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Biocatalysts has evolved and turned to a technology which is standard in the fine chemicals industry and is reflected by the number of biotransformation processes running on a business scale. An incremental number of books and review articles have elaborated industrial biotransformation processes in the past few years. They have debated how the technology can be utilized on a scale at the level of generation. Nonetheless, so far there has been no clear indication of the technical standards necessary to be met by biotransformation processes in order to be of commercial weight.
Within the last ten years, there has been a sharp rise in the whole number of biotransformation processes being conducted on an industrial scale (Figure 2.2). The continuation of this increase is anticipated. Utilizing numerous sources, a quantitative analysis of these industrial transformations has been conducted in order to obtain the necessary data. Cheetham and Liese et al., were specifically worthwhile. They point out most of industrial biotransformation processes. The process is expected to explain a reaction or a set of concurrent reactions in which a preformed precursor molecule is converted, rather than a fermentation process with de novo production from a carbon and energy source such as glucose by means of primary metabolism; it is expected to entail the utilization of enzymes and/or whole cells, or combinations thereof, either free or immobilised; also it is expected to result in generating a fine-chemical or commodity product that is typically recovered after the reaction; and it should have been reported to be operated on a commercial scale, or have been effectively scaled-up and announced to be commercialised, generally at a scale of >100 kg per year. In case of some processes, sources in the public domain provide imperfect or contradictory data; and it is possible that other processes have not been explained at all. Nevertheless, it is assumed that the gathered data will show all trends rightly.
The Analysis of the industrial bio transformations that were identified indicates that they commonly result in natural compounds or their derivatives (see Figure 2.3). None of the categories of natural compounds is more noticeable. Carbohydrate and fat derivatives are mainly utilized in the food sector; however the other kinds of compound are chiefly exploited in the pharma and agro sectors. On the whole, application in the pharma sector is profoundly dominant, as shown in Figure 2.4. Notice that the proportions shown in Figure 2.4 refer to the number of processes in each sector. The other sectors would gain in importance if the scale of the processes were taken into account. None of the nine products that are produced on a main scale (subjectively defined as >20 000 tons per year) originates from pharma usages; most of them are carbohydrates. A few products made in bulk appear to show that biotransformations are principally significant in the sector of fine-chemicals. Chirality is manifestly a significant issue, however majority of the products, which are enantiomerically pure, the chiral formation stems from the precursors in a straight line (Figure 2.5). If the enantiomeric purity stems from the biotransformation, both kinetic resolution and asymmetric synthesis recurrently take place. In most cases, Asymmetric syntheses are chiefly performed by oxidoreductases and yases; on the other hand, while kinetic resolutions nearly completely involve hydrolases. In about 25 per cent of these kinetic resolutions, the hydrolases are utilized in the synthetic instead of in the hydrolytic mode.
Overall, the yield of kinetic resolutions is only 50% at most, which is unappealing in terms of being economical and ecological. There are diverse solutions to this problem, for instance both enantiomers can occasionally be applied in the successive chemistry; on the other hand, it has been reported that it is possible to racemise the unwanted enantiomer (Figure 2.6). In situ racemisation was enzyme-catalysed in most cases; however, occasionally happened impulsively, resulting in racemisation circumstances that were well-matched with the circumstances necessary for kinetic resolution. On the other hand, chemical techniques rather than biocatalysis are being utilized for external racemisation, with ensuing recycling of the racemate. The destiny of the unwanted enantiomer (the distomer) has not been reported regarding numerous industrial kinetic resolution processes. Only in one case was it clearly pointed out that it was cast aside. Most biocatalytic research includes hydrolytic enzymes; just 25% of the researches are related to oxidoreductases and less than 15% with the other classes (Faber K 2000). In industrial biotransformations (Figure 2.7), the supremacy of hydrolases is less noticable. For instance, processes that are catalysed by leases and transferases are logically desirable as well. The number of processes including redox biotransformations is more essential. These occasionally involve two separated oxidoreductases - one for the biotransformation and one for cofactor regeneration.
nevertheless, most of redox biotransformations involve metabolizing cells with enzymes from all categories being active together with the oxidoreductases. This can still be valid even when there is no cell growth, as shown for reductions mediated by baker's yeast (Chin-Joe I 2001).
