Research Journal of Chemical Sciences ______ __________________________________ ISSN 2231 - 606X Vol. 1(5), 22 - 28 , Aug. (2011) Res.J.Chem.Sci. International Science Congress Association 22 Selection of P igment ( M elanin) production in Streptomyces and their application in P rinting and D yeing of W ool F abrics Ali M. Amal 1 , Keera A. Abeer 1 , Helmy M. Samia 1 , Abd El - Nasser H. Nadia 1 , Ahmed K.A. 2 and El - Hennawi H.M . 2 1 Microbial Chemistry Dep t . , Genetic Engineering and Biotechnology Division, National Research Center, Dokki, Cairo, EGYPT 2 Chemistry of Dyeing, Printing and Auxiliaries Department, Textile Division, National Research Centre, Cairo, EGYPT Available online at: www.isca.in (Received 2 nd June 2011, revised 6 th June 2011, accepted 14 th July 2011) Abstract Four strains among 30 Streptomyces were isolated producing a diffusible different pigment in eight different media. Streptomy ces virginiae was the most producer of a diffusible dark brown pigment on both peptone - yeast extract and tyrosine liquid m edium. Some factors affecting on the pigment production were studied. The optimum pH was observed at 6 with 10 days of the Streptomyces a ge and five days of incubation on a rotary shaker (180 rpm) at 30 â—¦C. The intensity of the pigment was affected by addi tion of carbon, nitrogen and phosphorus sources. Co, Mg and pb SO 4 were increased the intensity of the pigment production. The pigment produced at optimum condition had been used to dye and print the wool fabrics, the color strength values and fastness pro perties of samples were investigated. At the optimum conditions the dyed and printed wool fabrics posses color strength values 12, 10.5 respecti vely and very good fastness properties of washing,, perspiration and light fastness. The antimicrobial activity of the extracted pigment has been investigated. Keywords: Streptomyces , pigment , melanin , optimum , production , application on wool fabrics , antimicrobial potentialities. Introduction Actinomycetes are characterized by the production of various pigments on natural or synthetic media. These pigments are usually described in terms of various shades of blue, violet, red, rose, yellow, green, brown and black. The pigments maybe dissolved in to the medium or it may be retained in the mycelium. Actinomycetes had known to be produced various kinds of antibiotics and moreover these antibiotics include many pigments 1 . Production of pigments by actinomycetes has been utilized as an important cultural characteristic in describing the organisms. Nevertheless, very little is known a bout the exact chemical nature of pigments, because the formation of pigment is influenced by the pH of the medium, aeration, temperature of the growth and carbon and nitrogen sources. Actinomycetes also synthesized and excrete dark pigments melanin or mel anoid, which considered to be useful criterion for taxonomical studies 2 - 4 . These pigments are not sensitive to change in pH 5 . The actinomycetes, designated strain GIMV4.0001 produced a white aerial mycelium and violet - blue diffusible pigment on Gause's sy nthetic agar 6 . Another actinomycete designated as strain GIMN4.002T, produced white aerial mycelium and dark - blue diffusible pigment on Gause's synthetic agar 7 . The textile industry produces and uses approximately 1.3 million tonnes of dyes, pigments and d ye precursors, valued at around $23 billion, almost all of which is manufactured synthetically. However, synthetic dyes have some limitations, primarily, (i) their production process requires hazardous chemicals, creating worker safety concerns, (ii) they may generate hazardous wastes, and (iii) these dyes are not environment friendly 8 . Considering the toxic effects of the synthetic dyes there has been a renewed effort to study and implement the various natural dyes in the dyestuff industry. Primarily th ere are three categories of natural