Folia Mierobiol. 24, 473--477 (1979)
Degradation of Organic Nitrogenous Wastes by a Soil Streptomycete S. DE and A. L. CHANDRA .Department of .Microbiology, Bose Institute, Calcutta 700 009, lndia Received November 24, 1978
ABSTRACT. A soil strep~omyeete degraded hair, silk, wool, feather and leather which were collected from solid wastes. The organism was identified taxonomically and designated Streptomyces sp. A956. It degraded leather to the maximum extent and solubilized 35.9 ~o of the total nitrogen. 2.32 mg of glyeine equivalent amino nitrogen could be obtained by degradation of 100 mg leather.
Although microbial decomposition of silk, hair, feather (Nakanishi and Yamamoto 1974), wool (Noval and Nickerson 1959) and leather (Thompson et al. 1972; Welton and Woods 1973) have been reported, little a t t e m p t has been paid to use these microorganisms in composting various organic protein wastes. During a search for protein-degrading microorganisms from soil, a streptomycete which liquefies gelatin in 1 d was isolated. In the present s t u d y the streptomycete was taxonomically identified and its ability to attack and degrade common nitrogenous waste materials was initially tested with silk, wool, hair, feather and leather. Finally the extent of its leather-degrading properties was determined. MATERIALS AND METHODS
Taxonomic identification of the organism. Most of the criteria accepted by the International Streptomyces Project (Shirling and Gottlieb 1966, 1972) and proposed in other literature (Waksman 1961; Bergey's Manual of Determinative Bacteriology 1974) are used for the taxonomic identification of the organism. Micromorphological characteristics of the streptomycete were studied on slide cultures with yeast e x t r a c t - - m a l t extract, oat meal, inorganic salts--starch and glycerol-asparagine-agar media. Scanning electron microscopic studies were carried out according to the method of Williams and Davies (1967). For cell wall analyses purified cell walls and hydrolyzates were prepared according to Boone and Pine (1968). Two-dimensional chromatography was carried out using W h a t m a n No. 1 paper. The primary solvent used for separating both sugars and amino acids was p h e n o l - - w a t e r (4 : 1). The second solvent for sugar separation was 1-butanol--pyridine--water ( 6 : 4 : 3 ) and for amino acids 1-butanol=-acetic a c i d - - w a t e r (25 : 6 : 25). Migration for each run was at 30 ~ for 6 h. The sugars were detected by dipping the chromatograms in a 50 % solution of AgNO3 in 0.5 KOH. Amino acids were detected by spraying with ninhydrin solution. The presence of diaminopimelic acid (DAP) was confirmed according to Hoare and Work (1957).
4~4
S. DE and A. L. CHANDRA
V,,I. 24
Preparation of inoculum. Streptomyces sp. A956 was growll at 28 ~(', for 5 d (,'a y e a s t e x t r a c t - - m a l t e x t r a c t (Shirling and G o t t l i e b 1966) agar slants a , d the si>c,res were s u s p e n d e d in 10 ml sterile distilled w a t e r a n d used as i n o c u l a m . Cultivation of the org(~nism on nitro qeuo~ts ~,'o~'te.s'. F r a g m e n t s of u;cd ~.,! worn silk a n d wool g a r m e n t s , h u m a n hair and ehieke> f e a t h e r were c,.~l!ected frr ([Q.:.?'ct,,;,tGi(>il. '~:,,