Figure 2.3 the type of compounds produced using biotransformation processes
Whole cells are utilized in various non-redox biotransformations as well. It is worth mentioning that the cells are not metabolising in such cases and only one or two important enzymes necessary for the transformation are active. Generally speaking, in comparison with (partially) seperated enzymes (Figure 2.8), whole cells are more popular. Compared to isolated enzymes, immobilisation is less common when whole cells are utilized. It is possible to at least partially attribute this trend to the propensity to apply whole cells for redox biotransformations. In this case, oxygen is generally necessary for continual metabolic activity, and immobilisation is eschewed to hinder the restriction of oxygen diffusion (Freeman A 1998). Otherwise no correlation was found between the kind of reaction and the kind of catalyst utilized(i.e. cell or enzyme; free or immobilised).
Only in several cases were ultrafiltration membranes utilized in order to maintain the biocatalyst (Woltinger J 2001). Binding the catalyst on or within particles is far more common. Immobilisation is extremely recurrently exploited in combination with constant reactors; nevertheless, in comparison to batch reactors, constant reactors are less common especially for usages with smaller scale (Figure 2.9). Fed-batch reactors mainly involve free whole cells. In some cases, reactor information was imperfect; in some other cases there was no information for many processes, and coming to more exhaustive results was perilous.
In spite of the fact that the application of organic solvents has been a major research subject in the area of biotransformations, less than 10 industrial processes are presently being conducted regarding organic solvents (without a separate water phase). These usages include (trans) esterification or amidation reactions. In addition to an organic phase, there will be an aqueous phase in many cases. The existence of an aqueous brings about either a monophasic or a biphasic mixture. The biphasic category is more frequent and involves both processes where an organic solvent is utilized as substrate and product reservoir, and also a considerable number of processes where the organic phase is literally made of liquid substrate or product only, without needing an organic solvent to be added. High concentration is needed for industrial processes; it is likely that such concentration at this level results in multiphase reactions in which the substrate or product is not a liquid but a solid with a low aqueous solubility. The outcome is aqueous suspension reactions. Such reactions already take place at least 13 industrial processes. Approximately in 50 per cent of these cases, a suspension of substrate is converted into a suspension of product.
Figure 2.4 Industrial sectors in which the products of industrial biotransformations are used
Figure 2.5 Source of chirality for the products of industrial biotransformations
Figure 2.6 Enantiomer use in industrial biocatalytic kinetic resolutions
Figure 2.7 Enzyme types used in industrial biotransformations
Figure 2.8 Use of enzymes or whole cells in industrial biotransformations
Figure 2.9 Reactor types used in industrial biotransformations
22.214.171.124 Biotransformations in the chemical industry
Biotransformations are utilized by Chemical companies with the aim of generating products on a scale of a few hundred to occasionally over ten thousands of tons annually. The range of examples of processes, which operate on the scale of several thousands of tons annually, is from the acrylamide production process by Mitsubishi Rayon, Japan (Thomas SM 2002), to the generation of penicillin G/V-derived 6-aminopenicillanic acid by several manufacturers (Van de Sandt EJAX 2000). There are a few companies recurrently generating the same product and as a result, there is high pressure on production expenses. It is natural that the larger scale products are normally made in committed installations; these products are purchased by many customers. The buyers in turn process the compound more. In the meantime, there is a probability for the committed plants to be exploited for the purpose of fabrication of a group of compounds. The production of ChiPros (Hieber G 2001), a pool of chiral products manufactured with one infrastructure/technology platform, can be taken into account as an example. Generally speaking, the product lifetime is more than ten years, accordingly there is a likelihood for creating a comparatively stable flow of research finances which paves the ground for a certain process to be incessantly optimised. On the other hand, it is possible to utilize the finances in order to redesign specified processes entirely providing that progress in academic and industrial research causes this to be appealing. It is meaningful if the product is on the market long enough until it returns the research investment. The shift made by DSM (the Netherlands) from chemical to biocatalytic synthesis of the Î²-lactam antibiotic cephalexin (Van de Sandt EJAX 2000) can be taken into account as an example. Owning to the same reason, the larger chemical companies too are where research into elemental changes in process design for a certain product occurs. An example of an Metabolic engineering is an elemental change in process design; the new 7-ADCA process presented by DSM, which is on the basis of the expansion of the metabolic qualities of the penicillin G producer Penicillium chrysogenum, may be used as an illustration (Van de Sandt EJAX 2000).
