CHAPTER -II ISOLATION AND DIVERSITY OF ACTINOMYCETES

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1 CHAPTER -II ISOLATION AND DIVERSITY OF ACTINOMYCETES 2.1 INTRODUCTION Actinomycetes are heterogenous group of microorganisms holding unicellular as well as filamentous organisms. Actinomycetes are present in huge numbers in soil. They form the majority of microbial load of the agricultural, compost and garden soils. They encompass highly useful enzyme and antibiotic producers to harmful disease causing bacteria also. Actinomycetes are known to produce humic acid like substances which improve the fertility of soil. These are Gram positive microorganisms placed in Group 22 to 29 in Bergey s Manual of Determinative Bacteriology [1984]. All Actinomycetes are placed in Class Actinobacteria of Phylum 14 in Domain II, Bacteria in Volume 4 of Bergey s Manual of Systematic Bacteriology [2001]. They have high GC content (around 70%). Soil is a complex environment providing nutritional, physical and biological variability. It serves as a complete source of nutrients for the survival of microbes. Streptomycetes are among the most numerous and ubiquitous soil bacteria. They are crucial in this environment because of their broad range of metabolic processes and biotransformation. These include degradation of the insoluble remains of other organisms, such as lignocellulose and chitin (among the world s most abundant biopolymers), making Streptomycetes central organisms in carbon recycling. Actinomycetes have huge adaptability to survive in different environments. Numerous actinomycetes were isolated from termite s gut and their abundance was more than bacteria [Watanabe et al., 2003]. Although they are present in large numbers in all habitats but their number varies with season. In some parts of the world they are more prevalent in dry season as compared to rainy season as in lakes of the Middle Plateau, Yunnan, China. [Jiang and Xu, 1996]. Investigators all over the world have isolated Actinomycetes for various applications and they have found immense potential and diversity in all the 46

2 continents. These filamentous bacteria have been able to colonize geographically distinct locations like plain lands, agricultural soils, compost soil, river waters, estuaries, oceans, mountains, snow covered Arctic regions etc. Their presence is observed by classical screening methods and also based on 16S rrna gene sequence analysis [Lyutskanova et al., 2009; Dhanasekaran et al., 2009; Jeffrey et al., 2008; Lo et al., 2002; Song et al., 2001]. Microbiologists believe that there is an equal amount if not more of microbial diversity in deserts. Actinomycetes with motile spores and easy dispersal are widespread in arid regions. Molecular ecology study indicates that only 1-5% of these microbial species have been isolated [Hunter et al., 1998]. Marine environment has emerged as unexplored treasure for novel Actinomycetes. The strains isolated from benthic waters are usually rare and uncultured. The marine actinobacteria have proved to be potential sources of novel natural products. The advent of molecular techniques and metagenomics has accelerated the explorations. Actinobacteria emerge as an often significant, sometimes even dominant, environmental clade in marine samples. We are yet to understand their growth requirements. Many of the marine isolates require seawater for growth showing high degree of marine adaptation. Exploring marine samples has led to the isolation of more number of novel actinobacteria. Actinomycetes are also present in many free-swimming marine vertebrates and invertebrates, as well as in sessile ones. Cultivation of Actinomycetes sampled from a range of depths and sediments has expanded the diversity that has been detected, extended the data available for actinomycete classification and biogeography, and for the estimation of actinomycete numbers [Ward and Bora, 2006]. Bredholt et al., (2008) reported the dominance of Streptomycetes and Micromonospora in Marine water samples from Norway. Takizawa et al., (1993) also noticed the dominance of Micromonospora in Chesapeake Bay. The extreme variety in oceanic environments of pressure, salinity, temperature and nutrients enable marine microorganisms to develop unique biochemical and physiological competence for survival. This potentially offers an abundance of secondary metabolites that might vary from the metabolites produced by terrestrial 47