m e d i u m was sterilized at I00 k P a for 15 rain a~:d the s~bst.r~t (.s at 35 kl:'a 17,,' I*~ ~..i . F i f t y m g of t h e substrat, es were add,:d to , O m l of the basal sal-{.s >~c~:.:~.~ lit i~:l. ~>] conical flasks and i n o c u l a t e d with 1 0 7 _ IC s spvres. The mixtur,. ~. x~4.;.+ f2:('t:!:,,f >,I ~,i 28 ~ on g y r a t o r y shakers at 4.4 H z for I0 d a n d ce>trii'uged at a3 )tz t,,?' : b. 'Fhc~ s u p e r n a t a n t s were used for all analyse~ al~d ,.:>zyme a~s,~vs. A~alyses. Amino n i t r o g e n was e s t i m a t e d b y t]~(" nh'~l~5=dri~ (:o?.:":nl(:~'( ~'.-;~.,] ( Y e m m a n d Cocking 1955) a n d expressed as glycine eq~.~iv~,lt-~t,-'., h',,~,l ~i:~:~,.,: i> t h e s n b s t r a t e s and s u p e r n a t a n t s was os~imatcd b 3- the 1 were e x a m i n e d . Effects of inorganic salts ( K e H P O 4 , MgSOa.7H~O, CaCl~, FeSO~.7It,~O a n d Z n S O a . 7 H 2 0 a d d e d i n d i v i d u a l l y / c o m b i n a t i o n s at t h e basal m e d i u m cc)nce>t r a t i o n s ) , c a r b o n sources (glucose, fructose, m a n n i t o l , sucrose, maltose, lactose aml starch), p H (5--8), t e m p e r a t u r e ( 2 5 - - 4 0 ~ a n d i n o c u l u m size ( 4 - - 9 0 000 spores p e r nl) were noted. E s t i m a t i o n s were m a d e b y t h e release of a m i n o n i t r o g e n in the supernatant. B E S U L T S A N D DISCUSSION
The presence of glueosamine, alanine, muramic and glutamie acids in the cell wall of the organism indicates that it is an aerobic aetinomycete (Becker et al. 1965), the presence of L-DAP and glyeine (Lechevalier and Leehevalier 1967) the inability of the vegetative myeelium to be fragmented into bacillary and eoecoid forms and also the presence of eonidia borne on sporophores (Waksman 1961) justify the placement of the organism in the family Streptomycetaceae. Absence of arabinose, galaetose, lysine and DL-DAP and presence of L-DAI? showed the organism could be placed under the genus Streptomyces. Sporophores were formed in loose spirals (Plate 1) and the organism could be placed under the morphological section JRetinaculum apertura of Pridham et al. (1958); for cultural and physiological characteristics
1979
DEGRADATION OF ORGANIC NITROGENOUS WASTES
475
TABLE I. Cultural characteristics of Streptomyees sp. A956 a Medium
Sucrose-- nitrate agar Glucose-- asparagine agar Egg albumin agar Nutrient agar Glycerol-- calcium malate agar Stm'ch agar Glucose yeast, extract agar Emerson's agar Nutrient salt agar Potato ghleose agar Potato plug Carrot plug Yeast e x t r a c t - - m a l t extract agar Oat meal agar Inorganic salts starch agar Glycerol-- asparagine agar
Growth of substrata mycclium good, spreading scanty
Aerial myeclium
Reverse side colony eolour
abundant, pastel grey none
white white
scanty moderate spreading good spreading good spreading good spreading good spreading good spreading eopious copious folded good spreading
scanty, white moderate brownish orange abundant, greyish brov, n moderate, yellowish moderate, yellowish grey abunda~,t, greyish brown ahnndant ~,r,.)wnish grey moderato, pale grey moderate, brownish grey abundant, pearl grey
white white wh,te wlule x~hire ~ hire white pale yellow pals yellow greyish yellow
good spread ing good spreading good spreading
abundant brownish grey abundant reddish grey abundant brownish grey
white white ~ hite
nolle
a No aoluble pigments. Colour determinations according to Methucn Handbook of Colour (1967).