dyes. Firstly those are derived from plants like indigo. Second, the ones that are obtained from animal sources called Cochineal, and those that are got from minerals (Ocher). Natural dyes can provide the much needed alt ernative to the complex world of chemical dyes 9 - 10 . The global interest and demand in application of the fungal pigments such as carotenoids, flavonoids, betalains, quinones and some tetrapyrroles in dyeing of cotton, silk and wool has been reported in se veral studies and increased due to the toxicity problems caused by those of the synthetic origin 11 . The production and evaluation of microbial pigments as textile colorants is currently being investigated 11 . Fungi are more ecological interesting source of pigments, since some fungal species are rich in stable colorants such as anthraquinone 12 - 13 .This research explores methods where natural dyes are produced from fungal species The pigment producedfrom Sclerotinia sp.was applied on cotton shades, which were confirmed using the standard color code index 14 . Melanins are frequently used in medicine, pharmacology and cosmetics preparations 4. However, no reports concerning the production of brown pigment produced by Streptomyces strains is available. Aim of Work: The purpose of this study is to select the Streptomyces strain which is capable of producing brown pigment. This work deals with the fermentation, selection of the brown pigment and cultural condition, nutritional requirements for brown pigme nt production by Streptomyces . The efficiency of brown pigment produced from Streptomyces strain in dyeing and printing of wool fabrics and fastness properties have been investigated. The antibacterial activity of the extracted pigment against bacteria, ye ast and fungi.has been studied Material and Methods Organisms and culture conditions: A total thirty streptomycetes isolated from soil samples and identified by Keera were revived by inoculating 1 ml of heavy spore suspension (7 days old slants) into a 250 ml conical flask containing 50 ml of culture medium (g %: 2, starch; 0.2, KNO 3 ; 0.1, K 2 HPO 4 ; 0.05, NaCl and 0.05, MgSO4.7H 2 O, pH 7.0 adjusted before autoclaving) followed by incubating at 28ºC in a rotary shaker (180 rpm) for 5 days at pH 7.0. The cultures were screened for ability to grow and produce pigment in a defined eight different media such as starch nitrate, glycerol asparagine, dox, fish meal extract, oat meal extract, malt extract, tyrosine and peptone - yeast extract - iron media as mentioned in International Streptomyces Project (ISP) 15 . Streptomyces strain of high pigment producer was identified as S. virginiae 16 which had hooked spore chain, smooth spore surface, dark red grey aerial mycelium, red grey substrate mycelium melanin +ve and anti - fungal antibiotic ( t able 1). Table - 1 Antimicrobial Potentialities of the broth produced by S. virginiae Test Org anisms Zone of inhibition (mm) Aspergillus niger 21 Aspergillus terreus 15 Macrophomina phaseoli 15 Helminthosporium turcicus 26 Fusarium oxysporium 24 Diplodia oryzae 16 Rodotorula minuta 17 Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ __ ISSN 2231 - 606X Vol. 1(5), 22 - 28 , Aug. (2011) Res.J.Chem.Sci. International Science Congress Association 23 Aspergillus flavus , Botrytis allii , Trichoderma viride , Sacchromyces carlsbergensis , Sacchromyces cerevisiae , Candida albicans, Candida Tropicalis and Candida pseudotropicalis gave negative results. Some optimum conditions for pigment produced by the best Streptomyces strain were investigated. The effect of age of microorganism, incubation periods, aeration and initial pH, different concentration of peptone, different carbon sources and heavy metals on the pigment produced by the best strain of Streptomyces were determined. Melanin estimation : Melanin pigment was estimated from peptone - yeast extract iron medium according to Dastager et al . U V/Visible spectrophotometry : The absorption spectra of the dyes were recorded using Perkin Elmer Double Beam Spectrophtometer. The maximum absorption peaks of each dye were then detected in the visible region for determination of lambda max. and for further comparison with other dyes. Dyeing experiment : Lab scale dyeing experiments were carried out on wool fabrics using the produced dyes without mordanting. The dye bath was prepared with the produced dye at a liquor ratio 50: 1 and with 1 g/l amphoteric leveling agent (Albegal B), pH was adjusted to pH 1.0. Dyeing was started at 50°C for ten min., then the dye bath temperature was raised to boil over 30 min and the dyeing continued for 45 min. After dyeing the temperature was lowered to 60 °C, then the dyed samples were rinsed and washed off in an aqueous solution 2 g/l non - ionic detergent (Hospital CV) at 60°C using a liquor ratio 50: 1 for 30 mm, rinsed and dri ed. Printing experiment : Lab scale printing experiments were carried out on wool fabrics using the produced dyes without mordanting. The printing paste 100 gm. consists of 3gm. of sodium alginate as thickener, 3 ml of produced dye and 96 ml of water Color strength : The reflectance values of the dyed fabrics were measured using a Data Color SF 600+. Relative color strengths (K/S values) were determined using the Kubelka - Munk equation 17 . K/S = (1 - R) 2 /2R Where R is the decimal fraction of the ref lectance of dyed fabric, K is the absorption coefficient, and S is the scattering coefficient. Fastness testing : The dyed samples were tested according to ISO standard methods 8 . The specific tests were as follows: ISO 105 - C 02 , color fastness to washing; ISO 105 - E04, color fastness to perspiration, and ISO 105 - B02, color fastness to light (carbon arc) 18 . Antimicrobial Potentiality : The antimicrobial activities were measured as described by 19 . The pigment was precipitate by ammonium sulphate at different concentrations 20%, 25%, 30%, 35% and 65%. The antimicrobial activities of all precipitated were determined against bacteria (gram – ve and +ve), yeast and fungi. Results and Discussion A comparative study was performed between some available cultures of g rey series of Streptomyces to assess their ability to produce melanin pigment on eight different media such as starch nitrate, glycerol asparagines, dox, fish meal extract, oat meal extract, malt extract, tyrosine and peptone - yeast extract - iron liquid med ia. The results are summarized on t able 2. The studied Streptomyces strains could not produce any pigment on starch nitrate, glycerol asparagines, dox and oat meal extract media. The highest level of the pigment formation was detected in culture of Strepto myces virginiae with peptone - yeast extract - iron followed by tyrosine liquid medium. Therefore, S. virginiae was selected for further studies using peptone - yeast - extract - iron liquid medium. The optimum conditions Effect of Streptomyces virginiae age on pig ment formation and color strength value (K/S): The age of Streptomyces virginiae (1 - 10 day's) was detected for pigment formation on peptone - yeast - extract - iron liquid medium. We observed that the fourth day of S. virginiae age was the best day for pigment formation (figure 1). The color strength value (K/S) of wool dyed sample increased by increasing the age of S. virginiae till it reached to value (5.20) at age 4 th day, then slightly decrease during the period from 5 th to 10 th day figure 2, the result represents a good relationship between pigment production expressed as O.D. (Optical Density) and color strength of dyed wool sample. Table 3 illustrates the fastness properties of dyed wool fabrics to washing, perspiratio n (acidic and alkaline) and light. The results indicate that all the dyed samples showed good to very good ratings. The 2 nd day exhibited the best results of fastness properties Table - 2 Screening for melanin pigment produced