Microbial Biomaterials and Their Applications

23 Microbial Biomaterials and Their Applications Se-Kwon Kim, Ira Bhatnagar, and Ramjee Pallela CONTENTS 23.1 Introduction......................................................................................................................... 457 23.2 Microbial Biomaterials....................................................................................................... 458 23.2.1 Polyhydroxyalkanoates.......................................................................................... 458 23.2.2 Chitosan................................................................................................................... 459 23.2.3 Biosorbents............................................................................................................... 460 23.3 Concluding Remarks..........................................................................................................463 References......................................................................................................................................463

23.1 Introduction Microbes have been long considered as producers of secondary metabolites with pharmaceutical applications. Avenues are now opening in the less explored potential of microorganisms in the field of probiotics and biomedical sciences including tissue engineering. However, most of the microbial species studies employed for such biomedical studies are terrestrial strains. Little attention is given to the microbial flora and fauna of the oceanic environments despite the fact that oceans have been a rich source of natural antioxidants, photoprotective (Pallela et al. 2010), anti-inflammatory (Himaya et al. 2010), anticancer (Bhatnagar and Kim 2010a), and antimicrobial or general pharmaceutical agents with varied actions (Bhatnagar and Kim 2010b). Polyhydroxyalkanoates (PHAs) have the potential to replace petroleum-based plastics as biomedical materials for use in surgical pins, sutures, staples, blood vessel replacements, bone replacements and plates, medical implants, and drug delivery devices owing to their superior biodegradability and biocompatibility (Khanna and Srivastava 2005). Microbes are excellent producers of PHA and deserve to be studied further in this area. One of the present trends in implantable applications requires materials that are derived from nature. The impetus is twofold. First, such “natural” materials have been shown to better promote healing at a faster rate and are expected to exhibit greater compatibility with humans. Second, new concepts in implantable medical devices, especially in tissue engineering, derived from a combination of biomaterial onto which cells are seeded require the biomaterial to be biodegradable. Among the many other candidate biomaterials available from nature is chitin. Fungal cell wall is made up of chitin, and chitosan (CS) may be easily isolated from the fungal biomass. Yet, less attention is given in this field, and proper utilization of this fungal biomaterial is still not achieved.

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With increasing environmental awareness and legal constraints being imposed on discharge of effluents, a need for cost-effective alternative technologies is essential. In this endeavor, microbial biomass has emerged as an option for developing economic and ecofriendly waste water treatment process. Biosorption is considered a potential instrument for the removal of metals from waste solutions and for precious metals recovery, an alternative to the conventional processes, such as those based on ion exchange or adsorption on activated carbon (Veglio and Beolchini 1997). This chapter would try to focus some of the aspects of these biomaterials and the use of microbial entities as a source for the same.

23.2  Microbial Biomaterials 23.2.1 Polyhydroxyalkanoates Polyhydroxyalkanoates (PHAs) are biodegradable materials, which are accumulated to store carbon and energy in various microorganisms (Keshavarz and Roy 2010; Reddy et al. 2003). Certain nutritional factors, such as nutrient deficiency or the presence of excess carbon, limit their accumulation (Brandl et al. 1990; Reddy et al. 2003). Based on the number of carbon atoms in their monomers, PHAs are classified as “short-chain-length” PHAs, where the number of carbon atoms in the monomer is 3–5, such as polyhydroxybutyrate (PHB) and polyhydroxyvalerate, and “medium-chain-length” PHAs, with 6–16 carbons in the monomers. PHB is the most commonly used PHA, and the metabolic pathways of PHB have been elucidated in detail (Khanna and Srivastava 2005). The properties of PHB are similar to those of various synthetic thermoplastics such as polypropylene. Various microorganisms completely degrade PHB to water and carbon dioxide under aerobic conditions and to methane under anaerobic conditions. In general, PHAs are polyesters that are synthesized by various microorganisms such as Cupriavidus necator, Alcaligenes latus, Aeromonas hydrophila, Pseudomonas putida, and Bacillus spp. Several halophilic microbes (including Haloferax mediterranei, Vibrio spp., V. natriegens, V. nereis, and V. harveyi) have been reported to produce PHB (Higgins and Sharp 1989; Sun et al. 1994; Weiner 1997). The identification of the gene that is involved in PHA synthesis, polyhydroxyalkanoic acid synthase, was verified using V. parahaemolyticus and V. cholera. The benefit of halophilic microbes in PHA production is that they produce PHA with high molecular weight, such as P(3HB-co-3HV). Therefore, they have potential industrial applications and may reduce the cost by some fermentation strategies, such as the immobilization of NaCl onto the walls of bioreactors. However, the disadvantage of halophilic microbes in PHA production is their low productivity (Chen et al. 2006; Don et al. 2006; Huang et al. 2006). Few reports on marine PHA-producing microorganisms have been published in the recent past. A PHB-producing Gram-negative bacterium, identified as a Vibrio sp. BM-1 by the phylogenic analysis of its 16S rDNA, has been isolated from a marine environment in the north of Taiwan, which may be developed industrially for the production of PHB on a larger scale. Mineral salts such as Na2HPO4, KH2PO4, and MgSO4.7H2O are believed to be important for supporting bacterial life and as critical elements for synthesizing metabolites (Ghanem et al. 2005; Mokhtari-Hosseini et al. 2009). The productivity of PHB may be enhanced by carefully studying the effects of mineral salts on PHB production and utilizing the modern biotechnological advancements.