see Tables I and II. Comparative studies of the organism with Streptomyces spp. reported in the literature showed similarities with S. pseudogriseolus (Okami etal. 1955), both the streptomycetes have similar C-utilization patterns and produce no soluble and melanin pigments. But there are differences, Streptomyces sp. A956 has
TABLE II. Physiological characteristics of Streptomyces sp. A956 Test Melanin formation (Waksman 1961; Shirling and Gottlieb 1966) Decomposition of L-tyrosine (Gordon and Smith 1955) Diastatic activity (Lyons and Pridham 1962) Proteolytic activity (Gordon and Mihm 1957) Urease activity (Nitseh and Kutzner 1968) Production of If2S (Kuster and Williams 1964) NaC1 tolerance (Tresner etal. 1968) Utilization of carbon sources (Shirling and Gottlieb 1966) ])-Glucose
Reactiona
b + _ +++ +++ lO% +++
D-Fructose
I,-Arabinose n-Xylose L-Rhamnose D-Mannitol
myo-Inositol Sucrose Rafflnose Cellulose a
+++ § +++ +_L+ q-+ + ++
+ , __ + , + + .: . indicate reaction/utilization in increasing order, -- indicates no reaction/utilization.
476
S. D E a n d A. L. C H A N D I ~ A
Vol. 24
TABLE I I I , D e g r a d a t i o n of organic n i t r o g e n o u s w a s t e s b y Streptomyces sp. A t 5 6
Substrate
Vegetable-tanned calf l e a t h e r Human hair Silk Wool Chicken feather
Solubilized n i t r o g e n % glycine equivalent a
35.94 7.18 3.66 4.28 12.34
20.85 2.82 4.05 2.90 6.03
Proteolytic activity AA2s0b tyrosine units c
0.222 a 0.26 0.686 0.2 0.56
97 19 57 27 55
a Amino nitrogen, mg per g substrate. b D i l u t i o n 1 : 1. e n m o l p e r m l per 30 rain. d D i l u t i o n 1 : 200.
loose spirals, is weakly diastatic and produces no antibiotic. S. pseudogriseolus has closed spirals, is strongly diastatic and produces a xanthomycin-like antibiotic. Since the differences are more pronounced than the similarities the organism is given a separate identity and designated as Streptomyces sp. A956. This streptomycete degraded wool, hair, silk and feather and was remarkable in solubilizing leather proteins by 35.9 ~o (Table III). Initial studies showed that K2HPOa at 1.5 g/1 increased leather degradation over distilled water at pH 8.0. Combinations of the phosphate with CaCle, MgS04.TH20, ZnSO4.7H20 and FeSO4.TH20 at the concentrations suggested by Noval and Nickerson (1959) or doubling the concentrations produced no better results. So for maximum degradation of leather 1.5 g KeHP04 was added to 1 litre distilled water. None of the carbon sources tested could increase the leather-degrading capacity of the organism. The optimum temperature range for leather degradation was found to be 28--32 ~ An inoculum size of 12• 107 spores was needed for degradation of 100 mg leather. Under these optimal conditions Streptomyces sp. A956 solubilized vegetable-tanned calf leather and 2.32 mg glycine equivalent amino nitrogen could be obtained from 100 mg leather in 8 d. Microscopic examination revealed the growth of Streptomyces sp. A956 within the substrates causing changes in external appearance. Silk and wool fibres were swollen, hair was ruptured at the outer layers, feather barbs were shredded into pieces and leather was turned into a gel-like substance. This paper presents evidence that Streptomyces sp. A956 is capable of attacking various organic nitrogenous waste materials that accumulate in city garbage. Of special interest is its leather solubilizing property which could be used for degrading waste leather into simpler nitrogenous products of agricultural use. REFERENCES
ANSON M. L. : T h e e s t i m a t i o n of p e p s i n , t r y p s i n , p a p a i n a n d c a t h e p s i n w i t h h a e m o g l o b i n . J.Gen.Microbiol. 22, 79 (1938). B~CKER B., LECHEVAL~ER M. P., LECm~V~_~IER H . A.: C h e m i c a l c o m p o s i t i o n of cell wall p r e p a r a t i o n s f r o m cells o f v a r i o u s f o r m g e n e r a o f aerobic a c t i n o m y c e t e s . Appl.Microbiol. 13, 236 (1965). Bergey's Manual of Determinative Bacteriology (Sth ed.). W i l l i a m s & Wilkins, B a l t i m o r e 1974. B o o ~ . C. J . , PINE L.: R a p i d m e t h o d for c h a r a c t e r i z a t i o n o f a e t i n o m y c e t e s b y cell wall c o m p o s i t i o n . AppL Microbiol. 16, 279 (1968).