by different strains of stre ptomycetes on eight different liquid media incubated on a rotary shaker (180 rpm) for five days at 30 °C and pH 7.0 No. of Streptomyces Liquid medium O.D at 480 nm s. virginiae Fish meal extract 0.000 Soy - bean meal extract 0.000 Tyrosine 1.500 Peptone - yeast - extract - iron 2.248 S. X2 Fish meal extract 0.198 Soy - bean meal extract 1.113 Tyrosine 0.150 Peptone - yeast - extract - iron 0.377 S. 19 Fish meal extract 0.243 Soy - bean meal extract 0.000 Tyrosine 1.492 Peptone - yeast - extract - iron 1.863 S. N7 Fish meal extract 2.064 Soy - bean meal extract 0.000 Tyrosine 1.500 Peptone - yeast - extract - iron 0.272 Starch nitrate, glycerol asparagines, dox, oat meal extract liquid media gave negative results. Figure - 1 Effect of S.virginiae age on pigment formation Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ __ ISSN 2231 - 606X Vol. 1(5), 22 - 28 , Aug. (2011) Res.J.Chem.Sci. International Science Congress Association 24 Figure - 2 Effect of S.virginiae age on the color strength Effect of aeration on pigment formation by Streptomyces virginiae and color strength (k/s) : It is clear from results represented in f igure 3 that, maximum O.D of the pigment formation was observed in the volume 50 ml fermented medium of S. virginia / 250ml conical flask, then it decreases by increasing the medium volume. Figure 4 represents that the highest color strength value is detec ted at 50 ml of S. virginiae fermented area/250 ml then it decreases with increasing the medium volume. Results presented in t able 4, illustrate fastness properties of the dyed wool samples (washing, perspiration and light). The samples showed good to very good fastness properties. Wool samples dyed with dye sample obtained at volume 50 ml fermented medium of S. virginiae / 250ml conical flask, exhibited the best fastness properties. These features indicate that there was a good relation between K/S value and fastness properties of dyed wool samples. The results represent that there was a relation between K/S value and pigment pro duction presented as optical density. Effect of initial pH on pigment formation by Streptomyces virginiae: It is evident from results represented in figure 5 that, the optimum pH of pigment formation was detected at pH 6 by S. virginiae . Low pigment formation was observed with acidic pH while, alkaline pH gave moderate pigment formation. Table 5 represents the fastness properties and color strength of dyed wool sample at different pH, it is regarded that the dyed samples showed good to very good ratin g of fastness properties, acidic medium shows low color strength, the highest value of the color strength (k/s) is shown at pH 6 and 7, at higher pH slightly decreasing of K/S is observed (Figure 6). Effect of incubation period and color strength on pigme nt production by Streptomyces virginiae: The time course of pigment formation by Streptomyces virginiae was studied in twelve days. The maximum level of pigment formation was observed at 10 th day of incubation period and then slowly declined (Figure 7). It is noticed from figure 8 that high color strength value (K/S) of dyed wool fabrics is observed at the 10 th day of incubation period, then no remarkable increase in K/S value with 11 th and 12 th days. Table 6 represents that, the results of all the dyed s amples showed good to very good ratings, the period from 6 th to 10th days exhibited the best results of fastness properties. This observation may be attributed to increasing the dye fiber affinity as well as the dye - fiber stability relative to the produced dye at the 10 th day of incubation period. As a fiber, wool is heterogeneous, both chemically and physically. In case of wool without the damage in cuticle cells, it is assumed that the dye uptake in the early stage of adsorption is subject to the dyeing b ehavior of intracellular regions of cuticle - cuticle (surface) layer 17 . Dye most