Table 2.1 Efficiencies of biocatalytic processes for the production of fine chemicals*.
Stressing the process effectiveness, it seems rational to suppose that the higher volume products in particular will allow research into the diverse facets of a process. Process complexity, productivity, and product volume recurrently augment during the lifetime of a process. In line with the arguments described above, research activities could be activated into the diverse facets of a process once more. Again, the Î²-lactam antibiotics are utilized as an illustration that this is certainly the case. The group of biocatalysts has remained steady over numerous years; however, an elemental amount of research has been invested into the conditions of enzymatic synthesis and downstream processing (Wegman MA 1999). It is supposed that the major technical parameters (which have an influence on the expenses of a biotransformation process) are productivity (because there is a connection between higher productivity and lower capital costs); product concentration (which influences easiness of product recovery and purification); yield (which determines the cost of raw materials and the amount of by product which needs to be considered); and biocatalyst consumption. Out of the aforesaid 134 industrial biotransformation processes, those whose products are principally utilized in the chemical and pharmaceutical industries rather than in the food or the cosmetics industries, were chosen. Considering these essential parameters, these processes were analysed (Table 2.1). For most reported processes, ultimate product concentrations are well above 50 g/L, normally more than 100 g/L (total volume) (generally as reported for amino acids and carboxylic acids). They may be over 200 g/L for carbohydrates and amides. These distinctions associate with product relative degree of being poisonous and/or constancy, as the lowest ultimate product concentrations were reported for the production of epoxides, which necessitates competent in situ product removal techniques (Lye GJ 1999). For instance, it is possible to produce Epoxides in an effective way in organic/aqueous two-phase systems at product concentrations of over 15 g/L, as it was lately illustrated on pilot scale (Panke S 2002). High product concentrations can be restricted by the solubility of target compounds. Once more it is possible to solve this problem mainly through utilizing multiphase reaction media like organic/ aqueous reaction mixtures (Matsumae H 1999). Alternatively, solid adsorbers can be utilized as another solution, which has been used, for instance, for manufacturing an Epivir precursor (two tons per year) (Mahmoudian M 2001). Meanwhile, the fairly high product concentrations reported indicate that the frequent prejudice that bioprocesses yield relatively low product concentrations (Blaser HU 2001) is not defensible. It has been reported that volumetric productivities of biotransformation processes are normally well over 1 g/(L.h). The configuration of epoxides, which is possibly because of the fact that wild-type cells have low particular actions for abnormal substrates, is considered as an exception. On the other hand, amino acids and amides are generated with much higher productivities of up to 130 and 80 g/(L.h), correspondingly. These reactions are catalysed by cofactor-independent enzymes with high turnover rates and are carried out in entire cells in principle. This results in high catalyst titres in small volumes. These products are recurrently in the lower price range; as a result of this, the cost of raw material is considerable, in particular when production volumes augment. For instance, This directs the development of processes for generating natural amino acids away from biotransformations and towards fermentations of inexpensive carbon sources like glucose.
On the basis of the reports, yields for commercialised processes are normally well over 80% and principally over 90%. Kinetic resolutions, frequently including thorough alteration of one enantiomer and giving yields near 50%, are among exceptions. This way, the free utilization of kinetic resolutions at the multi-thousand tons per year scale due to the large amounts of by product formed has been hindered. Of course, there is an exception: when this restriction has been surmounted by the accessibility of appropriate racemisation measures. The quantity of consumed catalyst is another crucial parameter. It has an influence on expenses of catalyst. Data on the product to catalyst proportion are rare. The values of 102-104 kg/kg which have been attained in the course of chemical processes appear only to be found in bioprocesses that utilize enzymes or whole cells which have been made immovable (Thomas SM 2002). The main reason is the low amount of active sites per kg of biocatalyst.
Considering these numbers, a rudimentary idea of the existing problems is posed; problems that need to be eliminated providing that laboratory-scale biotransformations are to get competitive.
2.1.2 Selection of Biocatalyst
A significant sector in the chemical industry is formed by aroma production. The aromas compounds are used in the generation of cosmetics, perfumes, cleaning products and food processing. Conventionally, aromas have been extracted from plants; however, these measures are ordinarily low yield processes. Another way to generate aromas is through chemical synthesis. Nonetheless an obvious consumer taste exists for products having an organic originality. Therefore, a mounting scientific interest exists for searching alternatives of aroma production, dissimilar to processes which are dependant on extractive or chemical synthesis. As a consequence, several biotechnological viewpoints have been taken into account as real alternatives for aroma production (Berger 1995).