3 microorganisms. Metagenomics enables direct access to the genome complex of marine ecosystems for heterogeneous gene expression and functional exploitation of uncultivable marine microorganisms [Li and Qin, 2005]. Cotarlet et al., (2010) demonstrated that Actinomycetes are not only adapted to distinct geographical locations but also take care of their role as decomposers by producing enzymes like protease and amylase in polar regions. Handling, treatment and selective isolation procedures can enhance the recovery of majority of actinomycetal types present in the given samples. The samples used for isolation should be properly dispersed as the microorganisms showing mycelial growth may be bound to aggregate soil particles. Actinomycetes are slow growers as they have a typical life cycle. They produce mycelium from spores which anchors in the substratum. The substrate hyphae are approximately 0.5 to1.0 µm in diameter and often lack cross-walls during the vegetative phase. Growth occurs at the hyphal apices and is accompanied by branching, thus producing a complex tightly woven matrix of hyphae during the vegetative growth phase. These then form aerial mycelium which bears spores. The aerial hyphae are hydrophobic in nature. There exists a correlation between the appearance of aerial mycelium and production of secondary metabolites such as antibiotics. Various kinds of sample treatments can also be done for selective isolation. Investigators have reported selective isolation of Actinomycetes by treatment with phenol, calcium carbonate and various antibiotics. Drying, heating and addition of bacterial inhibitors like antibiotics are commonly used strategies for suppressing the growth of other categories of microorganisms. Isolation of rare Actinomyces was done by Srinivasan et al. (2001) by dry heating the sample at 120 C for 1 hr and plated on a medium containing inorganic salts, arginine, glucose and antibiotics such as penicillin and nystatin to isolate. Aerial spore mass is resistant to desiccation by heat treatment. Streptomycetes are characterized by abundant sporulation but the spores of some rare genera [Microbispora and Streptosporangium] survive heating more than the abundantly sporulating Streptomycetes. 48

4 Li et al., (2002) isolated Actinomycetes from soil using extremely high frequency radiations. This increased the frequency of isolation of rare genera by 2 to 7 times. The rare actinomycete genera were represented by Actinomadura, Microtetraspora, Nonomuraea, Micromonospora, Amycolatopsis, Pseudonocardia, Saccharotrix, and Streptosporangium. Antibiotics are incorporated in the media to restrict the growth of commonly appearing Streptomyces. Different genera are known to respond at different concentrations of antibiotics formed the basis for the selective isolation. About 10 to 25 μg of oxytetracycline inhibited Streptomyces sp., but Streptoverticillium were resistant to this concentration. Most of them are aerobic and spore forming bacteria which can grow on common laboratory media like nutrient agar with tough and leathery colonies without aerial mycelium. They can grow on very poor media like water agar also. Their isolation can be done by using aspargine or proline rich media. Some Actinomycetes genera are anaerobic or require very specialized growth media and incubation conditions eg. Frankia. They exhibit different pattern of growth on different media. Seong et al., (2001) used hair hydrolysate vitamin agar for isolation of rare Actinomycetes. Macromolecules such as casein, chitin, hair hydrolysate, and humic acid can be used as carbon and nitrogen sources of rare Actinomycetes. Phenol treatment of soil suspension also lowers the number of fungi and other bacteria, but the Actinomycetes are less affected, thus 65% of the colonies belonged to rare Actinomycetes. Actinomycetes require special media to allow differentiation and development of characteristic spores and pigments. The components present in the media or the quality of agar also influences the good expression of taxonomic characteristics by Streptomycetes. Highly purified agar can be devoid of growth promoters like salts of magnesium, iron, manganese, and zinc which slow down the growth and proper differentiation [Okami et al., 1963]. Some medium ingredients also enhance the production of melanin pigment [Lakshmipathy et al., 2010]. Actinomycetes produce a variety of pigments. This ranges from yellow, orange, red, purple, blue, olive green etc. Investigators have studied them for commercial production of pigments. A well 49

