APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1988, p. 2058-2063 Vol. 54, No. 80099-2240/88/082058-06$02.00/0Copyright © 1988, American Society for Microbiology

Identification and Characteristics of Actinomycetes Useful forSemicontinuous Treatment of Domestic Animal Feces

SHINSAKU HAYASHIDA,l* NOBUYA NANRI,1 YUJI TERAMOTO,' TOSHIAKI NISHIMOTO,'KAZUYOSHI OHTA,l AND MASAO MIYAGUCHI2

Department of Agricultural Chemistry, Kyushu University, Fukuoka 812,1 and Department of Agricultural Chemistry,Saga University, Saga 840,2 Japan

Received 22 January 1988/Accepted 23 May 1988

Selected strains of actinomycetes useful for practicing semicontinuous treatment of swine and poultry feceswere identified as Streptomyces antibioticus S-4, S. puniceus N-50-2, S. nigrifaciens N-9-3, Thermoactinomycesvulgaris HIR-60, and Thermomonospora viridis HIR-50. These five obligately aerobic strains grew preferably onnonsterilized fresh swine feces in 24 h without any additives. They assimilated offensive volatile fatty acids inthe swine and poultry feces. Cultures of these five strains were mixed and used as seed for the practicaltreatments of 1 ton of swine feces over the wide temperature range of 15 to 60°C. Strain HIR-50 grew mostpredominantly on both fresh swine and poultry feces at 50 to 55°C and decomposed uric acid. For the efficientpenetration of mycelia into the feces, manures were mixed once a day so as not to break the solid mass, andthe dehydration rate of feces had to be controlled in proportion to the mycelial growth rate. The actinomycetebiofertilizer thus manufactured in 10 days was odorless and promotive of plant growth.

Animal wastes were traditionally treated by compostingunder anaerobic and thermophilic conditions for 2 to 6months in Japan. The resulting manure created odor nui-sance due to volatile fatty acids, amines, ammonia, andhydrogen sulfide. The increase in livestock industry caused awaste disposal problem. Recent interest in the use of organicmatter for improvement of soil fertility urged the develop-ment of a proper treatment system for a large amount offeces before reuse as plant nutrients.

Previous studies (6, 14) reported that swine or poultryfeces were deodorized in practical semicontinuous treatmentby using a mixed culture of mesophilic and thermophilicactinomycetes. The semicontinuous treatment of swine fe-ces was carried out in an open-top plastic structure (2 by 30m) equipped with an automatic moving agitator and providedwith air ventilation. One ton of freshly excreted swine feceswas mixed with 10 kg of the seed culture, piled up to a depthof 20 to 30 cm on the concrete floor of the plastic house, andincubated. After 10 days of incubation, a matured bioferti-lizer was produced and the biochemical oxygen demandreached its minimal value. A dry product was obtainedbecause of the increase in fermentation temperature up to60°C and the action of sunlight and air. A portion (usually10% and more than 30% in winter) of the finished productwas used as seed for the next batch of feces to be treated.The amount of volatile fatty acids, a major source ofmalodor, was significantly reduced in 24 h.The objectives of the present study were isolation and

identification of coprophilous actinomycetes useful for thesemicontinuous treatment of domestic animal feces and theoptimization for composting on the basis of their culturalcharacteristics and physiological properties.

MATERIALS AND METHODSPreparation of swine and poultry feces extract agar media.

Swine feces (100 g) or poultry feces (400 g) were added to 400ml of tap water. The mixture was left to stand at roomtemperature for 10 min and filtered through two layers of

* Corresponding author.

gauze. After the addition of 1.5% agar, pH was adjusted to8.3 with 2 N Na2CO3 and the mixture was sterilized at 121°Cfor 30 min.