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23.2.2 Chitosan Current tissue engineering strategies are focused on the restoration of pathologically altered tissue architecture by transplantation of cells in combination with supportive scaffolds and biomolecules. In recent years, considerable attention has been given to CS-based materials and their applications as wound-healing agent, bandage material, skin grafting template, hemostatic agent, hemodialysis membrane, dental implants, and drug delivery vehicle (Chandy and Sharma 1993; Hirano 1996; Muzzarelli et al. 1993; Patel and Amiji 1996; Vasudev et al. 1997). CS has been applied to conduct the extracellular matrix (ECM) formation in tissue regenerative therapy (Laurencin et al. 1996; Muzzarelli et al. 1994; Yaylaoglu et al. 1999). Chitin, together with its variants, especially its deacetylated counterpart CS, has been shown to be useful as a wound dressing material, drug delivery vehicle, and increasingly a candidate for tissue engineering (Khor and Lim 2003). CS, a natural polymer obtained by alkaline deacetylation of chitin, is nontoxic, biocompatible, and biodegradable, and it has recently gained more interest due to its applications in food and pharmaceutics (Ramya et al. 2012). Interesting characteristics that render CS suitable for this purpose are a minimal foreign body reaction, an intrinsic antibacterial nature, and the ability to be molded in various geometries and forms such as porous structures, suitable for cell ingrowth, and osteoconduction. Due to its favorable gelling properties, CS can deliver morphogenic factors and pharmaceutical agents in a controlled fashion. Its cationic nature allows it to complex DNA molecules, making it an ideal candidate for gene delivery strategies (Figure 23.1). The ability to manipulate and reconstitute tissue structure and function using this material has tremendous clinical implications and is likely to play a key role in cell and gene therapies in coming years (Di Martino et al. 2005). Much of the potential of CS as a biomaterial stems from its cationic nature and high charge density in solution. The charge density allows CS to form insoluble ionic complexes or complex coacervates with a wide variety of water-soluble anionic polymers (Francis Suh and Matthew 2000). One of CS’s most promising features is its excellent ability to be processed into porous structures for use in cell transplantation and tissue regeneration. Porous CS structures can be formed by freezing and lyophilizing CS–acetic acid solutions in suitable molds (Madihally and Matthew 1999). CS/collagen composite scaffolds have also been made for skin tissue engineering (Ma et al. 2003). Chitosan + Chitosan-DNA gels OH O HO

O NH2

Chitosan

In vivo characterization

O

n

In vivo implantation

Composite scaffold generation

In vitro characterization

FIGURE 23.1 Schematic representation for application of CS in bone tissue engineering.