5 studied member of this group Streptomyces coelicolor produces blue pigment [Marroquin et al., 1954]. We have attempted the isolation from a variety of samples because we wanted to understand the diverse population of Actinomycetes in western region of Madhya Pradesh (India) and built a broad base for screening of glucose isomerase producing Streptomyces sp. from the nature. Our main objective is to develop glucose isomerase production technology using indigenous isolate. 2.2 MATERIALS AND METHODS Sampling Samples were collected from various compost pits, open fields, agricultural soil and natural vegetation soil. Altogether 26 samples (Table. 2.1) were screened for isolation of Actinomycetes. These samples were mainly picked up from western region of Madhya Pradesh (India) to study the actinomycetal diversity of this region. The soil samples were picked up in commercially available sterile polythene bags from a depth of 10 inches from surface. The samples were named according to the sources from which they were obtained. The isolates were named by the initial letter of the sample and numbered as subscript. The details of samples are listed in Table 2.1 Table 2.1: List of sampling sites and number of isolates obtained Sample Number Name Abbreviated Name No. of Isolates Sample No. 1- Parawadi P 2 Sample No. 2- Vadelao V 8 Sample No. 3- Gamna Ga 4 Sample No. 4- Gyanpura Gy 2 Sample No. 5- Gunavad Gu 5 Sample No. 6- NRCS N 2 Sample No. 7- Biofertiliser from nearby rural area R 2 50

6 Sample No. 8- Krishi Vigyan Kendra[Kasturba Gram] KV 1 Sample No. 9- Krishi Kshetra[Kasturba Gram Compost pit] KC 8 Sample No. 10- Krishi Kshetra[Kasturba Gram Biogas plant] KB 4 Sample No. 11- Maharaja Ranjit Singh College, Biofertiliser M 3 Sample No. 12- Monica Jain Garden Biofertiliser MJ 2 Sample No. 13- Bhaislay BI - Sample No. 14- Bhaislay BII 1 Sample No. 15- Nandini Phanse Ma m NPI 5 Sample No. 16- Nandini Phanse Ma m NPII 5 Sample No. 17- Nandini Phanse Ma m NPIII - Sample No. 18- Kirti Singh [Farm Compost] K 1 Sample No. 19- Maharaja Ranjit Singh College, Biofertilizer MR 1 Sample No. 20- Satya Narayan [Compost pit soil] S 3 Sample No. 21- Vikas Jat [Compost pit soil] VJ 2 Sample No. 22- Abhishek Gautam [Compost pit soil] AI 2 Sample No. 23- Abhishek Gautam [Compost pit soil] AII 5 Sample No. 24- Kunjbihari Nagar [Compost pit soil] KNI 2 Sample No. 25- Kunjbihari Nagar [Compost pit soil] KNII 4 Sample No. 26- Plant soil in the lab LP Isolation Procedure The samples were sun dried and treated with calcium carbonate. The soil samples were diluted in a ratio of 1:10 and 0.1 ml of the suspension was streaked on Actinomycete isolation agar and Bennett s agar (Appendix - I). The isolation plates were incubated at 28 C for a week and observed daily. The Actinomycetal colonies were purified further and observed. Their cultural characters were studied by growing 51

7 them on different media. Aerial spore mass colour, colony reverse (colour of substrate mycelium), soluble pigmentation were the main features used for differentiation among the isolates. The purified isolates were preserved on Actinomycete isolation agar and Bennett s agar slants Morphological analysis The spore arrangement pattern of actinomycetes was studied by performing slide culture technique [Williams et al., 1989]. The pure cultures were spot inoculated on Bennett s agar plates. A cover slip rinsed with ethanol was inserted in the centre of the spot in inclined position. On development of the colonies, the cover slip was removed and observed under the microscope at high power magnification (40x) Sugar utilization and enzyme characterization The isolates were screened for the production of glucose isomerase simultaneously and the efficient GI producers were taken up for sugar and enzyme characterization. The cultures selected were P1, AII4, NPI2, KB1, V5. Multiwell tissue culture plates were used for checking sugar utilization. Phenol broth (Appendix - I) without any sugar source was prepared and 1mL of this was poured in all the wells aseptically. Different sugar discs were added in the wells. The cultures were inoculated and plates incubated at 28 C. The sugar utilization was observed by the presence of growth in the respective wells. The capacity of isolates to produce enzymes was determined by incorporating respective substrates in Bennett s agar medium. The isolates were spot inoculated in the centre of the plates. Amylase production was checked by adding starch in Bennett s agar and cellulase by adding cellulose in Bennett s agar [Appendix - I], the zones were visualized by flooding the plates with iodine solution [Kar and Ray, 2008; Kasana et al., 2008]. Gelatinase production was checked by adding gelatin in the Bennett s agar medium and the hydrolysis zones were visualizing by pouring Coomassie brilliant blue on the plates [Vermelho et al., 1996]. Caseinase production was observed by incorporating casein in the medium [Appendix - I]. Pectinase production was checked by adding pectin in the medium and the hydrolysis zones 52