An economic substitute to the complex and costly extraction from raw materials like plants, is the utilization of biotechnology for the production of natural aroma compounds by fermentation or bioconversion by means of microorganisms (Harlander 1994; Janssens 1992). For the purpose of commercial cheese ripening, a fungus, Geotrichum candidum, has been selected and utilized. This fungus has aromatic properties and is often referred to as yeast (Jollivet 1994). It is possible that some strains generate fatty acids esters which are frequently linked to particular fruit aroma (Koizumi 1982; Latrasse 1987). G. candidum is exceedingly lipolytic and has a complete variety of substrate specificity (Jacobsen 1990; Sidebottom 1991). In the meantime, It is possible that Its proteolytic activity constitutes aroma compounds. This type of activity has been partially characterized by Gueguen and Lenoir (Gueguen and Lenoir 1975, 1976). For the aroma of the food products, like alcohols, phenolic compounds and sulphur compounds, aldehydes, esters, short to medium-chain free fatty acids, methyl ketones, lactones, dicarbonyls, an enormous range of compounds might be accountable (Gatfield 1988; Urbach 1997). Traditionally, it has been common to obtain aroma compounds, ranging from single to intricate substances, from plant sources. After clarification of their structure, synthetic aroma was ultimately generated through chemical synthesis.
At the present time, aroma represents more than 25 per cent of the world market for food additives. There are two ways for the manufacture of most of the aroma compounds: through chemical synthesis or via obtaining from natural materials. Nevertheless, on the basis of current market surveys, consumers prefer foodstuff that can be branded as natural. It is possible to produce aroma via chemical alteration of natural substances; nevertheless, it is not lawful to label the obtained products as natural. What is more, chemical synthesis frequently leads to production processes which are harmful in terms of protecting environment. Furthermore, it does not have substrate selectivity, which may results in the formation of unwanted racemic mixtures; as a result, process effectiveness reduces and downstream expenses augment. Moreover, exploiting direct extraction from plants as a way to produce natural aroma, includes a variety of problems. The most important problem is that in most cases these raw materials contain low concentrations of the required compounds; therefore, extraction method becomes costly. What is more, utilization of these kind of materials involves factors which are relatively uncontrollable; factors like climatic conditions and plant diseases. There are some downsides for both methods; on the other hand there is a growing interest in natural products. Consequently, many researchers have been persuaded to search other strategies in ortder to generate natural aroma.
There is another option for flavour synthesis. This option depends on microbial biosynthesis or bioconversion (Aguedo 2004; Janssens 1992; Krings and Berger 1998; Vandamme and Soetaert 2002). According to the most accepted views, the utilization of microbial cultures or enzyme preparations is recommended. Meanwhile, it has been reported that plant cell cultures is an appropriate production system. (Figure 2.10). Aroma can be synthesized by Microorganisms as secondary metabolites during fermentation on nutrients like sugars and amino acids. It is possible to use this quality in two dissimilar ways:
Flavour production in place, as an essential part of food or beverage production processes (i.e. cheese, yogurt, beer, wine) which determines the organoleptic features of the ultimate product
Utilization of Microbial cultures which have been particularly designed to produce aroma compounds that can be separated and exploited later on as additives in food production. Using this method, it is possible to regard the resulting aroma as natural
Figure 2.10 Biotechnological production of Aroma compounds
In both cases, it is possible to add precursors or intermediates to the culture medium with the aim of promoting the biosynthesis of particular aroma. Besides, in order to develop appropriate production systems for specific aroma additives, it is possible to use the data gained via the investigation of microbial metabolism in food fermentation processes. Alternatively, Enzyme technology makes a very hopeful way accessible for natural flavour biosynthesis. The production of aroma-related compounds from precursor molecules could be catalysed by some enzymes such as, proteases, glucosidases and lipases (Adinarayana 2004; Asther 2002; Kamini 1998; Macris 1987; Miranda 1999). There is an outstanding merit for utilization of enzyme-catalysed reactions: providing higher stereo selectivity than chemical routes. Additionally, the resulting products in this way may have the lawful status of natural substances. A substantial quantity of present investigation concentrates on the production of aroma compounds; however, at this point in time, only a few of these compounds are obtained by biotechnological routes. The main problem is that a source of substrate which is naturally rich is put in contact with specific enzymes which are exceedingly active. In satisfactory circumstances, this can lead to the production of aroma compounds in mass fractions of the order of several g/kg, instead of mg/kg encountered in raw materials. The obtained aroma compounds are called natural because they are generated from agro-products via natural biological actions. As a result, the proportion of isomers or isotopes is analogous to what can be found in extracted products and not to what can be obtained from chemical synthesis. The productivity of some of these processes is good; however, the resulting products are typically more costly than those obtained from chemical synthesis (Benjamin and Pandey 1997; Besson 1997; Beuchat 1982).
