A REVIEW ON ACTINOMYCETES AND THEIR BIOTECHNOLOGICAL APPLICATION

A REVIEW ON ACTINOMYCETES AND THEIR BIOTECHNOLOGICAL APPLICATION

Chavan Dilip V.*, Mulaje S. S.¹, Mohalkar R.Y.¹

Department of Quality Assurance, Maharashtra College of Pharmacy, Nilanga, Dist. Latur- 413 521, Maharashtra, India

ABSTRACT: Recent findings from culture-dependent and culture independent methods have demonstrated that indigenous marine actinomycetes exist in the oceans and are widely distributed in different marine ecosystems. There is tremendous diversity and novelty among the marine actinomycetes present in marine environments. Progress has been made to isolate novel actinomycetes from samples collected at different marine environments and habitats. These marine actinomycetes produce different types of new secondary metabolites. Many of these metabolites possess biological activities and have the potential to be developed as therapeutic agents. Marine actinomycetes are a prolific but underexploited source for the discovery of novel secondary metabolites.

Keywords:

Actinomycetes, Enzymes, Antibiotics, GC content etc

INTRODUCTION: Actinomycetes are high GC, Gram-positive bacteria with fungal morphology. They are rich source of secondary metabolites with diverse biological activity. The Gram-positive bacteria fall into two major phylogenetic divisions, “low-GC” and “high-GC". GC content is an abbreviation for the percentage of GC base pairs in an organisms DNA. Those that have a low GC content, have more AT base pairs in their DNA.

GC content is a crude measure of the relatedness of microorganisms, but is still useful for differentiating large phylogenetic divisions. They exhibit a wide range of life cycles, which are unique amongst the prokaryotes.

Gram-positive bacteria that have been placed within the phylum Actinobacteria, class Actinobacteria, subclass Actinobacteridae, order Actinomycetales which currently consists of 10 suborders, more than 30 families and over 160 genera. Being a large group of microbial resources of wide practical use and high commercial value, actinomycetes contribute to around 70% of the source of antibiotics and also produce numerous non-antibiotic bioactive metabolites, such as enzymes, enzyme inhibitors, immunological regulators, anti-oxidation reagents, and so on. Actinomycetes are widely distributed in natural habitats, especially soil and ocean¹⁵.

The marine environment harbors millions of species of microorganisms that play important role in mineralization of complex organic matter, degradation of dead plankton,  plants, animals, degradation of pollutants and toxicants and primary and secondary productivity. Marine microorganisms have a diverse range of enzyme activity and capable of catalyzing various biochemical reaction with novel enzymes such as amylase, lipase, deoxyribonuclease, lipase and protease.

Among the marine microorganisms actinomycetes comprises an important group. They have tremendous potential to synthesize bioactive secondary metabolites.

Great strides have been made in understanding the microbes of the seas around India, and these include research in India with the objective of understanding the both mycological and bacteriological aspects, covering near, offshore, and deep-sea waters. India has seas on three sides and is bestowed with long coastline of about 7500 km. This represents a vast potential of exploitable resources. The new ocean regime enabled India to control about 2.01 million square kilometers of sea as its exclusive economic zone (EEZ) ns in India that concentrated on marine microbiology. This is due to the lack of adequate facilities, trained manpower and limited financial support, therefore their work was restricted to coastal areas.

Even though people initially concentrated on studies related to microbial participation in biogeochemical cycles, slowly they become involved in other area of research. A considerable amount of work has been carried out to understand the role of microbes in phosphate utilization, nitrogen fixation and silicate solubilization. Some of the extreme halophiles also received attention during this period, but studies were mainly to understand their roles in salt pans and deterioration of dried fish.

Another area that attracted attention of the researchers during this period is the production of bioactive compounds by marine microbes. The major group studied in detail was the marine Streptomyces ¹⁶.

Several hundred species of Streptomyces were isolated from sea water, marine sediments including mangroves, marine mollusks and detritus. One of the significant observations was that nearly 70%of Streptomyces sp. isolated from marine mollusks was antagonistic, whereas only 20-25% of cultures isolated from sediments showed antagonism towards the test microorganism. During this period a novel marine Vibrio sp. was isolated from the marine sediments (east coast) which produced an antileukemic agent (L-asparginase). This was found superior to commercial preparations at that time in the treatment of tumors ¹⁷.