readily enters the fibers by diffusion through the intracellular region between the scale cells of the wool fibers, penetrates fairly rapidly into the non keratinous endocuticle region of the surface layer, which soon reaches equilibrium with the dye in the outer solution. Within the cuticle cells, the endocuticle and then the exocuticle becomes colored, as the dye migrates through the intracellular cement and penetrates into the cells from their undersides 20 . Effect of carbon sources supplemente d with the utilized medium on pigment formation by Streptomyces virginiae and color strength: Supplementation of the peptone - yeast - extract - iron medium with different carbon sources on pigment formation by S. virginiae was studied. The results are summarize d in t able 7. The highest level of pigment formation By S. virginiae was detected with L - arabinose followed by glycerol, fructose, mannose, manitol and glucose. The lowest level of the pigment formation was observed with galactose. The effect of carbon sou rces supplemented with the utilized medium has no remarkable change on the color strength of dyed wool sample all the color strength values lay between 11. - 11.8 Table - 3 Effect of pigment formation by Streptomyces virginiae age on color strength and fastness properties of dyed wool fabrics using produced pigment S. virginiae age 1 2 3 4 5 6 7 8 9 10 Color Yellow brown Yellow brown Brown R. brown R. brown R. brown brown Brown R. brown R. brown λ Max. (nm) 472 473 474 477 476 475 474 475 467 468 K/S Values 3 5.2 4.4 5.0 4.2 4.7 4.9 4.8 4.8 4.9 Wash fastness *Alt 3 3 - 4 3 - 4 4 4 4 4 4 3 - 4 3 - 4 *SC 3 - 4 3 - 4 4 4 4 4 4 4 3 - 4 3 - 4 *SW 3 3 3 - 4 4 - 5 4 - 5 4 4 4 3 - 4 3 - 4 Perspiration fastness Acid Alt 3 3 3 4 - 5 4 4 4 4 4 4 SC 3 3 3 4 - 5 4 3 - 4 4 4 4 4 SW 3 3 - 4 3 - 4 4 - 5 4 - 5 4 3 - 4 4 4 4 Alkali Alt 3 3 4 5 4 - 5 4 - 5 4 4 4 4 SC 3 - 4 3 - 4 4 4 4 3 - 4 4 4 4 4 SW 3 3 - 4 4 4 4 3 - 4 4 4 4 4 Light fastness 3 - 4 4 4 5 4 - 5 4 4 - 5 4 3 - 4 3 - 4 *Alt.=Alternation, *SC= Staining on cotton, *SW=Staining on wool Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ __ ISSN 2231 - 606X Vol. 1(5), 22 - 28 , Aug. (2011) Res.J.Chem.Sci. International Science Congress Association 25 Figure - 3 Figure - 4 Effect of aeration on pigment formation by S. virginiae Effect of S. virginiae aeration on the color strength K/S Table - 4 Effect of aeration on pigment formation by Streptomyces virginiae on the color strength K/S and fastness properties of dyed wool fabrics using produced pigment Table - 5 Effect of initial pH on color strength and fastness properties of dyed wool fabrics using produced pigment by Streptomyces virginia initial pH 1 2 3 4 5 6 7 8 9 10 Color Light brown Light brown yellow Brown Brown Brown Dark brown Dark brown R. brown R. brown R. brown λMax. (nm) 472 473 475 474 477 480 479 479 475 477 K/S Values 6.5 7.1 7.3 7.68 8.9 11.5 10.9 9.6 9.8 10.1 Wash fastness Alt 3 - 4 3 - 4 4 4 3 - 4 4 - 5 4 4 - 5 4 - 5 5 SC 3 3 - 4 3 - 4 4 4 4 4 4 4 4 - 5 SW 3 - 4 3 - 4 3 - 4 4 4 4 4 4 4 - 5 4 - 5 Perspiration Fastness Acid Alt 4 4 4 4 4 4 - 5 4 3 - 4 5 4 SC 4 4 4 4 - 5 4 - 5 4 3 - 4 4 4 - 5 4 - 5 SW 4 4 4 4 - 5 5 4 - 5 4 4 - 5 5 4 Alkali Alt 4 4 4 4 5 4 4 - 5 4 - 4 4 4 SC 4 4 4 4 4 4 - 5 4 4 4 - 5 4 SW 4 4 4 4 4 4 4 4 4 - 5 Light fastness 3 - 4 4 4 4 4 - 5 4 - 5 5 4 - 5 4 5 Streptomyces virginiae aeration volume 25 50 100 125 150 λ Max. (nm) 470 477 475 473 472 K/S Values 6.1 6.5 6.2 6.3 6.1 Wash fastness Alt 3 4 3 - 4 4 4 SC 3 - 4 4 3 - 4 4 4 SW 3 - 4 4 4 4 4 Perspiration Fastness Acid Alt 4 4 - 5 4 3 - 4 4 SC 4 4 - 5 4 - 5 4 4 SW 4 4 - 5 4 - 5 5 5 Alkali Alt 4 - 5 4 4 4 4 SC 4 4 4 - 5 4 - 5 4 SW 4 4 - 5 4 4 - 5 4 Light fastness 4 5 4 - 5 5 5 Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ __ ISSN 2231 - 606X Vol. 1(5), 22 - 28 , Aug. (2011) Res.J.Chem.Sci. International Science Congress Association 26 Effect of different concentration of peptone on