2.2 Microorganisms as biocatalyst
Screening a wide diversity of microorganisms has been utilized in order to obtain active biocatalysts. Microorganisms are far-flung all through nature, and numerous habitats exist. It is feasible to use these habitats to detect new microbial species. Bio prospection for new microorganisms from all biotopes discovered on our planet, including those featuring intense environmental circumstances (such as hyper saline and super cooled sea ice, geothermal ecosystems and hydrothermal vents), could also bring about finding new enzymes which have capability to catalyze different sorts of reactions.
Since distant past, Fungi have conventionally been considered as one of the most studied whole cell systems for microbial natural product isolation and also for biotransformation reactions. The isolation of fungi from the environment has triggered the interest of investigators because it is thought that only a very small number of existing fungal species have been literally identified. Natural infection in the environment favoured by damp climates results in incidence of fungi in plants. The isolation of fungi can be taken into account as a first step in comprehending the emergence of secondary metabolites in plants and the activation of specific enzymes in fungi.
Among the fungi existing in plants, Pathogenic and endophytic fungi are noteworthy because they can be taken into account as hopeful sources of biocatalysts with copious usages. In the literature, there are various definitions for the term endophytic fungi (Wilson D 1995, Redman R, Ganley R 2004). This term has been used to describe those fungi that can be detected at a specific moment within seemingly healthy plant host tissue. These fungi, for all or part of their life cycle, can live in the tissue of living plants. There are different forms for the colonization: inter- or intracellular, localized, or systemic (Schulz B 2005). The tissue of living plants is attacked by Endophytes. The invasion results in imperceptible and asymptomatic infections (Wilson D 1995). Colonizing parts of animals and plants internally or externally is the way that Pathogenic fungi can cause malady. Furthermore, it is possible for Pathogenic fungi to show long periods of being latent; in other words, this kind of fungi can occupy host tissue without causing it to show any symptom (Redman RS 1999). The differences between pathogenic and endophytic fungi are not very noticeable, and there are fungi which are pathogenic to one plant species on the one hand, but able to live as mutualistic endophytes in another host on the other hand. It is possible to designate the dissimilarity between pathogenicity and endophytic behavior by means of a single gene.
An unfamiliar or at least under-explored source for microbial biotransformations is Endophytic fungi. Endophytes were initially pointed out at the start of the 19th century, however the distinction between endophytes and phytopathogens. Nevertheless was cited for the first time by DeBary (1866). Nevertheless, endophytic fungi became noticeable by degrees only in last century, at the end of the 1970s. They were turned out to be able to protect plants against invasion from, diseases, insects, and mammalian herbivores. What is more, it is possible for them to generate metabolites similar to those generated by the host plant. In addition, they can be utilized in chemical and drug biotransformation processes (Stierle A 1993, Zikmundova M 2002, Shibuya H 2003-5, Agusta A 2005 and Borges KB 2007-8). A significant example of this is the fungus Taxomyces andreanea discovered inside the plant Taxus brevifolia. This plant produces taxol, which is an intricate anticancer diterpenoid of high benefit to the pharmaceutical industry.
2.2.1 Aspergillus spp
Aspergillus composes of filaments, and is cosmopolitan and ever-present fungus found in nature. Normally, it is separated from soil, plant remains, and indoor air environment. Only for some of the Aspergillus spp., has a teleomorphic state been explained; however, others have been considered to be mitosporic, devoid of any known sexual spore production. The genus Aspergillus contains over 185 species. Approximately 20 species have so far been reported as contributing agents of opportunistic infections in man. Aspergillus fumigatus is the most normally secluded species among them. It is followed by Aspergillus flavus and Aspergillus niger. There are other species which are less frequently separated as opportunistic pathogens, such as Aspergillus clavatus, Aspergillus glaucus group, Aspergillus nidulans, Aspergillus oryzae, Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor. You can refer to the list of outdated names and synonyms for older names of these species.