CLASSIFICATION OF ACTINOMYCETES ^{15, 16, 18}

During the first half of second phase (1976-1985) studies on microbiological pollution of coastal areas received attention because of its importance in coastal zone management. Several reports on the distribution of human pathogens and indicator bacteria in water, sediments, plankton, and faunal samples along with the Indian coast become available. Researchers reported the occurrence of three new serotypes of Salmonella: S. irumu, S. panama and S. lexington in the environmental samples from Pitchavaram mangroves and velar estuary.

Another bacterium of medical significance in the marine environment that received considerable attention is the halophilic Vibrio parahaemolyticus. This bacterium is an etiologic agent of seafood-borne gastroenteritis. A detailed and systematic survey conducted over a period of two years at Parangipettai indicated the ubiquitous presence of Vibrio parahaemolyticus in water, sediment, flora and fauna. In the last decade (1986-1995) investigations were under taken in many areas and on several groups of organisms¹⁸.

Occurrence & habitats of Actinomycetes:

1. Soil: Actinomycetes constitute a significant component of the microbial population in most soils. It has been estimated that counts of actinomycetes over 1 million per gram are commonly obtained. Over twenty genera have been isolated from soil. Lechevalier and Lechevalier (1967) found that 95% isolates belonged to streptomycetes. Environmental factors influence the type and population of actinomycetes in soil. Most actinomycete isolates behave as neutrophiles in culture, with a growth range from pH 5.0 to 9.0 and an optimum pH around 7.0.

The pH is a major environmental factor determining the distribution and activity of soil actinomycetes. Neutrophiles occur in less number in acidic soils below pH 5.0, whereas acidophilic and acidoduric streptomycetes are numerous in acidic soils. However, there are few reports of to 9.5 was isolated from soil near a salt lake. Most actinomycetes behave as mesophiles in the laboratory, with optimum growth temperature at 25 to 30^oC. Many mesophilic actinomycetes are active in compost.

However, the capacity of self-heating during decomposition often provides ideal conditions for obligate or facultative thermophilic actinomycetes able to grow at temperatures above 40^oC. Actinomycetes play an important role in the decomposition of plant and other material especially in the degradation of complex and relatively recalcitrant polymers. They degrade lignin, cellulose and lignocellulose. There is evidence that actinomycetes are involved in the degradation of many other naturally occurring polymers in soil such as hemicellulose, pectin, keratin, chitin and fungal cell wall material. Actinomycetes from rhizosphere suppress the growth of pathogens. Isolates from rhizosphere hydrolyses starch. Some rhizosphere isolates can synthesise gibberellin like substances.

Some actinomycetes produce indole acetic acid in culture and when inoculated into soil. Actinomycetes, particularly Streptomyces play a major role in antagonistic interactions in soil. Rhizosphere Streptomyces have been known as agents for control of fungal root pathogens. Many antagonistic interactions other than antibiotics may occur between Streptomyces and fungi. Reduced incidence of root infection has been correlated with an increase in number of Streptomyces in the rhizosphere which inhibits the pathogen by production of antifungal antibiotics. Actinoplanes can act as biological control agents of plant diseases ⁵.

2. Compost and related materials: Many mesophilic actinomycetes are active in compost in the initial stages of decomposition. However, the capacity for self-heating during decomposition provides ideal conditions for obligate or facultative thermophilic actinomycetes. Some genera like Thermo-actinomycetes and Saccharomonospora are strictly thermophilic. Thermophilic actinomycetes grow well on animal manure. They have been active in fermentation of pig faeces, straw and deodorization of pig faeces.

Thermomonospora species particularly grow during the second indoor phase of preparation of manure for mushroom cultivation, whereas Streptomyces diastaticus and Thermo actinomyces vulgaris predominate in the spent, steamed compost and its dust. Thermomonospora curvata was shown to be active in decomposition of municipal waste compost and to produce thermostable C1 and Cx cellulose. Actinoplanes and related organisms are common in soils, rivers and lakes and can grow on plant litter in rivers. The Micromonospora were considered to be indigenous to fresh water ecosystem and they had a role in the turnover of cellulose, chitin and lignin. Several workers confirmed the presence of Micromonospora in streams, rivers and lake sediments. Streptomycetes spores are also being continually washed into freshwater and marine habitats. Some investigators have claimed the existence of aquatic streptomycetes and growth on the chitinous exoskeletons of Procambarus versutus immersed in a woodland stream. Actinomycetes change taste and odour of potable water rendering it unpalatable. Earthy taste and odour is caused because of compounds like geosmin and methyl iso-borneol the products produced by Streptomyces. Plant and animal debris at water margins can provide substrates for actinomycetes growth and geosmin production ⁶.