8 were visualized by pouring alcoholic CTAB [Appendix - II]on the plates and incubating it for 10 minutes [Saadoun et al., 2007; Kobayashi et al. 1999]. Nitrate reduction test was performed in peptone nitrate broth (Appendix - I) to check the ability of the isolate to reduce nitrate to nitrite and other nitrogenous compounds. The tubes were incubated to observe the colour change by microorganisms. Catalase test was performed by adding a few drops of hydrogen peroxide on the organism s growth on Bennett s agar plate. 2.3 RESULTS AND DISSCUSSION Isolation A total of 75 cultures were isolated from 26 samples of compost pit, garden soil and agricultural areas. The screening plates inoculated with soil samples showed 30 to 40 colonies per plate on an average. The appearance frequency of actinomycetal colonies from the calcium carbonate treated samples was higher than those from non treated samples. Their characterization was done on the basis of cultural characters, biochemical characters, spore arrangement pattern and 16S rrna sequence. Actinomycetes grew not before 4 days of incubation at 28 C on primary screening soil plates. Once isolated, they grew fast as pure cultures. Isolates were identified as Actinomycetes by their chalky, velvety and powdery appearance on primary screening plates. Their substrate mycelium could be observed developing within 48 hours. The appearance of colonies between 2 nd to 4 th day seems to be like a typical bacterial colony. This is because of the formation of substrate and aerial mycelia but no sporulation. Confirmation of an actinomycetal colony can be done by observing the leathery texture of the colony. The colonies are tightly held on the agar surface like a plant on the soil surface. Many researchers have suggested different methods for selective isolation of Actinomycetes [Williams and Davies, 1965], where as we did not incorporate any antibiotic in the media to restrict the growth of any kind of Actinomycetes. We wanted to develop a collection of diverse Actinomycetes emphasizing on Streptomycetes. Treatment of sun dried samples with calcium carbonate gave better counts as also reported by earlier investigators [El-Nakeeb and Lechevalier, 1963]. These two 53

9 pretreatments must be decreasing the vegetative cell number and enriching the alkalophillic spore bearing organisms. Hirsch and Christensen, (1983) proposed membrane filter method for selective isolation of Actinomycetes where only the mycelial growing forms will penetrate through the membranee filter and reach the agar surface. They observed that the filters of pore size range of 0.45 to 0.22 allowed the exclusive penetration of Actinomycetes only. Fig. 2.1a is a primary screening plate which is showing numerous Actinomycete colonies with some nonmycelial bacteria also. Streptomycetes dominated in all the samples used for isolation which supports earlier reports of Streptomycetes being fast growing among other Actinomycetes and predominating in the soil microbial load. They are reported to dominate the counts in marine waters also [Ramasamy et al., 2007]. Fig. 2.1b is showing isolated culture picked up from a primary screening plate. The isolates were picked up and transferred repeatedly to get pure cultures on Bennett s agar. The samples were rich in diverse actinobacteria which is evident from the collection of isolates. Some of the representatives are shown in Fig 2.2. Fig. 2.1a Fig. 2.1b Fig. 2.1a: Primary screening plate showing mixed colonies from soil sample, 2.1b: Isolation plate of Streptomycete isolate P1. 54

