CHAPTER V TAXONOMIC STUDIES OF THE SELECTED ISOLATE C 9

CHAPTER V TAXONOMIC STUDIES OF THE SELECTED ISOLATE C 9 Selection of media for taxonomic studies: Culture media used for taxonomic studies on actinomycetes comprise: 1) Media used for characterization and identification of species, consisting of both synthetic and organic forms; the synthetic media have found extensive application in the study of the morphology, physiology and cultural properties of the organisms. While the organic media are used for obtaining supplementary cultural evidence. 2) Media used for obtaining maximum yield of the metabolite of interest. Waksman et al., 1948 recommended the inclusion of the following media for characterization of actinomycetes. 1. At least three synthetic media, preferably sucrose, sodium nitrate, salt agar or sucrose ammonium salt, agar, glucose or glycerol asparagine agar and calcium malate or citrate agar. 2. Two or three organic media such as nutrient agar, yeast extract malt extract agar, potato glycerol glutamate agar, or oatmeal agar. 3. Three or four complex natural media such as potato plug, gelatin or milk. 4. Peptone iron yeast extract agar for H 2 S production. 5. Tyrosine medium for tyrosine reaction. 6. A synthetic medium for carbohydrate utilization. In the present work, morphological studies and colour determination of the selected isolate was studied in accordance with the International Streptomycetes Project (ISP) procedures (Shirling and Gottlieb, 1966). 91

The following media as recommended by the ISP (Shirling and Gottlieb, 1966) were used for morphological studies and colour determinations. 1. Yeast extract malt extract agar (ISP 2) 2. Oat meal agar (ISP - 3) 3. Inorganic salts starch agar medium (ISP 4) 4. Glycerol asparagine agar medium (ISP 5) The following biochemical tests were carried out employing the prescribed media: 1. Nitrate reduction test 2. Carbon source utilization 3. Catalase production test 4. Urease test 5. H 2 S production test 6. Starch hydrolysis test 7. Casein hydrolysis test 8. Gelatin hydrolysis test 9. Citrate utilization test 10. IMViC tests 11. Ability to produce bioactive metabolite factors 12. Sodium chloride tolerance test 13. Growth temperature range 14. Growth ph range 15. Oxidase activity test 92

Preparation of inocula: In general, the agar media favouring abundant sporulation are those with C/N ratio as jowar starch agar, oatmeal agar (ISP) and starch casein agar medium. In the present study starch casein agar was used for isolation of actinomycetes from soil samples. These slants were inoculated from the stock culture and incubated at 28 o C for 7 to 10 days for maximum sporulation. Spore suspension was prepared by transferring a few loopful of spores from these slants into sterile distilled water and shaken thoroughly. Fresh spore suspension was prepared for each test. For gelatin liquefaction, starch hydrolysis and casein hydrolysis, a loopful of spores taken from the stock culture was used for inoculation. For all other tests spore suspensions prepared as above, were used employing equal volumes of the suspension in each case. Preparation of media: Detailed composition of all the media employed in this work were given in the Appendix-II, unless otherwise stated, 20 ml of each sterile medium was poured into each Petri dish of 9 cm diameter. After holding the dish for 24 h at 28 o C, these were inoculated with the organism under investigation, distributing the inoculum uniformly and incubated at 28 o C. 93

BIOCHEMICAL TESTS 1. Nitrate reduction test: Nitrate broth of 5ml was inoculated with a loopful of spores of selected isolate and incubated at 28 o C for 7 days. Controls were also run without inoculation. On 7 th day, the clear broth was tested for the presence of nitrate in the following way: Regent a) α naphthylamine test solution: α - naphthylamine Conc. H 2 So 4 Distilled water : 5.0 g : 8.0 ml : 1.0 l - naphthylamine was added to dilute sulphuric acid and stirred until solution was effected. Reagent b) Sulphanilic acid Sulphanilic acid Conc. H 2 So 4 Distilled water : 8.0 g : 48.0 ml : 1.0 l Sulphuric acid was added to 500 ml of water. Then sulphanilic acid was added, followed by water to make upto volume. Procedure: To 1 ml of the broth under examination and 1 ml of control, two drops of sulphanilic acid followed by two drops of α napthylamine solution were added. The presence of nitrate was indicated by a pink, red or orange colour and absence of colour change was considered as nitrite negative. In the latter case, the presence or absence of nitrate in the broth under examination was confirmed by adding a pinch of zinc dust after the addition of the reagents. When the unreduced nitrite, if present gave a pink, red or orange colour. Result: This test gave positive result with the selected isolate. 94