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Venkatesan et al. (2010) have nicely reviewed the use of CS composite scaffolds in tissue engineering applications (Venkatesan and Kim 2010). Our group has further developed carbon nanotube-grafted CS and natural hydroxyapatite composite scaffolds for bone tissue engineering and achieved profound results (Venkatesan et al. 2011). Not only this, but CS–hydroxyapatite and marine collagen scaffolds have also been synthesized and characterized for bone tissue engineering usage (Pallela et al. 2012). Use of CS in cartilage tissue engineering has also been explored with studies on the feasibility of CS-based hyaluronic acid hybrid biomaterial scaffold generation (Yamane et al. 2005). A detailed review on the application of CS-based polysaccharide biomaterials in cartilage tissue engineering has been published by a leading research group of America in recent past (Francis Suh and Matthew 2000). The direct use of in situ chitin with fungal mycelia from the fungus Ganoderma tsugae to produce wound-healing sacchachitin membranes has also been demonstrated (Su et al. 1997). A nonwoven mat obtained by first processing the mycelia to remove protein and pigment, followed by isolation of fibers in the 10–50 mm diameter range and final consolidation into a freeze-dried membrane under aseptic conditions, was used in a wound model study. The in vitro cell culture using rat fibroblasts and in vivo immunogenicity evaluations indicated no adverse responses (Hung et al. 2001). The wound healing of this fungal-based nonwoven mat as surmised from wound contraction measurements on two different animal model studies was favorable (Su et al. 1999). Fungal CS has also been reported to enhance disease resistance in case of fungal–pea interactions. CSs, (1) derived chemically from the chitin of fungal cell walls, (2) accumulated in Fusarium solani/pea interactions, or (3) released from chitinase and β-glucanase digestion of sporelings, were used to determine if these fungal polymers had the biological activity of the CS chemically derived from crustaceans. The biological activity of the cell wall chitin-derived CS from F. solani f. sp. phaseoli mimicked that of shrimp CS and was somewhat superior to that from f. sp. pisi. CSs derived from F. solani f. sp. phaseoli inhibited germination of F. solani macroconidia at concentrations as low as 8 μg mL−1 (Kendra et al. 1989). Apart from this, the food industry is also adopting the use of fungal CS for various processes including apple juice clarification. A study published a couple of years ago reported the use of fungal CS as a clarifying agent of apple juice. They reported that the clarity and color changes of the apple juice correlated closely for both fungal and shrimp CS treatment. However, the fungal CS proved highly effective in reducing the apple juice turbidity and gave lighter juices than the sample treated with shrimp CS (Rungsardthong et al. 2006). The use of fungal CS as a growth stimulator in orchid tissue culture has also been investigated and found to have profound effect on the growth of orchid plantlets (Nge et al. 2006). When examined for their antioxidant properties, fungal CS proved to be good antioxidant agents with an antioxidant activity of 61.6%–82.4% at 1 mg/mL concentration (Yen et al. 2007). 23.2.3 Biosorbents Although the heavy metals occur in immobilized form in sediments and as ores in nature, a large deposition of heavy metals in terrestrial and aquatic environment may occur due to activities like ore mining and industrial processes disturbing the natural biogeochemical cycles. A proper predisposal treatment of these nonbiodegradable and persistent, detrimental heavy metals is mandatory as the release of these pollutants without proper treatment would pose a significant threat to both environment and public health. The phenomenon of biomagnification further deepens the threat where these heavy metals get accumulated in large numbers in food chains. Generally applied techniques

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for the treatment of heavy metal-contaminated water are reverse osmosis, electrodialysis, ultrafiltration, ion exchange, chemical precipitation, phytoremediation, etc. However, all these methods have disadvantages like incomplete metal removal, high reagent and energy requirements, and generation of toxic sludge or other waste products that require careful disposal (Ahalya et al. 2003). An emerging cost-effective and eco-friendly technology to treat heavy metalcontaminated water seems to be that of biosorption. This technology employs various types of biomass as source to trap heavy metals in contaminated waters. The biosorbent is prepared by subjecting biomass to various processes like pretreatment, granulation, and immobilization, finally resulting in metal entrapped in bead-like structures. These beads are stripped of metal ions by desorption which can be recycled and reused for subsequent cycles (Figure 23.2). Biosorption can be defined as “a non-directed physicochemical interaction that may occur between metal/radionuclide species and microbial cells” (Alluri et al. 2007). It is a biological method of environmental control and can be an alternative to conventional contaminated water treatment facilities. It also offers several

Selection of biomass Pretreatment of biomass

Entrapment in polymeric matrices

Adsorption on inert support

Immobilization

Biosorbent granules Granules dumped into metal contaminated solution

Desorbed biosorbent for recycle

Desorption

Metal recovery FIGURE 23.2 Utilization scheme for fungal and bacterial biomass for biosorption of metals.