Isolation and selection of actinomycetes for feces treatment.Approximately 1,000 strains of actinomycetes were isolatedfrom the manures collected in the northern part of Kyushu,Japan, and in the suburbs of Bangkok, Thailand, by repeatedplating on the swine and poultry feces extract agar media.The isolates were tested for growth on sliced fresh swine andpoultry feces. The selected strains have been applied to thepractical treatment of swine or poultry feces for 7 years.Finally, five strains (S-4, N-50-2, N-9-3, HIR-60, and HIR-50) of actinomycetes were reisolated from the treated fecesas the predominant strains. These strains were maintainedon slants of the swine feces extract agar medium.Taxonomic studies of actinomycetes. Cultural characteris-

tics and physiological properties of isolates were studied byusing media described by Shirling and Gottlieb (13) andWaksman (15). Isolates were grown at 30 or 50°C for 21days, and results were recorded every 7 days. Utilization ofcarbon sources was investigated by the method of Pridhamand Gottlieb (10). Identification was performed according tothe Bergey's Manual ofDeterminative Bacteriology (4) andBiseibutsu no Bunrui to Dotei (9).

Uric acid-decomposing activity. Actinomycete strains weregrown in uric acid medium (pH 7.0; glucose [5 g], peptone[10 g], yeast extract [10 g], NaCl [0.5 g], uric acid [5 g], tapwater [1,000 ml]). Portions of the medium (5 ml) weredispensed into test tubes, autoclaved at 121°C for 20 min,and inoculated with a loopful of spores from the slantcultures. The culture was incubated on a reciprocatingshaker at 30 or 50°C for 5 days. The culture was diluted to100 ml with 1 N NaOH, and the solutions were diluted 10times with 0.1 M borate buffer (pH 9.5). A293 of the solutionswas determined spectrometrically (2).

Volatile fatty acid-utilizing activity. The mesophilic strainswere grown at 30°C in liquid Waksman medium (glucose [10g], beef extract [5 g], peptone [5 g], NaCl [5 g], tap water[1,000 ml]) on a rotary shaker for 24 h. The thermophilicstrains were grown at 50°C in liquid medium A composed of

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yeast extract (5 g), peptone (1.5 g), NZ amine type A(Sheffield Chemical Co.) (3.5 g), and tap water (1,000 ml) for36 h. To determine the volatile fatty acid-assimilating activ-ity, oxygen uptake [Q(02); microliters of 02 per 100 mg (wetweight) of cells per hour] was measured at 30°C for meso-philes and 50°C for thermophiles with a Warburg manometer(12). Cells were harvested by centrifugation and washedtwice with 0.05 M phosphate buffer (pH 8.0). A 1-ml portionof actinomycetes suspension (100 mg of cells per ml) in 0.05M phosphate buffer (pH 8.0) was added to the main com-partment of the clean, dry Warburg flasks equipped with acenter well. A 0. 1-ml portion of 20% KOH was added to thecenter well, which contained a filter paper strip. To the sidearm, 0.8 ml of 0.05 M phosphate buffer (pH 8.0) and 0.1 mlof 0.1 M substrate solution were added. Substrates used ascarbon sources were glucose, acetic acid, propionic acid,butyric acid, valeric acid, caproic acid, and caprylic acid (allsubstrates were purchased from Ishizu Pharmaceutical Co.Ltd., Japan).

Volatile fatty acids in fresh swine feces, nontreated swinefeces (mixture of fresh swine feces and seed culture at thebeginning of treatment), and treated swine feces were ana-lyzed by gas-liquid chromatography. A 10-g sample of feceswas acidified to pH 1 with 1.0 N HCl, and free volatile fattyacids were extracted with 2.5 ml of CH2Cl2 for 3 h. Theextract was dehydrated with anhydrous Na2SO4 and concen-trated to 1 ml. Sample solution (2 ,ul) was analyzed with aHitachi model 163 gas chromatograph equipped with a flameionization detector. A glass column (2 m by 3 mm) waspacked with 2% diethylene glycol succinate (DEGS) plus0.5% H3PO4 on Chromosorb W AW. The column wasmaintained at 80°C with N2 as a carrier gas at a rate of 30 ml/min. Individual fatty acids were identified by comparingtheir retention times with those of standard fatty acids.