2. Carbon source utilization: The ability of different actinomycete isolate in utilizing various carbon compounds as source of energy was studied by following the method recommended by ISP. Chemically pure carbon sources certified to be free of admixture with other materials were used. The following carbon sources (at 1% level) were used for this test. D glucose, L (+) arabinose, sucrose, D-xylose, meso-inositol, D-mannitol, D-fructose, L (+) rhamnose, raffinose, galactose and salicin. The inoculated tubes were incubated at 28 o C and observed on 7 th day and 14 th day. Acid production from above carbon sources were studied Table 24: The results of carbon source utilization with the selected isolate The results were recorded as follows: Acid production from arabinose galactose glucose mannitol raffinose salicin xylose sucrose rhamnose meso-inositol fructose _ + + + _ + + Positive utilization (+): When growth on tested carbon was significantly better than on the basal medium without carbon Utilization negative (-): When growth was similar to or less than growth on basal medium without carbon. 95

3. Catalase production Test: During aerobic respiration in the presence of oxygen, microorganisms produce hydrogen peroxide (H 2 O 2 ) which is lethal to the cell. The enzyme catalase present in some micro organisms breaks down hydrogen peroxide to water and oxygen as shown below: 2 H 2 O 2 2 H 2 O + O 2 and helps them in their survival. Catalase test is performed by adding H 2 O 2 to trypticase soyagar slant culture. Release of free oxygen gas (O 2 ) bubbles is a positive catalase test. Procedure: Preparation of trypticase soyagar (ph 7.3) slants Composition of tripticase soyagar Trypticase - 15.0 gm Phytone - 5.0 gm Sodium chloride - 5.0 gm Agar - 15.0 gm Distilled water - 1.0 litre Trypticase soyagar slants were inoculated with the selected isolate. An uninoculated trypticase soyagar slant was kept as control. Then these tubes were incubated at 35 o C for 24-48 h. While holding the inoculated tube at an angle, allow 3-4 drops of hydrogen peroxide to flow over the growth of each slant culture. Results: A catalase positive culture will produce bubbles of oxygen within one minute after addition of H 2 O 2. Release of free oxygen gas bubbles was observed in the selected isolate, thus showing catalase positive test. 96

4. Urease activity test: Urea is a major organic waste product of protein digestion in most vertebrates and is excreted in the urine. Some microorganisms have the ability to produce the enzyme urease. The urease is a hydrolytic enzyme which attacks the carbon and nitrogen bond amide compounds (eg: urea) with liberation of ammonia as shown below: H 2 N H 2 N Urease C = 0 + H 2 O 2NH 3 + CO 2 Ammonia Urea Hydrolysis Urease test is performed by growing the test organisms on urea broth or agar medium containing the ph indicator phenol red (ph 6.8). During incubation, microorganisms possessing urease will produce ammonia that raises the ph of the medium/broth. As the ph becomes higher, the phenol red changes from a yellow colour (ph 6.8) to a red or deep pink colour. Failure of the development of a deep pink colour due to no ammonia production is evidence of a lack of urease production by the micro organisms. Results: Development of a deep pink colour due to ammonia production was observed. Urease production was observed by the selected isolate. 97

5. Hydrogen sulfide production test: Hydrogen sulfide (H 2 S) commonly called rotten egg gas because of the copious amounts liberated, when eggs decompose, by certain bacteria such as Proteus vulgaris through reduction (hydrogenation of sulphur containing amino acids (cystine, cysteine and methionine) or through the reduction of inorganic sulphur compounds such as thisulfates (S 2 O 2-3 ), sulfates (SO 2-4 ) or sulfites (SO 2 3 ). The hydrogen sulfide production can be detected by incorporating a heavy metal salt containing (Fe 2+ ) or (Pb 2- ) ion as H 2 S indicator to a nutrient culture medium containing cysteine and sodium thiosulfate as the sulfate substrates. The inoculated peptone-yeast extract-iron agar slants were incubated for 7 days. Hydrogen sulfide colourless gas when produced reacts with the metal salt (ferrous sulfide) forming visible insoluble black ferrous sulfide percipitate). Observations: The inoculated tubes were examined for the presence or absence of black colouration and they were compared with uninoculated controls. Results: No Colour change in the medium was observed. So the selected isolate gave negative test for hydrogen sulphide production test. 98