Aspergillus spp. have a fame to play a role in three diverse clinical settings in man: (1) opportunistic infections; (2) allergic states; and (3) toxicoses. The main factor prearrange to development of opportunistic infections is Immunosuppression (Ho, PL 2000). It is possible that these infections present in a wide range. The range can vary from local involvement to distribution; it is called aspergillosis as a whole. Generally speaking, out of all filamentous fungi Aspergillus is the most frequently separated one in invasive infections. Meanwhile, It is the second most normally recovered fungus in opportunistic mycoses following Candida. Nearly there is a probability for the involvement of all organs or systems in the human body. Onychomycosis, cutaneous aspergillosis, hepatosplenic aspergillosis, plus Aspergillus fungemia, and dispersed aspergillosis, sinusitis, cerebral aspergillosis, meningitis, endocarditis, myocarditis, pulmonary aspergillosis, osteomyelitis, otomycosis, endophthalmitis, are likely to develop (Collier, L 1998). In the meantime, there is a probability for Nosocomial occurrence of aspergillosis owing to catheters and other devices. A major peril for the development of aspergillosis in neutropenic patients in particular is construction in hospital environments. In addition, Aspergillus spp. are likely to be local colonizers in lung cavities which have been formerly developed because of ankylosing spondylitis or neoplasms, tuberculosis, sarcoidosis, bronchiectasis, pneumoconiosis, presenting as a distinctive clinical entity, called aspergilloma (Hohler, T 1995). Moreover, Aspergilloma are likely to take place in kidneys. Some Aspergillus antigens are fungal allergens; they might instigate allergic bronchopulmonary aspergillosis in atopic host in particular (Kurup, V.P 2000). A number of Aspergillus spp. generates a range of mycotoxins. These mycotoxins, by chronic ingestion, have turned out to own carcinogenic potential principally in animals. Aflatoxin is well-known among these mycotoxins; it is likely to bring about hepatocellular carcinoma. Aflatoxin is chiefly generated by Aspergillus flavus and is a source of contamination of foodstuff like peanuts (Mori, T 1998). Aspergillus spp. might be a cause of infections in animals and man. Meanwhile, Aspergillus is likely to lead to respiratory infections in birds. Furthermore, it is possible that Aspergillus brings about mycotic abortion in the cattle and the sheep (St-Germain, G 1996). Eating bg quantities of aflatoxin might bring about fatal effects in poultry animals which are fed with grain poisoned with the toxin. The growth rate, color of the colony, and thermotolerance are the main macroscopic characteristics which are outstanding in species identification (Larone, D 1295). The growth rate is swift to fairly swift but Aspergillus nidulans and Aspergillus glaucus are two exceptions. Aspergillus nidulans and Aspergillus glaucus grow gradually and reach a colony size of 0.5-1 cm following incubation at 25Â°C for 7 days on Czapek-Dox agar, but those of the residual species are 1-9 cm in diameter in the specified setting. These alterations in growth rate facilitate species identification. Aspergillus colonies are downy to powdery in texture. Depending on the species, the surface colour may differ. The reverse is uncoloured to pale yellow in most of the isolates. Nonetheless, reverse colour may be purple to olive in some strains of Aspergillus nidulans and orange to purple in Aspergillus versicolor (TABLE 2.2). Aspergillus fumigatus is a thermotolerant fungus. At temperatures more than 40Â°C, it grows satisfactorily. Among the Aspergillus species, this property is unique to Aspergillus fumigatus. Aspergillus fumigatus is likely to grow at a temperature from 20 to 50 Â°C.