Determination of treatment conditions. The batchwisetreatment of swine feces was carried out in a plastic house.

(i) Preparation of seed. The selected five strains wereinitially cultivated separately on 50 g of solid wheat branmedium [wheat bran, 50 g; Ca(OH)2, 1 g; swine feces, 10 g;tap water, 40 ml] in a 500-ml Erlenmeyer flask at 30°C formesophiles or 50°C for thermophiles for 10 days. Wheat brancultures rich in spores of these actinomycetes were mixedand used as a starter. For preparation of a larger amount ofseed culture, a nonsterilized mixture of fresh swine feces (5kg), dried swine feces (1 kg), and Ca(OH)2 (90 g) wasinoculated with 5% (wt/wt) of the wheat bran culture de-scribed above (1 x 1012 CFU/g) in an aluminum tray (900 by360 by 60 mm) with a lid and cultivated at 30°C for 10 days.

(ii) Procedure of treatment. Freshly excreted swine feces(100 kg) were piled up to a depth of 20 to 30 cm with 10 kg ofthe seed on the concrete floor of a plastic house andcultivated batchwise. Manures were mixed once a daywithout aeration.

Analytical methods. Moisture content was measured with amoisture meter (Kett Co., Japan). pH was measured with apH meter (model HM-5B; TOA Electronics Ltd.) in thefollowing way. A 1-g sample was suspended in 5 ml of tapwater and left to stand for 10 min at room temperature, andthen pH was measured. Room temperature and materialtemperature of manure were measured with a temperaturesensor (model R 005) and recorded with an ElectronicRecording Controller (model EH 200; CHINO Co., Japan).Total nitrogen was measured by the Kjeldahl methods (3).Carbon contents were measured by the procedure describedin Dojyo Hiryo-gaku Jikken Note (1). Biochemical oxygendemand was determined by the Winkler method (5). The cell

number of the actinomycetes was counted by plating onswine feces extract agar medium by the plate dilutionmethod. The cell number of coliform bacteria was countedby plating on desoxycholate agar Nissui medium (NissuiPharmaceutical Co., Japan). The cell number of anaerobicbacteria was measured with GasPak Anaerobic Systems(BBL Microbiology Systems, Div. Becton Dickinson andCo., Cockeysville, Md.).

Pot experiments. To assess the effectiveness of treatedfeces as fertilizer, pot experiments were carried out intriplicate. Humic volcanic ash soil (pH 4.7) collected atKuroishi near Kumamoto in Japan was used for pot exper-iments. The soil was passed through a 16-mesh (1-mm) sieve,and the pH was adjusted to 6.0 with CaCO3. A total of 600 gof soil per pot was supplemented with the treated swinefeces, dried swine feces, rapeseed meal, or ammoniumsulfate to give nitrogen contents of 0.1, 0.2, 0.4, 0.8, and 1.6g per pot. Komatsuna, a kind of chinese mustard (Brassicarapa var. previridis) (25 seeds), was placed on the soilsurface. Water was supplied every day to keep the 60% fieldcapacity content. The plants were grown at 25 + 1°C in theglass house of Biotron Institute at Kyushu University. After45 days, they were harvested and their fresh weights weremeasured. The pot experiments were repeated eight times bythe same procedure. The fertilizer effect was expressed asthe average fresh weights of the plant.

RESULTS

Identification of selected actinomycete strains. (i) Strain S-4.Vegetative hyphae produced a well-developed branchedmycelium. Mature spore chains showed a curved shape.Sizes of the spores and mycelium were uniform. The sporesurface was smooth and gray. Optimal pH and temperaturefor growth were pH 8.0 and 37°C, respectively. Carbonsource utilization was the same as that of Streptomycesantibioticus reported by Pridham and Tresner (11). How-ever, NaCl tolerance was 0 to 13%. On the basis of morpho-logical and cultural characteristics, strain S-4 was consid-ered to belong to the "Rectus Flexibilis, Smooth, Gray,C+" series described by Pridham and Tresner (11) andidentified as a strain of S. antibioticus.