6. Starch hydrolysis (amylase production test): Amylase is an exoenzyme that hydrolyses (cleaves) starch, a polysaccharide (a molecule which consists of eight or more monosaccharide molecules) into maltose, a disaccharide (double sugars, i.e. composed of two monosaccharide molecules) and some monosaccharides such as glucose. These disaccharides and monosaccharides enter into the cytoplasm of the bacterial cell through the semipermeable membrane and there by used by the endoenzymes. Starch is a complex carbohydrate (polysaccharide) composed of two Constituents-amylase, a straight chain polymer of 200-300 glucose units, and amylopectin, a large branched polymer with phosphate groups. Amylase production is known in some bacteria while well-known in fungi. Amylases commercially produced from various aspergilli. They are used in the initial steps in several food fermentations processes to convert starch to fermentable sugars. They are also used to partially predigest the foods for young children, to clarify fruit juices and in the manufacture of corn and chocolate scruples. The ability to degrade starch is used as a criterion for the determination of amylase production by a microbe. In the laboratory, it is tested by performing the starch test to determine the absence or presence of starch in the medium by using iodine solution as an indicator. Starch in the presence of iodine produces a dark blue colouration of the medium and a yellow zone around a colony in a blue medium indicates amylolytic activity. Results: On addition of iodine solution, it has not produced as dark blue colouration of the medium. So the selected isolate gave negative test for starch hydrolysis. 99

7. Casein hydrolysis test: Casein is the major protein found in milk. It is a macromolecule composed of aminoacids linked together by peptide bonds, CO-NH. Some microorganisms have the ability to degrade the protein casein by producing proteolytic enzyme, called proteinase (caseinase) which breaks, the peptide bond CO-NH by introducing water into the molecule, liberating smaller chains of amino acids called peptides, which are later broken down into free amino acids by extracellular or intracellular peptidases which are transported through the cell membrane into the intracellular amino acid pool for use in the synthesis of structural and functional cellular proteins. Casein hydrolysis can be demonstrated by supplementing nutrient agar medium with milk. The medium is opaque due to the casein in colloidal suspension. Formation of a clear zone adjacent to the bacterial growth, after inoculation and incubation of agar plate cultures, is an evidence of casein hydrolysis. Results: No Clear zone is formed around the growth zone. So the selected isolate gave negative test for casein hydrolysis. 100

8. Gelatin hydrolysis test: Proteins are organic molecules composed of aminoacids, in other words proteins contain carbon, hydrogen, oxygen and nitrogen, though some proteins contain sulphur too. Amionoacids are linked together by peptide bonds to form a small chain (a peptide) or a large molecule (polypeptide) of protein. Gelatin is a protein produced by hydrolysis of collagen, a major component of connective tissue and tendons in humans and other animals. It dissolves in warm water (gels) when cooled below 25 0 C. Large protein molecules are hydrolyzed by exoenzymes and the smaller products of hydrolysis are transported into the cell. Hydrolysis (liquefaction) of gelatin in the laboratory can be demonstrated by growing microorganisms in nutrient gelatin. Once the degradation of gelatin occurs in the medium by an exoenzymes, it can be detected by observing liquefaction, (i.e. even very low temperature 4 o C will not restore the the gel characteristic) or testing with a protein-precipitating material (i.e. flooding the gelatin agar medium with the mercuric chloride solution and observing the plates for clearing around the line of growth. The refrigerated isolate inoculated gelatin tubes were examined to see whether the medium is solid or liquid and the flooded plates for any clearing, around the line of growth. Results: No liquefaction of gelatin by the isolate was observed. The selected isolate gave negative test for gelatin hydrolysis. 101