pigment formation and color strength by Streptomyces virginiae : Different concentrations of peptone were studied on the pigment formation by Streptomyces virginiae . T he results obtained in f igure 9 show that, the maximum level of the pigment formation was observed with 1.5 g% peptone. Peptone concentration has no any effect on the color strength of dyed wool sample. t able 8 represents the fastness properties and color strength value of dyed wool fabrics at different concentration of peptone the result showed that all samples have good fastness properties the highest value of color strength are exhibited at 1.5 and 1.75 of peptone concentration. Effect of heavy metal io ns on pigment formation by Streptomyces virginiae : Some divalent metals salts were added to the utilized medium after sterilization each in concentration of 0.1 mM. As shown in f igure 10, Co ++ , Hg ++ and Pb ++ gave high concentration of the pigment formation by S. virginiae . Moderate concentration of the pigment formation was observed with Ni ++ and Cu ++ . Mn ++ gave low concentration of the pigment formation. Changing the heavy metal does not make significant eff ect on the color strength of dyed wool sample . Fastness properties of printed wool fabric at optimum conditions : Table 9 illustrates the fastness properties of printed wool fabric using dye produced by Streptomyces virginiae at the optimum conditions. The result indicated that, a good to very good fastness properties rating and the color strength value is 10.5 . Antimicrobial potentialities : The dark brown pigment produced by S. virginiae was precipitated by different concentration of ammonium sulphate from the fermented broth of peptone - yeast - extract - iron liquid medium produced by Streptomyces virginiae . Antimicrobial activities of the dark brown pigment are illustrated in Table 10. It showed activity with fermented br oth against Gram – v and +v bacteria ( Pseudomonase aeurogenosa and Staphylococcus aureuse ) as well as fungi ( Fusarium oxysporium, Botrytis allii , Diplodia oryzae , Aspergillus flavus, and Aspergillus niger ). Pigment precipitated at 20% - 35% concentration of ammonium sulphate gave antimicrobial activity with Aspergillus niger . Antimicrobial activity was observed at 20% ammonium sulphate with Botrytis allii , Diplodia oryzae , Aspergillus flavus . Some differences in our results observed with the data obtained wi th Keera, 2004 (Table 1). The obtained results suggest that antimicrobial substance is broad spectrum . The protective role of melanin against UV Radiation (UVR) : Human skin is repeatedly exposed to UVR that influences the function and survival of many cel l types and is regarded as the main causative factor in the induction of skin cancer. in some parts of the polymer molecule and a gradual loss UV radiation is one of the major causes of degradation of textile materials, which is due to excitations in some parts of the polymer molecule and a gradual loss of integrity, and depends on the nature of the fibers. It has been traditionally believed that skin pigmentation is the most important photo protective factor, as melanin, functioning as a broadband UV absor bent, so it is expected that, the extracted melanin pigment exhibits from good to excellent ultra violet protection factor (UV P) 21 - 22 . Table - 6 Effect of incubation period on color strength and fastness properties of dyed wool fabrics using produced pigment by Streptomyces Virginia initial pH 1 2 3 4 5 6 7 8 9 10 Color Light brown Light brown yellow Brown Brown Brown Dark brown Dark brown R. brown R. brown R. brown λMax. (nm) 472 473 475 474 477 480 479 479 475 477 K/S Values 6.5 7.1 7.3 7.68 8.9 11.5 10.9 9.6 9.8 10.1 Wash fastness Alt 3 - 4 3 - 4 4 4 3 - 4 4 - 5 4 4 - 5 4 - 5 5 SC 3 3 - 4 3 - 4 4 4 4 4 4 4 4 - 5 SW 3 - 4 3 - 4 3 - 4 4 4 4 4 4 4 - 5 4 - 5 Perspiration Fastness Acid Alt 4 4 4 4 4 4 - 5 4 3 - 4 5 4 SC 4 4 4 4 - 5 4 - 5 4 3 - 4 4 