Table 2.2 The colour of the colony in diverse Aspergillus species
White, brownish with age
Goldish to red brown
Blue-green or gray
White to tan
Green with yellow areas
Yellowish to brown
Green, buff to yellow
Purplish red to olive
White to yellow
Cinnamon to brown
White to brown
White at the beginning, turns to yellow, tan, pale green or pink
White to yellow or purplish red
All species have the same basic microscopic morphology. Nonetheless, there are some other microscopic structures which are unique to specific species. They make up the integral traits for species identification accompanied by the surface colour of the colony. Hyphae are septate and hyaline. The conidiophores stem from the basal foot cell situated on the supporting hyphae and end in a vesicle at the apex. Vesicle is the characteristic formation for the genus Aspergillus. The morphology and color of the conidiophore differ in various species. The flask-shaped phialides are either uniseriate and attached to the vesicle straight or are biseriate and attached to the vesicle through a supporting cell, metula. Phialides Cover the surface of the vesicle totally ("radiate" head) or partly only at the upper surface ("columnar" head). Over the phialides are the round conidia (2-5 Âµm in diameter); they form radial chains. Sclerotia, cleistothecia, aleuriconidia, and Hulle cells are among other microscopic structures. These structures are highly essential in terms of identifying some Aspergillus species. Cleistothecium is a round, closed structure which encircles the asci which bear the ascospores. When the cleistothecium bursts, the asci are spread to the surrounding. Cleistothecium is created during the sexual reproduction stage of some Aspergillus species. Aleuriconidium is a kind of conidium which is generated by lysis of the cell that supports it. The base is frequently shortened and carries bits and pieces of the lysed supporting cell. These bits and pieces form annular frills at its base. Hulle cell is an enormous sterile cell carrying a small lumen. It is similar to cleistothecium and is linked to the sexual stage of some Aspergillus species.
Figure 2.11 Aspergillus niger
Figure 2.12 Aspergillus terreus
2.2.2 Fusarium oxysporum
Composed of filaments, Fusarium is a fungus which is largely disseminated on plants and in the soil. Fusarium is found in typical mycoflora of some products, such as rice, bean, soybean, and other crops (Pitt J A 1994). At tropical and subtropical areas, various species are chiefly more common. In cold climates, some of them live in soil; some other species include a teleomorphic state (Sutton D A 1998). Fusarium spp. is a widespread contaminant and a completely familiar plant pathogen. In the meantime, this fungus is likely to cause a variety of infections in man. Also, one of the rising causes of opportunistic mycoses is Fusarium (Vartivarian S E 1993). At the moment, the genus Fusarium includes more than twenty species. Among these species, Fusarium solani, Fusarium oxysporum, and Fusarium chlamydosporum are the most widespread ones (de Hoog G S 2000). In order to access a far more comprehensive list of the presently acknowledged Fusarium spp., refer to the table of synonyms. Fusarium spp are common plant pathogens; in the meantime, they are contributing agents of surface and systemic infections in man. Fusariosis is a name for all Infections which result from Fusarium spp. Fusarium solani is the most dangerous and harmful Fusarium spp. (Mayayo E 1999). The most important predisposing factor for developing cutaneous infections resulting from Fusarium strains is Trauma. Alternatively, distributed opportunistic infections grow in immunosuppressed hosts, principally in neutropenic and transplant patients (Venditti M 1988). In comparison with Fusarium infections developing in patients suffering from hematological malignancies and also compared to bone marrow transplantation patients, it can be said that Fusarium infections following solid organ transplantation are likely to remain local and have a better consequence (Sampathkumar, P 2001). Mycetoma, sinusitis, pulmonary infections, endocarditis, peritonitis, central venous catheter infections, septic arthritis, disseminated infections, Keratitis, endophthalmitis, otitis media, onychomycosis, cutaneous infections principally resulting from burn injuries, and fungemia owing to Fusarium spp. have been reported (de Hoog G S 2000). Meanwhile, there have been some reports concerning outbreaks of nosocomial fusariosis. It is probable that existence of Fusarium in distribution systems of hospital water give rise to dispersed fusariosis in immunosuppressed patients. In the meantime, there is a probability for the existence of Fusarium in soil of potted plants in hospitals. These plants make up a perilous mycotic source for nosocomial fusariosis (Summerbell R C 1989). Mycotoxins are generated by Fusarium spp. Long-term consumption of grains which have been contaminated with these toxins might result in allergic symptoms or be carcinogenic. Fumonisins are the mycotoxins generated by Fusarium moniliforme and Fusarium proliferatum in maize. They might give rise to oesophageal cancer (Pitt J I 2000). zearalenones are another class of mycotoxins. They are also likely to be generated by some Fusarium spp. whose characteristic is their growth in grains (Schaafsma, A. W 1998). Some researches and investigations regarding how to decrease or remove Fusarium mycotoxins from contaminated agricultural and food commodities are under way.