9. Citrate utilization test: Citrate test is used to differentiate among enteric bacteria on the basis of their ability to utilize citrate as the sole carbon source. The utilization of citrate depends on the presence of an enzyme citrase produced by the organism that breaks down the citrate to oxaloacetic acid and acetic acid. These products are later converted to pyruvic acid and carbondioxide enzymatically as shown below. Citric acid Citrase Oxaloacetic acid + acetic acid enzymes Pyruvic acid + CO 2 The citrate test in performed by inoculating the microorganisms into an organic synthetic medium, Simmon s Citrate agar, where sodium citrate is the only source of carbon and energy. Bromothymol blue is used as an indicator. When the citric acid is metabolized, the CO 2 generated combines with sodium and water to form sodium carbonate an alkaline product, which changes the colour of the indicator from green to blue and this constitutes a positive test. CO 2 + 2Na + + H 2 O Na 2 CO 3 (produced during citric acid Alkaline ph metabolism) (blue Colour) Bromothymol blue is green when acidic (ph 6.8 and below) and blue when alkaline (ph 7.6 and higher). Results: In Simmon s citrate agar slants which were inoculated with the isolate, if was observed that there is no change in the colour of the medium. It was confirmed that the isolate was a citrate-negative where the medium remains green. 102

IMVIC TESTS The IMVIC tests consist of four different tests: (i) Indole production (ii) Methyl-red (iii) Voges-Proskauer and (iv) Citrate Utilization. The IMVIC Tests were designed to differentiate Gram negative intestinal bacilli (Family Enterobacteriaceae) particularly Escherichia Coli and Enterobacter-Klebsiella group, on the basis of their biochemical properties and enzymatic reactions in the presence of specific substrates. 10 (a) Indole production test Tryptophan, an essential amino acid, is oxidized by some bacteria by the enzyme tryptophanase, resulting in the formation of indole, pyruvic acid and ammonia. The indole test is performed by inoculating a bacterium into tryptone broth, the indole produced during the reaction is detected by adding Kovac s reagent (dimethyl aminobenzaldehyde) which produces a cherry-red reagent layer as illustrated. Tryptophan Tryptophanase Indole + Pyruvic acid + NH 3 Indole + Kovac s Reagent HCl Butanol Rosindole + H 2 O (Cherry-red compound) Results: Tryptone broth was inoculated with the isolate and one tube was kept as an uninoculated comparative control. After 48 h of incubation, there was no development of a cherry (deep) red colour in the top layer of the tube. It was confirmed that the isolate was indole-negative due to absence of red colouration. 103

10 (b) Methyl-red and Voges-Proskauer tests: The Methyl-red (MR) and the Voges-Proskauer (VP) tests are used to differentiate two major types of facultatively anaerobic enteric bacteria that produce large amounts of acid and those that produce the neutral product acetoin as end product. Both these are performed simultaneously because they are physiologically related and are performed on the same medium MR-VP broth. Opposite results are usually obtained for the Methyl-Red and Voges-Proskauer tests, i.e. MR+, VP- OR MR-, VP+. In these tests, if an organism produces large amounts of organic acids: formic acid, acetic acid, lactic and succinic acid (end products) from glucose, the medium will remain red (a positive test) after the addition of methyl red a ph indicator (i.e. ph remaining below 4.4). In other organisms, methyl red will turn yellow (a negative test) due to elevation of ph above 6.0 because of the enzymatic conversion of the organic acids (produced during the glucose fermentation) to non-acidic end products such as ethanol and acetoin (acetyl methyl carbinol). MR-VP broth tubes were inoculated with the isolate. After 48 h of incubation, to one tube 5 drops of methyl red indicator was added, to the second tube 12 drops of V-P reagent I and 2-3 drops of V-P reagent II were added and one tube was kept as uninoculated comparative control. Results: In the MR test, in the isolate inoculated broth, the colour of methyl red turned to yellow, is a negative test. In the VP test, in the isolate inoculated broth, there is no change in colouration, is a negative test. 104

11. Ability to produce antibiotic factor: The antibiotic producing capacity of the selected isolate was studied again for confirmation by submerged fermentation technique in PM 2 medium. The antimicrobial spectra of the selected isolate were checked again by agar well diffusion method. Table 25: Antimicrobial spectrum of promising isolate by submerged fermentation in selected medium Antimicrobial activity (Diameter of Microorganism zone of Inhibition in mm) 20 µl 30µl 40µl 50µl Bacillus megaterium 11 13 15 17 Bacillus subtilis 30 32 33 35 Staphylococcus aureus 21 25 26 28 Klebsiella pneumoniae 32 33 34 35 Pseudomonas fluorescens 24 24 25 28 Proteus vulgaris 31 34 35 37 Escherichia coli 10 10 11 12 105