1979
DEGRADATION OF ORGANIC N I T R O G E N O U S WASTES
4~
GORDOH R. E., SMITH M. M. : Proposed group of characters for the separation of Streptomyces and Nocardia. J.Bacteriol. 69, 147 (1955). GORDO.-~ R. E., MIgM J. M.: A ,comparative study of some strains received as nocardiae. J.Bacteriol. 73, 15 (1957). 1-Io.~E D. S., WORK E.: The stereoisomers of ~e-diaminopimelic acid. 2. Their distribution in the bacterial order Actinomycetales and in certain Eubacteriales. Biochem.J. 65, 441 (1957).' KORNERtr~ A., W~tNSCgER J. H.: Methuen Handbook of Colour (2nd ed.). Methuem & Co., London 1967. Ki2STER E., WILLIAMS S. T.: Production of hydrogen sulphide by streptomycetes and methods for its detection. Appl.Microbiol. 12, 46 (1964). LECgSVALIER H. A., LECHEV~IER M. P.: Biology of Actinomycetes. Ann.~ev.Mierobiol. 21, 71 (1967). LYoNs A. J., PRIDtIAM T. G.: Proposal to designate strain ATCC 3004 (IMRU 3004) as the neotype strain of Streptomyces albus (Rossi-Doria). J.Bacteriol. 83, 370 (1962). NAKANISHI T., YAMAMOTO T.: Action and specificity of a Streptomyces alkalophilie proteinase. Agr. Biol. Chem. 38, 2391 (1974). NITSCH B., KUTZ:NEEH. J.: Ureaseaktivit~t bei Streptomyeeten. Z.Naturforsch. 23b, 565 (1968). NovAL J. J., NICKE~SON W. J.: Decomposition of native keratin by Streptomyces fradiae. J.Bacteriol. 77, 251 (1959). OKAr~I Y., UT)~ARX R., OYAGI H., I~AKA~URAS., U~EZAWA H.: The screening of antitoxoplasmic substance produced b y streptomycete and antitoxoplasmie substance No. 534. J.Antibiot. (Japan) 8A, 126 (1955). PRIDHAM T. G., I-~ESSELTINE C. W., BENEDICT R. G..* X guide for the classification of streptomyeetes according to selected groups. Placement of strains in morphological sections. Appl.Microbiol. 6, 52 (1958). SB~RLING E. ]3., GOTTr,IEB D. : Methods for characterization of Streptomyces species. Intern.J.Syst.BacterioL 16, 366 (1966). SHIR~I~O E. B., GOTTLIEB D.: Cooperative description of type strains of Streptomyees. Intern.J.Syst.Bacteriol. 22, 265 (1972). T~OMPSON J. A., WOODS D. R., WELTON R. L.: Collagenolytic activity of aerobic halophiles from hides. J.Gen.Microbiol. 70, 315 (1972). TRES~ER H. D., HAYES J. A., BACKUS E. J.: Differential tolerance of streptomycetes to sodium chloride as a taxonomic aid. Appl.Microbiol. 16, 1134 (1968). VOG~.L A. J. : A textbook of quantitative inorganic analysis including elementary instrumental analysis. Longmans Green & Co. 1961. W.~KSMAN S. A.: The Actinomycetes, Vol. II. Baillier, Tindall & Cox, London 1961. WELTON R. L., WOODS D. R. : Holotolerant eollagenolytic activity of Achromobacter iophagus. J.Gen.Mierobiol. 75, 191 (1973). WILLIAMS S. T., DAVIES F. L. : Use of a scanning electron microscope for the examination of Aetinomycetes. J.Gen.Mierobiol. 48, 171 (1967). YE~r~ E. M., C o c K n ~ E. C.: Determination of amino acids b y ninhydrin. Analyst 80, 209 (1955). The plate will be found at the end of the issue.