(ii) Strain N-50-2. A well-developed branched myceliumwas produced, and mature spore chains were flexuous. Longspore chains were occasionally observed. Sizes of sporesand mycelium were uniform. The spore surface was smoothand yellowish. Carbon source utilization was the same asthat of S. puniceus (11), although NaCl tolerance (0 to 10%)was slightly stronger than that of the type strain. StrainN-50-2 was considered to belong to the series "RectusFlexibilis, Smooth, Yellow, C-." This actinomycete wasidentified as a strain of S. puniceus.

(iii) Strain N-9-3. Spore chains were short and flexuous.Helical chains were occasionally observed. Spore surfacewas smooth and gray. NaCl tolerance was 0 to 13%. StrainN-9-3 was considered to belong to the series "RectusFlexibilis, Smooth, Gray, C-." From the above results andthe carbohydrate utilization tests, this actinomycete was

identified as a strain of S. nigrifaciens (11).(iv) Strain HIR-60. Aerial mycelium was chalk-white.

Single spores were formed directly on the mycelium or on a

short sporophore. The diameters of hyphae were variable.Trunklike mycelium was thick, and branched mycelium was

getting thinner at the top. Spore surface was smooth. Solublepigment was not formed. Growth of this actinomycete oc-

curred at 40 to 60°C and at pH 6.0 to 10.0. Optimal

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TABLE 1. Cultural characteristics of strain HIR-50

Agar medium Growth of colony Color of colony Reverse color Soluble pigment

Sucrose-nitrate "Glucose-asparagine Scant Opaque yellow Colorless NoneGlycerol-asparagine Scant Opaque yellow Colorless NoneInorganic salts-starch Scant Opaque yellow Colorless NoneOatmeal Scant Translucent Colorless NoneYeast extract-malt extract Scant Watery yellow Colorless NoneTyrosine Moderate Yellowish gray Colorless NoneNutrient Abundant Dark grayish green Yellowish gray Dark greenPeptone-yeast extract-iron Scant Watery yellow Colorless NoneCzapek Scant Gray Colorless NoneWaksman Abundant Dark grayish green Yellowish brown Dark greenSwine feces extract Abundant Dark grayish green NDb Dark green

No growth.bND, Not detected.

temperature and pH for growth were 50°C and pH 7.5,respectively. Hydrolysis of starch and nitrate reduction wereobserved. These results indicated that this thermophilicactinomycete was classified as a strain of Thermoacti-nomyces vulgaris by the method of Kuster (8).

(v) Strain HIR-50. Cultural characteristics and physiolog-ical properties of the most predominant and impOrtant strain,HIR-50, are summarized in Tables 1 and 2. Aerial myceliumwas dark grayish green. Single spores and occasionally twospores were formed on aerial mycelium. The spore surfacewas smooth. Trunklike and branched mycelia were alsofound. Growth of this actinomycete occurred at 40 to 55°Cand pH 6.0 to 10.0. The optimal temperature and pH forgrowth were 50°C and pH 7.5, respectively. Dark-greensoluble pigment was produced on both nutrient agar andWaksman agar medium (15). This strain hydrolyzed starchand coagulated milk. These results indicated that this ther-mophilic actinomycete was a strain of Thermomonosporaviridis according to Kuster (8).Other physiological properties. All five strains were obli-

gately aerobic, because they grew abundantly under aerobicconditions but did not grow under anaerobic conditions(BBL GasPak Anaerobic Systems). The thermophilic strainsHIR-50 and HIR-60 hardly grew on the ordinary culturemedia but grew preferably on the feces extract media.Therefore, they belong to a group of coprophilous microor-ganisms. All five strains assimilated volatile fatty acids(Table 3). In particular, Ther-momonospora viridis HIR-50 assimilated n-valeric acid and Thermoactinormyces viIl-garis HIR-60 assimilated n-butyric acid. However, caprylicacid could not be assimilated as a sole carbon source by