Table 26: Antimicrobial spectrum of promising isolate by submerged fermentation in PM 2 medium Antimicrobial activity (Diameter of Microorganism zone of Inhibition in mm) 20 µl 30µl 40µl 50µl Bacillus megaterium 23 27 27 27 Bacillus subtilis 24 26 28 29 Staphylococcus aureus 23 26 27 28 Klebsiella pneumoniae 26 26 24 24 Pseudomonas fluorescens 26 31 33 33 Proteus vulgaris 25 26 28 28 Escherichia coli 21 21 24 26 12. Sodium chloride tolerance test: This test was carried out on Bennett s agar medium. Fifty ml of sterile molten Bennett s agar medium each contained different concentrations of sodium chloride (2%, 5%, 7% and 10.5%) was cooled to 40-45 o C and poured into sterile petriplates (6 dia) and allowed to solidify. The isolate was streaked on the agar medium incubated at 28 o C for 7 days and the presence or absence of growth was noted. Note: Results of this test were presented in Table 28. 13. Growth temperature range: The starch-casein agar medium slants were inoculated with the selected isolate and incubated at 12 o C, 25 o C, 28 o C, 37 o C, 42 o C and 50 o C. The extent of growth was recorded on 4 th day and 8 th days. Note: Results of this test were presented in Table 28. 106

14. Growth ph range: The starch-casein agar medium slants which were previously adjusted to various ph range starting from ph 5.2, ph 8.0, ph 9.0 and ph 10.5 and they were incubated at 28 o C for 7 days and the presence or absence of growth was noted. Note: Results of this test were presented in Table 28. 15. Oxidase activity of the selected isolate: To differentiate certain group of bacteria, oxidase activity is one of the tests. Certain bacteria are oxidase negative as found in most of the numbers of family. Enterobacteriaceae, while pseudomonas shows oxidase positive. To perform the test, a dye dimethyl-p-phenylene diamine hydrochloride is used. It donates the electron to cytochrome C, becomes oxidised, and produces a colour. Procedure: The selected isolate was inoculated on trypticase soyagar plate and growth was observed on 7 th day. Few drops of oxidase reagent dimethyl-pphenyline diamine hydrochloride were placed on the colonies, in such a way, it should cover the growth. Results: The selected isolate has produced oxidase as indicated by oxidation of reagent to a deep pink colour. The colonies first became pink, then changed to dark red and finally turned to black, it indicates that the selected isolate was oxidase positive. 107

Morphology: The aerial mycelium of isolate C 9 and Psuedonocardia compacta were white and appeared powdery they are characterized by acropetal budding of substrate and aerial mycelium leading to segmented hyphae. The colonies are composed of aerial hyphae which bear mostly apical and sometimes apical swellings or spores. The substrate mycelium was yellowish brown for both the cultures. Based on the biochemical and physiological characteristics, our isolate was identified as Psuedonocardia species and designated as RC 1714 and deposited with MTCC Acession number MTCC 10601. Our isolate was compared with the data based on cultural characteristics biochemical and physiological properties of reported species of Psuedonocardia which revealed that our isolate was similar in some aspects with Psuedonocardia compacta. Hence a detailed comparison of the morphological, physiological and biochemical properties of our isolate C 9 and Psuedonocardia compacta was carried out. Our isolate C 9 and P. compacta were similar with respect to the following biochemical properties. They were both negative for H 2 S, gelatin, starch and casein hydrolysis, NaCl tolerance and positive in ability to reduce nitrate. Both the cultures did not utilize arabinose, meso inositol, rhamnose and xylose. Our isolate C 9 and P. compacta were different in the following aspects. 108

Table 27: Similarities and differences between our isolate C 9 and P. compacta Biochemical and physiological properties isolate C 9 P.compacta H 2 S production - - Gelatin hydrolysis - - Starch hydrolysis - - Casein hydrolysis - - Nitrate reduction + + Sodium chloride tolerance at 7% - - Acid production from L(+)arabinose - - Meso- inositol - - Rhamnose - - D(+)xylose - - D- fructose + + D-glucose + + Sucrose + - Galactose + - Salicin + - D- mannitol - + Urease production + - Isolate C 9 utilized glucose fructose, sucrose, galactose, and salicin, while P. compacta utilized glucose and fructose only, mannitol was utilized by P. compacta while our isolate did not. The two cultures were similar to each other in morphology and most of the physiological and biochemical properties. However, some differences were observed in the utilization of sucrose, galactose, mannitol and salicin. In view of dissimilarities observed in the utilization of the above compounds, we proposed to assign our isolate C 9 as a new variant of P. compacta and designated it as P. compacta var.tirumala. 109