Degradation of Organic Nitrogenous Wastes by a Soil Streptomycete S. DE and A. L. CHANDRA .Department of .Microbiology, Bose Institute, Calcutta 700 009, lndia Received November 24, 1978
ABSTRACT. A soil strep~omyeete degraded hair, silk, wool, feather and leather which were collected from solid wastes. The organism was identified taxonomically and designated Streptomyces sp. A956. It degraded leather to the maximum extent and solubilized 35.9 ~o of the total nitrogen. 2.32 mg of glyeine equivalent amino nitrogen could be obtained by degradation of 100 mg leather.
Although microbial decomposition of silk, hair, feather (Nakanishi and Yamamoto 1974), wool (Noval and Nickerson 1959) and leather (Thompson et al. 1972; Welton and Woods 1973) have been reported, little a t t e m p t has been paid to use these microorganisms in composting various organic protein wastes. During a search for protein-degrading microorganisms from soil, a streptomycete which liquefies gelatin in 1 d was isolated. In the present s t u d y the streptomycete was taxonomically identified and its ability to attack and degrade common nitrogenous waste materials was initially tested with silk, wool, hair, feather and leather. Finally the extent of its leather-degrading properties was determined. MATERIALS AND METHODS
Taxonomic identification of the organism. Most of the criteria accepted by the International Streptomyces Project (Shirling and Gottlieb 1966, 1972) and proposed in other literature (Waksman 1961; Bergey's Manual of Determinative Bacteriology 1974) are used for the taxonomic identification of the organism. Micromorphological characteristics of the streptomycete were studied on slide cultures with yeast e x t r a c t - - m a l t extract, oat meal, inorganic salts--starch and glycerol-asparagine-agar media. Scanning electron microscopic studies were carried out according to the method of Williams and Davies (1967). For cell wall analyses purified cell walls and hydrolyzates were prepared according to Boone and Pine (1968). Two-dimensional chromatography was carried out using W h a t m a n No. 1 paper. The primary solvent used for separating both sugars and amino acids was p h e n o l - - w a t e r (4 : 1). The second solvent for sugar separation was 1-butanol--pyridine--water ( 6 : 4 : 3 ) and for amino acids 1-butanol=-acetic a c i d - - w a t e r (25 : 6 : 25). Migration for each run was at 30 ~ for 6 h. The sugars were detected by dipping the chromatograms in a 50 % solution of AgNO3 in 0.5 KOH. Amino acids were detected by spraying with ninhydrin solution. The presence of diaminopimelic acid (DAP) was confirmed according to Hoare and Work (1957).
4~4
S. DE and A. L. CHANDRA
V,,I. 24
Preparation of inoculum. Streptomyces sp. A956 was growll at 28 ~(', for 5 d (,'a y e a s t e x t r a c t - - m a l t e x t r a c t (Shirling and G o t t l i e b 1966) agar slants a , d the si>c,res were s u s p e n d e d in 10 ml sterile distilled w a t e r a n d used as i n o c u l a m . Cultivation of the org(~nism on nitro qeuo~ts ~,'o~'te.s'. F r a g m e n t s of u;cd ~.,! worn silk a n d wool g a r m e n t s , h u m a n hair and ehieke> f e a t h e r were c,.~l!ected frr ([Q.:.?'ct,,;,tGi(>il. '~:,,
m e d i u m was sterilized at I00 k P a for 15 rain a~:d the s~bst.r~t (.s at 35 kl:'a 17,,' I*~ ~..i . F i f t y m g of t h e substrat, es were add,:d to , O m l of the basal sal-{.s >~c~:.:~.~ lit i~:l. ~>] conical flasks and i n o c u l a t e d with 1 0 7 _ IC s spvres. The mixtur,. ~. x~4.;.+ f2:('t:!:,,f >,I ~,i 28 ~ on g y r a t o r y shakers at 4.4 H z for I0 d a n d ce>trii'uged at a3 )tz t,,?' : b. 'Fhc~ s u p e r n a t a n t s were used for all analyse~ al~d ,.:>zyme a~s,~vs. A~alyses. Amino n i t r o g e n was e s t i m a t e d b y t]~(" nh'~l~5=dri~ (:o?.:":nl(:~'( ~'.-;~.,] ( Y e m m a n d Cocking 1955) a n d expressed as glycine eq~.