10 Fig. 2.2: A few representatives of the Actinomycetal isolates 55

11 Aerial spore mass appearance The isolates produced a range of aerial spore mass colours, colony reverse and pigments. The aerial mycelium and spore mass were white (N1, N2, VJ2), creamish or ivory (V6, Ab, Gu3, KC4), yellowish cream (Gu1), orangish (V2, V3, NPII4), pink (Gu2, P) glossy green (NPI6), purplish grey (NPII1), dark brown (V1, V4), orangish brown (MR1), light grey (P1, P2, V5, MJ1) and dark grey (KB4, AII4), blue (NPI5, NPII5.) and black (S4) in colour. The colonies appeared leathery till the development of substrate mycelia but gained powdery, cottony, velvety or ash like appearance as aerial spore mass developed. A photographic representation of these categories have been shown in Fig. 2.3a-i and Fig. 2.4 a-i Fig. 2.3a Fig. 2.3b Fig. 2.3c Fig. 2.3d Fig. 2.3e Fig. 2.3f 56

12 Fig. 2.3g Fig. 2.3h Fig. 2.3i Fig. 2.3 Varied spore mass colour a: VJ2, white; b: Gu3, ivory; c: S4, black; d: LP, pink; e: pink; f: V4, dark brown; g: P1, ash grey; h: P2 light grey; i: NPII1, bluish grey. Fig. 2.4a Fig. 2.4b Fig. 2.4c Fig. 2.4d Fig. 2.4e Fig. 2.4 a: Dark velvety grey spore mass colour of AII4; b: Very dark grey spore mass colour of KB4; c: light blue spore mass colour of NPI5; d:blue spore mass colour of NPII5; e:glossy green colonies of NPI6. 57

13 Pigmentationn The collection of isolates was diverse with respect to growth pattern, aerial and substrate hyphae and pigments. Excessive to moderate pigment production was also observed among the isolates. Production of melanoid pigments was widespread among the isolates. Our collection had some isolates which exhibited light coloured aerial spore mass and produced abundant soluble pigment contrasting the jet black colour spore mass possessing isolate which did not produce any pigment. The isolates V1 and V4 showed heavy olive green pigmentation in the beginning which later turned the colour of the whole media to dark brown. As reported by earlier researchers, pigment production is influenced by medium composition and ph [Sanchez-Marroquin and Zapata, 1954]. The pigmentation was favored on Actinomycete isolation agar and Bennett s agar. The isolates V2 and V3 produced purplish pink pigment, NPI2, V6, Ab, KC3, KC4 pale yellow, KC1 light pink, M2, M3 light purple, KC5 brown, KNI1 dark brown KNII3 dark purlish black, NPII1 mustard [Fig. 2.5]. Majority of the cultures produced the earthy odor of Geosmins in pure cultures [Gerber and Lechevalier, 1965]. Fig. 2.5: Different coloured pigments produced by isolates. 58

14 Colony reverse Many cultures looked same from the colony surface appearance but they were different when observed from the reverse of the colony. The colony reverse showed the difference in the substrate hyphae. The cultures developed as rubbery, hard or chalky white colonies in the beginning which later on developed into their original forms showing velvety or powdery appearance with respective colours of aerial mass. The colony reverse of isolates were yellow (Ga), mustard (V6), orange (V3), reddish brown (P1, P2), grey (KB1) and white (Gu). This formed the basis of primary differentiation of cultures. A vast range of colony reverse among the isolates can be observed in Fig Fig. 2.6: Actinomycetal isolates showing different mycelial colour (colony reverse). The colonies developed concentric rings on aging [Fig. 2.7]. There was a slight difference in colour in the centre and in the concentric rings. The growing ends were lighter in colour. Most of the Streptomyces isolates exhibited white colonies which became grey on aging. 59

15 Fig. 2.7a Fig. 2.7b Fig. 2.7c Fig. 2.7d Fig. 2.7e Fig. 2.7: Appearance of concentric rings around the isolates on aging Colony surface appearance There was a huge variety in the texture of Streptomyces isolates which definitely had grey aerial spore mass, some had powdery ash like texture (P1), some dark velvety grey (AII4), some formed webbed colonies [Fig. 2.8a] and others showed cottony appearance. The isolated colonies of VJ1 [Fig. 2.8b] exhibited wavy appearance on the surface with depression in the centre of the colony. The isolate KB4 produced dew drops on the surface of the colonies [Fig. 2.8c]. 60