IDENTIFICATION AND CHARACTERIZATION OF THE SELECTED ISOLATE Identification and characterization of micro-organisms play a key role as it expands the scope for industrially important products. In the present investigation, a criteria laid down by the International streptomycetes project were followed for the identification and characterization of the selected isolate. To establish the novelty or otherwise of the present isolate with the reported organisms in the literature, the various morphological, cultural and biochemical characteristics of the isolate were compared with the description of the numerous Pseudonocardia sp and other species cited in the literature. The literature survey includes. Bergey s Manual of Determinative Bacteriology (Buchanan and Gibbons, 1974), Bergey s Manual of systematic Bacteriology (Williams, 1992-93), the Actinomycetes Vol II by Waksman 1961 (Waksman, 1961), Reports of the international streptomyces project (ISP) (Shirling and Gottlieb, 1968; 1969; 1972) and the information collected from the following journals: Journal of General Microbiology, Journal of Bacteriology, the journal of antibiotics, (Japan), International journal of systematic Bacteriology, Hindustan antibiotics Bulletin, Indian Journal of microbiology, journal of biotechnology, and bioengineering, biological and microbiological abstracts and chemical abstracts. Cell wall composition: The cell wall composition and whole cell sugar pattern of the isolates were determined using the following procedure: 110

Procedure: All the strains were grown in 25 ml of yeast extract malt extract medium for about 48h on a rotary shaker at 28 o C. After sufficient growth has been obtained, this inoculum was transferred into 75ml of the same media contained in 500 ml conical flasks. These flasks were kept on a rotary shaker at 28 o C for about 5 days. The cells were harvested at maximum growth. The cells were collected by centrifugation and washed three times with distilled water. These cells were then analyzed for the composition of amino acids and sugars. The cells were separated into the screw capped tubes. One tube was used to analyze amino acids and the other tube for sugars. Amino acids: About 10mg of cells were hydrolyzed for 2h with 1ml of 6N HCl in a closed screw capped tube held at 100 o C. After cooling, contents were filtered through a filter paper. The solid material on the paper was washed with 3 drops of distilled water. The liquid hydrolysate was dried three consecutive times on a steam bath in watch glass to remove the HCl. The residue was taken up in 0.3ml of distilled water for chromatography. Sugars: 10mg of cells were hydrolyzed for 2 hrs with 1ml of 2N HCl in a closed screw capped tube held at 100 o C and the rest of the procedure is same as for amino acids. 111

Chromatography: Amount of about 5-10 l of each sample was spotted on Wahatman No.1 Chromatography paper. The solvent system used for amino acids was butanol: acetic acid : water (4:1:5) and descending chromatography technique was used. The solvent system used for carbohydrates was butanol: pyridine: water: toluene (5:3:3:4) and descending chromatography technique was used. Developing reagents: For amino acids: 0.24% W/V Ninhydrin in 95% V/V Ethyl alcohol, were sprayed, the after spraying the paper was dried at 65 o C for 25 min and spots were recorded. For sugars: 0.1 ml of Aniline in 100 ml of 0.1 N oxalic acids was used. After spraying the paper was dried at 65 o C for 2-5 min and spots were recorded. Result: Cell wall composition analysis revealed that our isolate possessed type IV cell wall, which was inferred by the presence meso-dap, arabinose and galactose as characteristic sugars. The isolation of various antibiotics from actinomycetes species in the middle of this century stimulated extensive screening for antimicrobial compounds and every new antibiotic was produced by a new species. To establish the novelty the following literature was referred. Bergey s Manual of Determinative Bacteriology (Buchanan et al., 1974), Bergey s Manual of Systematic Bacteriology (Williams et al., 1992-93) and all other relevant journals. 112

Identification of the Selected Isolate: Micromorphology: The aerial mycelium developed moderately on most of the media. The aerial hyphae were short and extensively branched. The sporophores appeared straight. The spore chains araised by successive acropetal formation designated as Pseudonocardia type (Henssen and Schnepf, 1967). The spore chains arised terminally or laterally. The colonies were found to be white in colour and the aerial mycelium was powdery forming a thick cover. Our strain grows slowly on a variety of media and the aerial mycelium appears after 7-10 days of growth.good growth was observed in starch casein agar and glycerol aspargine agar media. Moderate growth was observed on yeast extract malt extract agar and belongs to the rectus-flexible (RF) type. Figure 10: Scanning electron microscopy of pseudonocordia species The data on cultural characteristics, physiological and biochemical properties and antimicrobial spectrum, carbon utilization pattern are given in Tables 28,29&30. 113