~iv~,lt-~t,-'., h',,~,l ~i:~:~,.,: i> t h e s n b s t r a t e s and s u p e r n a t a n t s was os~imatcd b 3- the 1 were e x a m i n e d . Effects of inorganic salts ( K e H P O 4 , MgSOa.7H~O, CaCl~, FeSO~.7It,~O a n d Z n S O a . 7 H 2 0 a d d e d i n d i v i d u a l l y / c o m b i n a t i o n s at t h e basal m e d i u m cc)nce>t r a t i o n s ) , c a r b o n sources (glucose, fructose, m a n n i t o l , sucrose, maltose, lactose aml starch), p H (5--8), t e m p e r a t u r e ( 2 5 - - 4 0 ~ a n d i n o c u l u m size ( 4 - - 9 0 000 spores p e r nl) were noted. E s t i m a t i o n s were m a d e b y t h e release of a m i n o n i t r o g e n in the supernatant. B E S U L T S A N D DISCUSSION
The presence of glueosamine, alanine, muramic and glutamie acids in the cell wall of the organism indicates that it is an aerobic aetinomycete (Becker et al. 1965), the presence of L-DAP and glyeine (Lechevalier and Leehevalier 1967) the inability of the vegetative myeelium to be fragmented into bacillary and eoecoid forms and also the presence of eonidia borne on sporophores (Waksman 1961) justify the placement of the organism in the family Streptomycetaceae. Absence of arabinose, galaetose, lysine and DL-DAP and presence of L-DAI? showed the organism could be placed under the genus Streptomyces. Sporophores were formed in loose spirals (Plate 1) and the organism could be placed under the morphological section JRetinaculum apertura of Pridham et al. (1958); for cultural and physiological characteristics
1979
DEGRADATION OF ORGANIC NITROGENOUS WASTES
475
TABLE I. Cultural characteristics of Streptomyees sp. A956 a Medium
Sucrose-- nitrate agar Glucose-- asparagine agar Egg albumin agar Nutrient agar Glycerol-- calcium malate agar Stm'ch agar Glucose yeast, extract agar Emerson's agar Nutrient salt agar Potato ghleose agar Potato plug Carrot plug Yeast e x t r a c t - - m a l t extract agar Oat meal agar Inorganic salts starch agar Glycerol-- asparagine agar
Growth of substrata mycclium good, spreading scanty
Aerial myeclium
Reverse side colony eolour
abundant, pastel grey none
white white
scanty moderate spreading good spreading good spreading good spreading good spreading good spreading eopious copious folded good spreading
scanty, white moderate brownish orange abundant, greyish brov, n moderate, yellowish moderate, yellowish grey abunda~,t, greyish brown ahnndant ~,r,.)wnish grey moderato, pale grey moderate, brownish grey abundant, pearl grey
white white wh,te wlule x~hire ~ hire white pale yellow pals yellow greyish yellow
good spread ing good spreading good spreading
abundant brownish grey abundant reddish grey abundant brownish grey
white white ~ hite
nolle
a No aoluble pigments. Colour determinations according to Methucn Handbook of Colour (1967).
see Tables I and II. Comparative studies of the organism with Streptomyces spp. reported in the literature showed similarities with S. pseudogriseolus (Okami etal. 1955), both the streptomycetes have similar C-utilization patterns and produce no soluble and melanin pigments. But there are differences, Streptomyces sp. A956 has
TABLE II. Physiological characteristics of Streptomyces sp. A956 Test Melanin formation (Waksman 1961; Shirling and Gottlieb 1966) Decomposition of L-tyrosine (Gordon and Smith 1955) Diastatic activity (Lyons and Pridham 1962) Proteolytic activity (Gordon and Mihm 1957) Urease activity (Nitseh and Kutzner 1968) Production of If2S (Kuster and Williams 1964) NaC1 tolerance (Tresner etal. 1968) Utilization of carbon sources (Shirling and Gottlieb 1966) ])-Glucose
Reactiona
b + _ +++ +++ lO% +++
D-Fructose
I,-Arabinose n-Xylose L-Rhamnose D-Mannitol
myo-Inositol Sucrose Rafflnose Cellulose a
+++ § +++ +_L+ q-+ + ++
+ , __ + , + + .: . indicate reaction/utilization in increasing order, -- indicates no reaction/utilization.