Fusarium spp. develop speedily on Sabouraud dextrose agar at 25Â°C and generate woolly to cottony, flat, spreading colonies. Fusarium dimerum is the only species which grows gradually. The color of the colony is likely to be white, cream, tan, salmon, cinnamon, yellow, red, violet, pink, or purple from the front. The colour might be colorless, tan, red, dark purple, or brown from the reverse. It is likely to observe a sclerotium (the organized mass of hyphae that remains latent during undesirable circumstances macroscopically. The normal colour of the mass is dark blue. In contrast, sporodochium (the cushion-like mat of hyphae bearing conidiophores over its surface) is typically absent in culture; however, whenever it is present, it is likely to be observed in cream to tan or orange colour. But, Fusarium solani is an exception and brings about blue-green or blue sporodochia. It is possible to microscopically observe hyaline septate hyphae, conidiophores, phialides, macroconidia, and microconidia. Besides these fundamental elements, chlamydospores could also be generated by Fusarium oxysporum, Fusarium semitectum, Fusarium solani, Fusarium chlamydosporum, Fusarium napiforme, and Fusarium sporotrichoides (Sutton D A 1998). Phialides have a cylindrical shape, with a small collarette. They are occasionally single but are sometimes generated as a component of a intricate branching system. It is possible to observe Monophialides and polyphialides (in heads or in chains). Phialides on conidiophores, which might be unbranched or branched, are a source of the production of Macroconidia (3-8 x 11-70 Âµm). They are 2- or more celled, thick-walled, smooth, and cylindrical or sickle- (canoe-) shaped. Macroconidia have a distinctive basal foot cell and pointed distal ends. They have a tendency to gather in balls or rafts. Microconidia (2-4 x4-8 Âµm), on the other hand, are formed on long or short simple conidiophores. They are 1-celled (at times 2- or 3-celled), smooth, hyaline, ovoid to cylindrical, and arranged in balls (a times occurring in chains). There are different shapes for them. When Chlamydospores are present, they are sparse, in pairs, clumps or chains. They are thick-walled, hyaline, intercalary or terminal. Essential characteristics for the differentiation of Fusarium species are macroscopic and microscopic features, such as, color of the colony, length and shape of the macroconidia, the number, shape and arrangement of microconidia, and presence or absence of chlamydospores. It is feasible to utilize Molecular methods, such as 28S rRNA gene sequencing, for swift identification of Fusarium strains to species level (Hennequin C 1999). Fusarium is considered as one of the most drug-resistant fungi. Generally, the most resistant of all Fusarium spp., is Fusarium solani. Fusarium strains result in relatively high MICs for flucytosine, ketoconazole, miconazole, fluconazole, itraconazole, and posaconazole. The novel triazole, has no activity against Fusarium (Johnson E M 1998). Fusarium spp. is inherently resistant to the new glucan synthesis inhibitors, caspofungin, anidulafungin, and micafungin. In spite of the fact that it is not active alone, the combination of caspofungin with amphotericin B seems to be synergistic against some Fusarium isolates. Amphotericin B (Rotawa N A 1990), voriconazole, and natamycin (Reuben A 1989) are the only antifungal drugs that bring about comparatively low MICs for Fusarium. In comparison with, itraconazole, voriconazole brings about markedly lower MICs. Terbinafine are likely to act well in vitro activity against some isolates. It is hard to treat Fusarium and the invasive forms usually result in death. The most frequently utilized antifungal drug for treatment of systemic fusariosis is Amphotericin B alone or in combination with flucytosine or rifampin. In the meantime, Lipid formulations of amphotericin B, such as liposomal amphotericin B and amphotericin B lipid complex can be exploited. Nonetheless, most cases remain resistant and do not react to amphotericin B treatment. In some immunosuppressed patients with disseminated fusariosis, Granulocyte and GM-CSF transfusions related to amphotericin B therapy are likely to save patients' lives. In spite of the fact that it is restricted in vitro activity, it seems that posaconazole is effective in murine fusariosis. Human data are awaited for verification of this finding. Topical natamycin is utilized for the purpose of treatment of keratitis resulting from Fusarium. Besides antifungal therapy, keratoplasty is essential for several patients. Patients with mycetoma resulting from Fusarium are likely to respond to itraconazole. On the other hand, Onychomycosis resulting from Fusarium are likely to be treated with itraconazole and ciclopirox nail lacquer.