16 Fig. 2.8a Fig. 2.8b Fig. 2.8c Fig. 2.8a: Webbed colonial appearance; b: depressions on the top of the colonies; c: dew drops produced on the colony surface Growth response on different media The isolates were grown on a different media (Actinomycete isolation agar, Bennett s agar and Wheat bran agar) to study their growth pattern. The media composition was found to influence the extent of sporulation and appearance of aerial mycelia which is in accordance with earlier researchers [Okami et al., 1963; Sanchez-Marroquin, 1962]. They also differed in the time required for sporulation and attaining the spore colour on maturity. Bennett s agar medium was helpful in understanding the cultural characters like aerial spore mass colour, colony reverse and pigmentation satisfactorily while wheat bran agar was useful for fast growth of the organism as it is an enriched medium. Sporulation occurred very fast with dense growth on this medium which is in accordance with earlier reporters [Manhas and Bala, 2004; Srinivasan et al., 1983]. Fast and luxurious growth of the cultures on wheat bran medium must be due to the elaborate nutrients provided by wheat bran. Graying of the Streptomycetes colonies was also accelerated on this medium. A comparison of growth of isolates on different media is shown in Fig The detailed cultural characters of 75 actinomycetal isolates are mentioned in the Table

17 i ii i ii iii iii Fig. 2.9a Fig. 2.9b Fig 2.9 Different appearance of same isolate on different media; a(i): NPII2 grown on Actinomycete isolation agar; a(ii): NPII2 grown on Wheat bran agar, a(iii) NPII2 grown on Bennett s agar; b(i): NPI2 grown on Actinomycete isolation agar; b(ii): NPI2 grown on Wheat bran agar; b(iii) NPII2 grown on Bennett s agar. Table 2.2: Cultural characters of actinomycetal isolates Isolate Aerial Spore Mass Texture Colony Reverse Soluble Pigment P1 Ash Grey Powdery Blakish brown Light grey P2 Dark Grey Powdery Brownish grey - V1 Dark Ash Grey Ashlike Black Dark Olive Green V2 Purplish Grey Velvetty Purplish Black Light purple V3 Purplish Grey Velvetty Purplish Black Light purple V4 Dark Ash Grey Ashlike Black Dark Olive Green V5 Light Grey Cottany Mustard brown Mustard grey 62

18 Isolate Aerial Spore Mass Texture Colony Reverse Soluble Pigment V6 Dirty White Chalky Mustard Pale Ab Dirty White Chalky Mustard Pale KB1 Light Grey Ashlike Brown Grey KB2 Whitish Grey Powdery Dark Brown Light Grey KB3 Light Ash Grey Chalky Creamish - KB4 Greenish Grey Ashlike Greenish Mustard Mustard black M2 Dark Grey Powdery Dark Brown Greyish Purple M3 Light Grey Powdery Brick Brownish Blakish Grey M4 Light Grey Powdery Blakish Brown Blakish Grey Ga1 Dirty Yellowish White Powdery Yellow - Ga2 White Fibrous White - Ga3 White Chalky White - Ga4 White Chalky Brownish Purple - Gy1 Light Grey Powdery Blackish Brown - Gy2 Light Grey Powdery Mustard Light Orange Gu1 Creamish Yellow Chalk Powder White - Gu2 Purplish pink Powdery Peach Violet Gu3 Yellowish Grey Velvety Greenish yellow - 63