3. Marine habitats: Actinomycetes were mentioned incidentally in early studies on the microbial community of marine habitats. The selective isolation procedures and reliable diagnostic tests were not used in such pioneering surveys. There is evidence that actinomycetes usually form a small fraction of the bacterial flora in marine habitats and counts are low compared with those from terrestrial and freshwater sites. Some workers considered actinomycetes to be part of an indigenous marine microflora, whereas others saw them primarily as wash-in components that merely survived in marine and littoral sediments as spores. This latter view is supported by the observation that the numbers of actinomycetes in marine habitats decrease with increasing distance from land.

It has been suggested that the isolation of organisms from marine sites far removed from the possibilities of terrestrial contamination could be used as evidence of a marine origin but it is now evident that the endospores of Thermoactinomyces can be transported very long distances by ocean currents. Sediment collected from a depth of 4920 meters in the Atlantic Ocean 500 miles from land was found to contain small numbers of thermo actinomycetes. Okami and Okazaki (1978) observed that actinomycetes were widely distributed in the marine environment. The occurrence of Streptomyces, Nocardia and Micromonospora growing on dead marine algae and contact slides suspended in the sea has been reported. Study of actinomycete distributions in sediments is depending upon the depth from which samples are collected. The best marine source of actinomycetes is sediment and also reported from water, sand, rocks, seafood's, marine plants, mangrove sediment and deep sediment.

Actinomycetes from marine sources have been reported to decompose agar, alginates, cellulose, chitin, oil and other hydrocarbons. They have been also implicated in the decay of wood submerged in seawater. Atlas (1981) included the genera Arthrobacter, Brevibacterium, Corynebacterium and Nocardia among the microorganisms important in the degradation of petroleum hydrocarbons in aquatic habitats. Actinomycetes have been shown to degrade cellulose, starch and lignin in seawater under laboratory conditions ²¹.

Characteristics and nutrition of Actinomycetes: Actinomycetes are heterotropic in nature. Most of them are strict saprophytes, while some from parasitic or mutualistic associations with plants and animals. Actinomycetes are commonly believed to have a role in the recycling of nutrients. They are aerobic and some like Actinomyces are anaerobic. The species like Frankia require very specialized growth media and incubation conditions ⁵. Many actinomycetes are growing on the common bacteriological media used in the laboratory such as nutrient agar, trypticase agar, blood agar, brain heart infusion agar and starch casein agar. Sporoactinomycetes require special media to allow differentiation and the development of characteristic spores and pigments.

Some of these media are not available commercially and must be prepared in the laboratory using colloidal chitin, soil extract and decoctions of plant materials. Pale, shiny, hard colonies of Streptomyces species on nutrient agar can be transformed into bright yellow colonies with a powdery white aerial mycelium and spirals of arthrospores when the organism is subcultured on a more suitable growth medium, such as oatmeal or inorganic salts starch agar. Outgrowths from a spore or fragments of mycelium develop into hyphae that penetrate the agar (substrate mycelium) and hyphae that branch repeatedly and become cemented together on the surface of the agar to form a tough, leathery colony. The density and consistency of the colony is depending on the composition of the medium.

Nocardioform actinomycetes exhibit fragmentation, the hyphae breakup into rods and cocci and form soft or friable colonies ⁶.

In strains of certain genera such as Streptomyces, the colony becomes covered with free, erect hyphae surrounded by a hydrophobic sheath that grow into air away from the colony (aerial mycelium). These hyphae are initially white but forms different colours when spore formation begins. Colonies appear powdery or velvety and readily distinguished from the more typical bacterial colonies. Streptomyces species have chains of spores on the aerial mycelium, which are normally absent from the substrate mycelium. These spores are arthrospores, regular segments of hyphae with a thickened spore wall surrounded by a hydrophobic sheath that may bear spin or hairs ⁷.

Novel approaches in isolation of Actinomycetes: Experience has shown that discoveries of previously unknown and important natural products occur when new screening systems are utilized. The isolation of actinomycetes from mixed microflora present in nature is complicated because of their characteristic slow growth relative to that of other soil bacteria. There are five basic stages for the isolation of industrially important actinomycetes.