4 - 5 4 - 5 SW 4 4 4 4 - 5 5 4 - 5 4 4 - 5 5 4 Alkali Alt 4 4 4 4 5 4 4 - 5 4 - 4 4 4 SC 4 4 4 4 4 4 - 5 4 4 4 - 5 4 SW 4 4 4 4 4 4 4 4 4 - 5 Table - 7 Effect of carbon sources supplemented with peptone - yeast - extract - iron medium pigment formation by Streptomyces virginiae Different carbon Sources O.D at 480 nm Color strength Value Glucose 2.165 11.3 Fructose 2.253 11.6 Cellobiose 1.994 11.4 Manitol 2.168 11.6 L - arabinose 2.379 11.8 Galactose 1.467 11.0 Mannose 2.183 11.4 Lactose 1.761 11.1 Sucrose 1.922 11.3 Glycerol 2.282 11.7 Starch 1.614 11.1 Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ __ ISSN 2231 - 606X Vol. 1(5), 22 - 28 , Aug. (2011) Res.J.Chem.Sci. International Science Congress Association 27 Figure - 7 Figure - 8 Effect of incubation period on pigment formation Effect of incubation period on color strength by S. virginiae by S. virginiae Figure - 9 Figure - 10 Effect of different concentration of peptone on Effect of divalent heavy metals on pigment pigment formation by Streptomyces virginiae formation by Streptomyces virginiae Table - 8 Effect of different concentration of peptone on color strength and fastness properties of dyed wool fabrics using produced pi gment by Streptomyces virginiae Peptone concentration g/l 0.25 0.5 0.75 1 1.25 1.5 1.75 2.0 Color Yellow Brown Yellow brown brown Reddish Brown Reddish brown R eddish brown Brown brown λMax. (nm) 452 473 474 477 476 475 474 475 K/S Values 4.1 5.0 6.1 9.2 9.5 12 11.3 11 Wash fastness *Alt 3 3 - 4 3 - 4 4 4 4 4 4 *SC 3 - 4 3 - 4 4 4 4 4 4 4 *SW 3 3 3 - 4 4 - 5 4 - 5 4 4 4 Perspiration Fastness Acid Alt 3 3 3 4 - 5 4 4 4 4 SC 3 3 3 4 - 5 4 4 4 4 SW 3 3 - 4 3 - 4 4 - 5 4 - 5 4 5 - 4 4 Alkali Alt 3 3 4 5 4 - 5 4 - 5 4 4 SC 3 - 4 3 - 4 4 4 4 4 4 4 SW 3 3 - 4 4 4 4 4 4 4 Light fastness 3 - 4 4 4 5 4 - 5 5 4 - 5 4 Table - 9 Fastness properties of printed wool fabric at optimum conditions Color Dark brown λMax. (nm) 482 K/S Values 10.5 Wash fastness *Alt 4 - 5 *SC 4 - 5 *SW 4 - 5 Perspiration Fastness Acid Alt 4 - 5 SC 5 SW 4 - 5 Alkali Alt 4 SC 4 SW 4 - 5 Light fastness 5 - 6 0 2 4 6 8 10 12 14 16 3 4 5 6 7 8 9 10 11 12 Colour Strength K/S incupation period (days) Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ __ ISSN 2231 - 606X Vol. 1(5), 22 - 28 , Aug. (2011) Res.J.Chem.Sci. International Science Congress Association 28 Table - 10 Antimicrobial spectrum of Streptomyces virginiae pigment precipitated at different concentration of ammonium sulphate Test organism Zone of inhibition (mm) Control broth Concentration of ammonium sulphate 20% 25% 30% 35% 40% Pseudomonase aeurogenosa 25 0 0 0 0 0 Staphylococcus aureuse 20 0 0 0 0 0 Fusarium oxysporium 25 0 0 0 0 0 Botrytis allii 12 17 0 0 0 0 Diplodia oryzae 14 20 0 0 0 0 Aspergillus flavus 14 28 0 0 0 0 Aspergillus niger 16 20 18 18 18 0 Bacillus cereus, Bacillus subtilis, Candida albicans and Sacchromyces cerevisiae gave negative results. Conclusion The above mentioned results indicate that the optimum conditions for production of dark brown dyes which give highest color strength value and very good fastness properties of dyed and printed wool fabrics at using peptone - yeast - extract - iron liquid medium are Streptomyces virginiae , age 2 days, in itial pH 6, incubation period 10 days and peptone concentration 1.5. Pigment precipitated at 20% - 35% concentration of ammonium sulphate gave antimicrobial activity with Aspergillus niger . From the previous results, the dye produced from S. virginiae is very suitable for dyeing and printing wool fabrics as a replacer of synthetic dyes which has big environmental problems. In future the antimicrobial substance will be applied in the medical fabrics. References 1. Miyaura J. and Tatsumi C. Studies on the Antibiotics from Actinomycetes. An Antibiotics Pigment from Streptomyces F - 23b , Bull: Univ. Osaka Pref ., Ser. B . 129 - 137 (1960) 2. Zonova G.M. 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