19 Isolate Aerial Spore Mass Texture Colony Reverse Soluble Pigment Gu4 Light Grey Velvety Yellow - Gu5 Light Grey Powdery White Creamish R1 Grey Powdery Blakish Grey Light Mustard R2 White Fibrous White - N1 White Chalky Mustard Mustard N2 White Chalky White - KC1 Creamish Yellow Fibrous Pinkish Brownish KC2 Grey Velvetty Blackish Brown Purpulish Grey KC3 Yellowish Grey Powdery Light Mustard - KC4 Dirty Yellowish Grey Powdery Light Mustard Light Mustard KC5 Bluish Grey Velvetty Blakish Grey Light Grey KC6 Grey Velvetty Black Greyish Purple KC7 Light Grey Cottany Pale - KC8 Yellowish White Powdery Light Mustard Pale MR1 Grey Velvetty Mustard Grey - NPI1 Dirty White Cottany Blakish Brown Greyish Purple NPI2 Grey Velvetty Purpulish Mustard Light Purple NPI3 Dirty White Cottany Blakish Brown Light Purple 64

20 Isolate Aerial Spore Mass Texture Colony Reverse Soluble Pigment NPI4 White Chalky White - NPI5 White Chalky White - NPI6 Bluish Grey Velvetty Grey - NPII1 Earthy Brown Powdery Blakish Brown Purplish Black NPII2 Grey Powdery Blakish Brown Light Purple NPII4 White Cottany Light Mustard - NPII5 Bluish Grey Velvetty Bluish Grey - NPII6 Green Glossy Grey - KNI1 Ash Grey Ashlike Black Greyish Brown KNI2 Light Grey Powdery Grey - KNII1 Earthy Brown Powdery Blakish Brown Purplish Black KNII2 Buff Grey Velvetty Light Mustard Light Purple KNII3 Dark Greyish Purple Powdery Dark Brown Brownish Purple KNII4 Buffy White Chalky White - AI1 Dark Ash Grey Powdery Grayish Black - AI2 Dark Grey Powdery Brown Brownish AII1 Grey Powdery Blakish Brown - AII2 White Chalky White - AII3 White Fiborous White - 65

21 Isolate Aerial Spore Mass Texture Colony Reverse Soluble Pigment AII4 Ash Grey Velvety Mustard with Grey Margin - AII5 Blue Chalky Grey Light Purple BII1 Dirty White Chalky Dark Grey - MJ1 Grey Powdery Greyish Mustard - MJ2 Grey Chalky Greyish Mustard - VJ1 Light Grey Velvety Orangish Mustard - VJ2 White Chalky White - S1 White Chalky White - S2 Grey Velvety Dark Grey - S4 Black Velvety Black - KV White Chalky White - LP Pink Velvety Pink - K White with grey spots Powdery Greyish Growth in liquid media The actinomycetes grew in liquid media (Bennett s broth and Medium No. 9) in the form of beads. The size and appearance of beads varied with the media composition and type of the organism. Fig shows the growth of the isolate P1 in different media combinations. The beads appear as orange and light brown in Medium No. 9 (Fig. 2.10a) whereas it appears white in Bennett s broth as in Fig. 2.10b. The fibrous outgrowth from the beads to varying degrees is visible in Fig. 2.10c, d and e. The beads in Fig. 2.10f are absolutely smooth. 66

22 Fig. 2.10a Fig. 2.10b Fig. 2.10c Fig. 2.10d Fig. 2.10e Fig. 2.10f Fig. 2.10: Actinomycetes growing in liquid media. 67

23 2.3.2 Morphological Analysis The mycelium and spore pattern can be easily studied by slide culture technique [Williams et al., 1989]. The isolate morphology can be easily understood during the initial phase of sporulation. At this stage the densely branched substrate mycelium can be differentiated from darker less branched aerial mycelia. The aerial mycelia are hydrophobic in nature and stand upright. There exists a correlation between the appearance of aerial mycelium and production of secondary metabolites such as antibiotics. Most of the antibiotic producing strains are known to be pigmented. The pattern of spore arrangement was studied and the isolates were grouped according to the features described in Bergey s Manual of Systemic Bacteriology Volume 4 [2001]. The isolates P1, P2, V1, V4, KB1, KB4, M2, Gy1, KC2, KNI1, MJ2 and AII4 exhibited typical spiral spore arrangement as of Streptomyces (Fig. 2.11a). The isolates Ab and KC3 were grouped as Kitasatosporia as they showed straight chains of spores. The isolates Ga3, Gu1, KC1 showed straight chains of spores with curled ends as Actinomadura. Pseudonocardia exhibited wavy chains of spores as in Ga4 and BII1. Saccharomonospora had a series of doubly placed spores as in the isolates NPI5, NPI6 and AII5 (Fig. 2.11g). The isolate AII3 had straight spore chains developing from branched ends and were placed in Streptoverticillium group. The mycelium of Micromonospora had one spore at the terminus as in the isolate MR1. Fig. 2.11a Fig. 2.11b 68