1. Choice of substrate: Isolation of actinomycetes from freshwater and marine environment has been reported. There must be some differences between organisms existing in marine and terrestrial environments. In the course of screening of actinomycetes isolated from shallow sea area, some antagonistic actinomycetes, such as xanthomycin producing actinomycetes have been isolated more frequently than from terrestrial soil. Few of these actinomycetes were found to be new and produce either new antibiotics or biologically active substances under specially devised conditions. Thus, the isolation of actinomycetes from marine areas gives us another source for finding new actinomycetes and new antibiotics.
2. Re-heat treatment: Pretreatment that allows the selective isolation of an actinomycetes component normally found to be rare or absent in soils. One such example is the rehydration technique applied to leaf litter from freshwater habitats, which has yielded many actinoplanetes and the novel genus ‘Cupolomyces. The aerial spores of most actinomycetes generally resist desiccation and show higher resistance to wet or dry heat. Such mild temperature treatments significantly reduce the numbers of Gram-negative bacteria. Drying plus mild heat treatments coupled with selective media can yield well-separated bioactive actinomycetes isolated from marine sediments.

Nonomura and Ohara (1969) used pre-treatment such as dry heating, specialized growth media and long incubation times to isolate new species of Actinomadura, Microbispora, Micro tetraspora, Streptosporangium, Thermom- onospora and Thermoactinomyces. Many antibiotics are tested against a range of actinomycetes, bacteria and fungi representing types found in soil. From these, nystatin (50 mg/ml), acetidione (50 mg/ml), polymyxin B sulphate (5 mg/ml) and sodium penicillin (1 mg/ml) are generally selected for incorporation into a starch casein medium to achieve selective growth of actinomycetes on soil dilution plates.

Recently, differences in sensitivity to antibiotics have been used to increase the selectivity of media for particular actinomycetes. Thus, tetracycline or its derivatives have been used for the isolation of Nocardia spp., novobiocin for Thermoactinomycetes spp. and rubomycin, bruneomycin, streptomycin, kanamycin and rifampicin for Actinomadura spp. novobiocin for Thermoactinomycetes spp. and rubomycin, bruneomycin, streptomycin, kanamycin and rifampicin for Actinomadura spp. Different actinomycetes species are isolated using different compounds are shown in figure 1 ¹⁹.

FIG. 1: COLONY FORMATION

3. Selective media: Bacteriostatic and fungistatic chemicals such as phenol and sodium propionate have been incorporated into isolation media to suppress growth of bacteria and moulds and thus favour actinomycetes. But such amendments at permissible concentrations frequently allow growth of contaminants and at higher levels may also suppress actinomycete. Chitin agar with mineral salts is more effective than that without mineral salts for isolating actinomycetes from water. Chitin agar showed selectivity superior to that of other media for isolating actinomycetes from water and soil ¹³.

Selective media:

 Incubation: The majority of antibiotic producing actinomycetes grow best between 25 to 30^oC. Thermopiles are incubated at 40 to 45^oC and psychrophiles at 4 to 10^oC.  Incubation times for isolation plates are usually from 7 to 14 days. Longer incubation times have often been disregarded because of the slow growing actinomycetes would be unsuitable candidates for economic fermentation. However, the early growth of some species of bacteria can modify the nutrient environment of the isolation plate by supplying growth factors. For isolation of novel actinomycetes incubation period may be extended for one month.

4. Colony Selection: Selection of a colony is the most time consuming method. It depends upon the aims of the screening programme. There might be much duplication of the colonies. For isolation of microorganisms, more rational ways must be utilized. The majority of researchers now select candidate colonies by using a stereomicroscope and transferring growth with the aid of a pointed wooden cocktail stick. Tiny colonies can be distinguished and chosen and the rough wooden points carry sufficient spores or hyphal fragments to give a successful transfer. The site of sample collection, knowledge of the secondary metabolite of an isolate, objective enrichment techniques and objective culture media formulations would lead to an isolation of novel and potential isolates¹⁹.

Biotechnology and Importance of actinomycetes: The attention given to the actinomycetes in biotechnological applications is a natural result of the great metabolic diversity of these organisms and their long association with the environment. Actinomycetes are a unique group of organisms in the prokaryotes having different morphological, cultural, biochemical and physiological characters. This group is a potential producer of antimicrobial substances, enzyme inhibitors, immunomodifiers, enzymes and growth promoting substances for plants and animals¹⁹.

1. Antibiotics: Actinomycetes have been known as the greatest source of antibiotics. Two third of today’s antibiotics are obtained from actinomycetes. The important antibiotics from actinomycetes include anthracyclines, aminoglycosides, b-lactams, chloramphenicol, macrolides, tetracyclines, nucleosides, peptides and polyethers. Until 1974 antibiotics of actinomycete origin were almost exclusively confined to Streptomyces. Recently efforts have been made to explore rare actinomycetes like Actinomadura, Actinoplanes, Ampullariella, Actinosynnema and Dactylosporangium for the search of new antibiotics. Target directed screening is being used for screening of antibiotic producing actinomycetes. Molecular biological techniques have helped on lar