476
S. D E a n d A. L. C H A N D I ~ A
Vol. 24
TABLE I I I , D e g r a d a t i o n of organic n i t r o g e n o u s w a s t e s b y Streptomyces sp. A t 5 6
Substrate
Vegetable-tanned calf l e a t h e r Human hair Silk Wool Chicken feather
Solubilized n i t r o g e n % glycine equivalent a
35.94 7.18 3.66 4.28 12.34
20.85 2.82 4.05 2.90 6.03
Proteolytic activity AA2s0b tyrosine units c
0.222 a 0.26 0.686 0.2 0.56
97 19 57 27 55
a Amino nitrogen, mg per g substrate. b D i l u t i o n 1 : 1. e n m o l p e r m l per 30 rain. d D i l u t i o n 1 : 200.
loose spirals, is weakly diastatic and produces no antibiotic. S. pseudogriseolus has closed spirals, is strongly diastatic and produces a xanthomycin-like antibiotic. Since the differences are more pronounced than the similarities the organism is given a separate identity and designated as Streptomyces sp. A956. This streptomycete degraded wool, hair, silk and feather and was remarkable in solubilizing leather proteins by 35.9 ~o (Table III). Initial studies showed that K2HPOa at 1.5 g/1 increased leather degradation over distilled water at pH 8.0. Combinations of the phosphate with CaCle, MgS04.TH20, ZnSO4.7H20 and FeSO4.TH20 at the concentrations suggested by Noval and Nickerson (1959) or doubling the concentrations produced no better results. So for maximum degradation of leather 1.5 g KeHP04 was added to 1 litre distilled water. None of the carbon sources tested could increase the leather-degrading capacity of the organism. The optimum temperature range for leather degradation was found to be 28--32 ~ An inoculum size of 12• 107 spores was needed for degradation of 100 mg leather. Under these optimal conditions Streptomyces sp. A956 solubilized vegetable-tanned calf leather and 2.32 mg glycine equivalent amino nitrogen could be obtained from 100 mg leather in 8 d. Microscopic examination revealed the growth of Streptomyces sp. A956 within the substrates causing changes in external appearance. Silk and wool fibres were swollen, hair was ruptured at the outer layers, feather barbs were shredded into pieces and leather was turned into a gel-like substance. This paper presents evidence that Streptomyces sp. A956 is capable of attacking various organic nitrogenous waste materials that accumulate in city garbage. Of special interest is its leather solubilizing property which could be used for degrading waste leather into simpler nitrogenous products of agricultural use. REFERENCES
ANSON M. L. : T h e e s t i m a t i o n of p e p s i n , t r y p s i n , p a p a i n a n d c a t h e p s i n w i t h h a e m o g l o b i n . J.Gen.Microbiol. 22, 79 (1938). B~CKER B., LECHEVAL~ER M. P., LECm~V~_~IER H . A.: C h e m i c a l c o m p o s i t i o n of cell wall p r e p a r a t i o n s f r o m cells o f v a r i o u s f o r m g e n e r a o f aerobic a c t i n o m y c e t e s . Appl.Microbiol. 13, 236 (1965). Bergey's Manual of Determinative Bacteriology (Sth ed.). W i l l i a m s & Wilkins, B a l t i m o r e 1974. B o o ~ . C. J . , PINE L.: R a p i d m e t h o d for c h a r a c t e r i z a t i o n o f a e t i n o m y c e t e s b y cell wall c o m p o s i t i o n . AppL Microbiol. 16, 279 (1968).
1979
DEGRADATION OF ORGANIC N I T R O G E N O U S WASTES
4~
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