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advantages over conventional treatment methods including cost-effectiveness, efficiency, minimization of chemical/biological sludge, requirement of additional nutrients, and regeneration of biosorbent with possibility of metal recovery. The unabated discharge of effluents by diverse industries constitutes one of the major causes of land and water pollution by chromium compounds and has become an obnoxious health hazard. Out of hexavalent and trivalent species of chromium which are prevalent in industrial waste solutions, the hexavalent form has been considered more hazardous to public health due to its mutagenic and carcinogenic properties. Cr(VI) causes severe diarrhea, ulcers, eye and skin irritation, kidney dysfunction, and probably lung carcinoma (Costa 2003). In reaching for these goals in pollution standards, many methods have been used for the removal of Cr(VI) from wastewaters including ion-exchange resins, filtration, and chemical treatment (Singh and Tiwari 1997). However, these methods are not completely satisfactory and have the following disadvantages: (1) generation of large amount of secondary waste products due to various reagents used in a series of treatments such as reduction of Cr(VI), neutralization of acidic solution and precipitation, and (2) the instability of ion-exchange resins due to serious oxidation by Cr(VI). Thus, there is a need for the development of new cost-effective methods that are more environmentally friendly (Park et al. 2005). The removal of hexavalent chromium from aqueous solution was studied in batch experiments using dead biomass of three different species of marine Aspergillus after alkali treatment. All the cultures exhibited potential to remove Cr(VI), out of which, Aspergillus niger was found to be the most promising one. This culture was further studied by employing variation in pH, temperature, metal ion concentration, and biomass concentration with a view to understand the effect of these parameters on biosorption of Cr(VI). Higher biosorption percentage was evidenced at lower initial concentration of Cr(VI) ion, while the sorption capacity of the biomass increased with rising concentration of ions. Biomass as low as 0.8 g L−1 could biosorb 95% Cr(VI) ions within 2880 min from an aqueous solution of 400 mg L−1 Cr(VI) concentration. Optimum pH and temperature for Cr(VI) biosorption were 2.0°C and 50°C, respectively (Khambhaty et al. 2009). Not only the dead fungal biomass of Aspergillus species but also other bacterial and fungal sp. has been employed for biosorption (Table 23.1). The removal of chromium from TABLE 23.1 Microbial Species Used as Biomaterials in Heavy Metal Biosorption Microbial Species Phanerochaete chrysosporium Aspergillus niger Aspergillus fumigates Aspergillus terreus Penicillium chrysogenum Saccharomyces cerevisiae, Kluyveromyces fragilis Saccharomyces cerevisiae Saccharomyces cerevisiae Bacillus polymyxa Bacillus coagulans Escherichia coli Escherichia coli Pseudomonas species

Heavy Metal

References

Ni(II), Pb(II) Cd Ur(VI) Cu Au Cadmium

Çeribasi and Yetis (2001) Barros Júnior et al. (2003) Bhainsa and D’Souza (1999) Gulati et al. (2002) Niu and Volesky (1999) Hadi et al. (2003)

Methyl mercury and Hg(II) Uranium Cu Cr(VI) Hg Cu, Cr, Ni Cr(VI), Cu(II), Cd(II), Ni(II)

Madrid et al. (1995) Volesky and May-Phillips (1995) Philip and Venkobachar (2001) Alluri et al. (2007) Bae et al. (2003) Churchill and Churchill (1995) Muraleedharan et al. (1991)

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aqueous solution was carried out in batch experiments using dead biomass of four fungal strains—A. niger, Rhizopus oryzae, Saccharomyces cerevisiae, and Penicillium chrysogenum. All of these dead fungal biomass completely removed Cr(VI) from aqueous solutions, that of R. oryzae being the most effective. The removal rate of Cr(VI) increased with a decrease in pH or with increases of Cr(VI) and biomass concentrations, thus supporting the mechanism that Cr(VI) is removed via a redox reaction. Park et al. have further concluded that from the practical view point, the abundant and inexpensive dead fungal biomass could be used for the conversion of toxic Cr(VI) into less toxic or nontoxic Cr(III) (Park et al. 2005).

23.3  Concluding Remarks Microbial systems have long been proven to have a storehouse of potentialities in the health-care arena, and they form an inevitable component of many of the food processing industries. The promise for fungal chitin and CS as a biomaterial is vast and will continue to increase as the chemistry to extend its capabilities and new biomedical applications are investigated. The microbial biomaterials may be even further explored for tissue engineering applications as well. An extensive research for the use of PHB to manufacture biodegradable polymers using marine microorganisms is also a good area to look up to in terms of biomedical advances (Wei et al. 2011). Heavy metal contamination has also been a major area of concern among the environmentalists. Conventional technologies to clean up heavy metal ions from contaminated waters have been utilized, but these technologies are not all cost effective. An alternative to these technologies are the bioremediation methods which are inexpensive. Hence, microbial biosorbent systems could be envisaged as one of the promising solutions toward biodetoxification of heavy metal pollution. In short, the microbial biomaterials should be given equal importance, and the marine microbial flora and fauna should be more deeply studied and discovered to get fruitful benefits from these unrevealed treasures of Mother Nature.

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