Table 28: Physiological and biochemical properties: S.No Reaction Response Result 1. Nitrate reduction Colour changes from brown to Positive orange 2. Catalase production Production of free oxygen gas Positive bubbles 3. H 2 S production No Colour change in the Negative medium 4. Urease production Colour changes from yellow to Positive deep pink 5. Starch hydrolysis Colour of the medium does not Negative changes to dark blue 6. Casein hydrolysis No clear zone is formed around Negative the growth zone 7. Gelatin hydrolysis No liquifaction of gelatin Negative 8. Milk coagulation and No Coagulation and no Negative peptonization peptonization 9. Growth temp. range a) at 12 o C b) at 25 o C c) at 28 o C d) at 37 o C e) at 42 o C f) at 50 o C - + +++ ++ + - Growth between 28 0-37 o C 10. Growth ph range a) at ph 5.2 b) at ph 8.0 c) at ph 9.0 d) at ph 10.5 11. Tolerance to NaCl Growth on NaCl 2% W/V NaCl 5% W/V NaCl 7% W/V NaCl 10% W/V NaCl 13% W/V + +++ ++ + The following grades were used to indicate the extent of growth throughout the present investigation. + + - - - -: no growth, +: poor growth, ++: moderate growth, +++: good growth. 114

The following Table-29 shows the carbon source utilization pattern. Table 29: Carbon source utilization pattern Utilization Carbon Sources Positive D-glucose (++), D-fructose (+++), sucrose (+++), galactose (++), salicin (++) Negative mannitol (-), raffinose (-), xylose (-), rhamnose (-), meso-inositol (-), L (+) arabinose (-) Table 30: Antimicrobial spectrum of the culture filtrate in selected medium Test organism A) Gram positive bacteria Bacillus megaterium Bacillus subtilis Staphylococcus aureus Diameter of zone of Inhibition in mm 20 µl 30 µl 40 µl 50 µl 11.0 13.0 15.0 17.0 30.0 32.0 33.0 35.0 21.0 25.0 26.0 28.0 B) Gram negative bacteria Pseudomonas fluorescens, 24.0 24.0 25.0 28.0 Klebsiella pnemoniae, 32.0 33.0 34.0 35.0 Proteus vulgaris 31.0 34.0 35.0 37.0 Escherichia coli. 10.0 10.0 11.0 12.0 Result: The culture filtrate exhibited significant broad spectrum antibacterial activity 115

Figure 11: Antimicrobial spectrum of promising isolate C 9 by submerged fermentation in selected medium 116

Discussion: The most significant characteristics of our isolate are summarized below: The strain grew well on starch casein agar medium, glycerol aspargine agar medium and moderate growth was observed on yeast extract-malt extract agar and oat meal agar. The sporophores occurred as cylindrical rods, they are arranged in chains. The aerial mycelium developed moderately to good on most of the media. It was white in colour. The strain was catalase, oxidase and urease positive. It didnot hydrolyze starch, casein and gelatin. It did not coagulate and peptonize milk. It showed strong nitrate reduction. It was negative for IMViC tests and H 2 S production. It exhibited good growth at 28 o C, no growth at 12 o C and 50 o C and showed poor growth at 37 o C. No growth was observed on meso-inositol, L (+) arabinose, D-mannitol, D-xylose, D-rhamnose and on raffinose and moderate growth on D- fructose, D-glucose, sucrose and on galactose. It exhibited good growth at ph 5.2 and ph 8.0 but moderate growth at ph 9.0 and at ph 10.5. It could tolerate 2% W/V sodium chloride and 5% W/V sodium chloride but failed to grow at 7%, 10% & 13%w/v NaCl. It exhibited excellent antibacterial activity against both Gram positive and Gram negative organisms. A detailed survey of the literature indicated that our strain belongs to the genera Pseudonocardia species. The culture was sent to IMTECH (Institute Of Microbial Technology) for further characterization and identification. Based on the biochemical and physiological characteristics, our isolate was identified as Psuenocardia species and designated as RC1714 and deposited with the MTCC accession number i.e. MTCC10601 117

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