatives [35]; saponins, flavonoids and cardenolides [36, 37]; cardioids such as calotoxin, calotropin, uscherin, uscchardin, choline, o-pyrocatechuric acid, glycoside calotropaginin, benzoyllineolone, benzoylisoloneolane, syriogenis and uzariganin etc. [38]. It has been traditionally used for various medicinal purposes such as treatment of animal worms, defense role in plants, acting against herbivorous insects, nematodes and phytopathogenic fungi [39]. Different parts of roots, leaves, flowers and latex from the plant are used in several medicinal preparations [40]. It was also reported to exhibit potent analgesic and weak antipyretic activity in various experimental model, possess antioxidant and anti-hyperglycemic property [41], antihelmintic activity [42, 43]; insecticidal and antifungal proteins and their enzymatic profiles have been characterized [44–46]; and there is an empirical association between antioxidant property and residual peroxidase activity. The milk weed has been established to be efficacious in the chemotherapy of malaria, menorrhagia, fever, leprosy and snake bites. Research works investigated on many biological activities of *C. procera* including osmotin proteins exert antifungal activity [47] and

anti-inflammatory potential in rats. *C. procera* latex dispensed to rats revealed pain-killing effects wound healing and toxic [48].

The leaf of *Calotropis procera* is a natural coagulant used traditionally in waste water treatment and it has also been reported that *Calotropis procera* leaf is effective in removal of environmental pollutant, polyphenolic crystal violet dye from aqueous solution of textile effluent [49] which presumed to be ascribed to the presence of peroxidase in *Calotropis procera* leaf that oxidized phenols to phenoxy radicals. Some studies suggest that the insoluble fraction of *C. procera* latex is associated with the noxious effects of this fluid [50]. Contrariwise, some constituents of this fluid cause toxicity in small ruminants [51].

From the investigation carried out by Mafulul *et al.,* [52] in the extraction, partial purification and characterization of peroxidase from *Calotropis procera* leaves, it was revealed that peroxidise purified from *Calotropis procera* leaves in primary purification procedures resulted in 1.613-fold purification of peroxidase from the crude extract. Subsequently, enzyme precipitation using ammonium sulphate with the dialyzed fraction showed 2.04 purification folds. *Calotropis procera* leaves peroxidase maintained above 50% over a temperature range of 20–70 with optimum temperature 50°C.

Furthermore, considering the availability and abundance of *Calotropis procera* fresh leaves in Nigerian distribution coupled with availability of advance purification method, this plant tends to provide a very cheap source of peroxidase for phenolic pollutants' bioremediation for waste treatment especially in oil spill region of Niger Delta. It provides potential alternative peroxidase that can compete with commercially available peroxidases for biotechnological applications.

#### **3.3 Imarsil - an inexpensive synthetic chromatographic absorbent**

Imarsil is a novel, inexpensive synthetic chromatographic absorbent and oxidized natural polymer of *Brachystegia nigerica. B. nigerica* is a legume used especially in the eastern states of Nigeria as condiment to thicken soup. Its thickening characteristics have been attributed to the presence of hydrocolloid property or gelling property [53]. Imarsil possesses quick and simple recovery approach more importantly in the clarification of microbial biocatalyst from fermentation broth [11]. Cherry and Fidants, [54] demonstrated the use of carboxymethyl cellulose, edible gum and tannic acid as precipitants and as well as organic solvents also poses the problem of product recovery. Gel filtration technique is also considered assiduous and expensive in the developing countries.

Several procedures of concentrating protein and dilute enzyme from fermentation cell extracts and media using agricultural residues as coagulants. Furthermore, Kareem *et al.* [55] investigated the use of Imarsil and activated charcoal to purify crude lipase in a two-step purification fold which brought about an increase in specific activity from 5.29 to 20.8 Umg−1 with protein reduction of 18.24% in the supernatant and ultimate 3.93-fold purification. The study on crude amylase purification showed that a 40-fold purification was attained with 50% final yield of the total fungi amylase in a 3-step purification technique. The elution pattern of *Rhizopus oligosporus* SK5 amylase on Sephadex G-100 column had peaks at fractions (19–22) and (34–38). This purification fold value is conceived greater than values obtained in previous work [56].

Osho et al. [57] studied on production and optimization of bacterial cellulase using agricultural cellulosic biomass by solid state cultivation where the enzyme was clarified with Whatman No 1 filter paper, partially purified with Imarsil (1% w/v) and incubated at 4°C for 3 h. It was reported that at temperature ranges of 40–90°C, enzyme activity increases in crude and partially clarified states as the relative

*Prospects of Biocatalyst Purification Enroute Fermentation Processes DOI: http://dx.doi.org/10.5772/intechopen.97382*

activity also increased to 50 and 60°C for both forms of cellulase respectively. A decline in activity was noticed as temperature increases for both solutions. However, 90% activity of the partially purified enzyme was retained between 50 and 55°C and activity peaked at 60°C. Partial clarification of enzyme is therefore needed to enhance their stability even at much temperature. Kareem and other coworkers [58] outlined that partial clarification of enzyme using activated charcoal preceding gel filtration will established a high purification fold thus preventing some awkwardness of plugging and scaling up when treating crude extracts that sometimes contain particulate and viscous materials. These studies have further substantiated the use of Imarsil as a coagulating-flocculating agent in purification of crude enzyme extracts.