24 Fig. 2.11c Fig. 2.11d Fig e Fig. 2.11f 69

25 Isolation And Diversity Of Actinomycetes Fig. 2.11g Fig. 2.11i Fig. 2.11h Fig. 2.11j 70

26 Fig. 2.11k Fig. 2.11l Fig. 2.11m Fig n Fig. 2.11: Microscopic details of spore arrangement pattern of a few isolates, Streptomyces (a-f): a: P1; b: AII4; c: V5; d: KB1; e: KB2; f: Gy2; g: Saccharomonospora - AII5; h: Streptoverticillium - AII3; i: Nocardioides - VJ1; j: Streptoverticillium- KC8; k: Streptomyces - KC5; l: Thermomonospora - P; m: Saccharomonospora - NPI6; n: Dermatophillus - S1. 71

27 2.3.4 Biochemical characterization Sugar utilization was checked for five good producers of glucose isomerase. Glucose, raffinose and terhalose, xylose and sucrose were found to be the sugars of choice of the isolates [Table 2.3] [Pridham and Gottlieb, 1948]. According to the description given in Bergey s Manual of Systemic Bacteriology [2001], most of the Streptomyces of gray aerial spore mass series utilize glucose and xylose whereas raffinose and sucrose is not very commonly utilized. The isolates P1, AII4, V5 and KB1 were positive for catalase whereas NPI2 was found to be negative. All the isolates exhibited cellulolytic, lipolytic and pectinolytic activity. The isolates P1, V5, KB1 and NPI2 also degraded starch but AII4 did not show amylase production [Table 2.4]. Table 2.3: Sugar Utilization tests for selected Streptomyces isolates Sugar P1 AII4 VPI2 KB1 V5 Maltose Mannitol Mannose Melibiose Raffinose Rhamnose Salicin Sorbitol Sucrose Trehalose Xylose Adonitol Arabinose Cellobiose Dextrose Dulcitol

28 Fructose Galactose Inositol Inulin Lactose [(+) = Sugar Utilized (-) = Sugar not Utilized] Table 2.4: Enzymatic characterization for selected Streptomyces isolates Test P1 A II4 NPI2 KB1 V5 Glucose isomerase production Amylase production Protease production Lipase production Cellulase production Gelatinase production Pectinase production Nitrate reductase Catalase (+) = Enzyme produced (-) = Enzyme not produced 2.4 CONCLUSIONS The isolation of morphologically and culturally diverse actinobacteria has revealed a treasure of bioresource in the western region of Madhya Pradesh. The presence of huge counts of actinomycetes in soil also indicates the fertility of soil, as these organisms are known to be efficient in mineralization and recycling of the organic matter. The samples yielded different group of organisms but the grey spore mass bearing Streptomycetes was dominant. The growth rate of grey series was quiet high as compared to white and blue spore mass bearing isolates. The sporulation was also better by Streptomycetes as compared to non-streptomycete isolates. 73

29 The objective behind screening diverse actinomycetes was to point out an efficient glucose isomerase producing Streptomycete strain. Streptomycetes are widely used for the production of glucose isomerase at industrial level. The biochemical characters exhibited by the selected isolates and the spore pattern arrangement studied by slide culture technique help us to conclude that these organisms belong to the Streptomycete group. The elaborate enzyme production capacity of these isolates accounts for the presence of all the macromolecules which are being degraded by them. All the selected isolates had typical spiral spore arrangement which is a characteristic feature of this group. Slide culture technique is a quick tool for determination of the actinobacterial group. Their confirmation will be done further by sequencing of 16s rrna. 74