TABLE 2. Physiological properties of strain HIR-50Property Result

Temperature range for growth (C) ........................... 40-55Optimal temperature for growth (°C) ........................... 50pH range for growth ........................... 6.0-10.0Optimal pH ........................... 7.5Coagulation of milk........................... PositivePeptonization of milk ........................... PositiveGelatin liquefaction ....... .................... NegativeHydrolysis of starch........................... PositiveNitrate reduction ........................... NegativeFormation of melanin pigment ........................... NegativeNaCl tolerance (%) ........................... 0-7Decomposition of uric acid........................... PositiveGrowth under anaerobic conditions ........................... Negative

any strain. None of these strains was able to produceantibiotics.

Optimization of conditions for feces treatment. Optimalculture conditions for growth of actinomycetes on nonsteri-lized fresh swine feces were determined. (i) Growth oc-curred at a pH range of 6.0 to 10.0 and optimally at pH 8.0.(ii) Optimal temperature was 30°C for three mesophiles and50°C for two thermophiles. (iii) For maintaining an aerobicenvironment for growth of actinomycetes, the average mois-ture content of feces was initially adjusted to below 65%.Higher moisture content caused an anaerobic environmentin the inner part of the feces. The mixture of cultures of thesefive strains used as seed enabled the practical treatment offeces over the wide temperature range of 15 to 60°C. Underthese conditions, the cell number (per gram) of the meso-philic and thermophilic actinomycetes increased from 8 x106 to 8 x 107 and from 4 x 106 to 3 x 109, respectively. Nosignificant growth of putrid clostridial bacteria was ob-served. The temperature of the manure was controlled bythe depth of the feces heaped up on the floor in the plastichouse (usually 40 cm during the first 24 h and 30 cm later on).In a ventilated plastic house, swine feces were heaped up toa layer about 30 cm deep. The temperature increased from 25to 50°C in 24 h and remained almost constant (50 to 60°C) forthe next 3 days (Fig. 1). The temperature was then graduallydecreased. Overheating of the manure caused by bacterialgrowth was prevented when the inoculated actinomycetesgrew predominantly. The dehydration rate had to be con-trolled for a complete treatment with actinomycetes. Theaverage moisture content decreased from 65 to 35% and pHranged from 8.0 to 8.5 during 10 days of treatment. Volatile

TABLE 3. Volatile fatty acid-assimilating activityof actinomycetes

Oxygen uptake [Q(02)]" by strain:Carbon source

S-4 N-9-3 N-50-2 HIR-50 HIR-60

Glucose 14.3 3.7 2.6 11.9 7.7Acetic acid 9.8 2.2 7.3 8.3 4.3Propionic acid 8.9 3.3 6.8 6.1 1.8n-Butyric acid 9.0 2.6 1.2 0.8 18.4n-Valeric acid 7.5 2.2 1.9 63.9 8.6Caproic acid 3.7 1.2 0.7 0.8 0.7Caprylic acid NDb ND ND ND ND

"Q(02) (microliters of 02 per 100 mg [wet weight] of cells per hour) wasmeasured by a Warburg manometer at 30°C for mesophiles (strains S-4, N-9-3,and N-50-2) and 50'C for thermophiles (strains HIR-50 and HIR-60).

" ND, Not detected.

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FIG. 1. Changes in pH, temperature, and moisture content dur-ing microbial treatment of swine feces. Cell numbers (CFU per

gram) of thermophilic actinomycetes at 50°C (-O-), mesophilicactinomycetes at 30°C (-0-), anaerobic bacteria (A), and coliformbacteria (A) are indicated. Other symbols: ---0---, biochemicaloxygen demand; ---0---, pH; V, temperature of manure; V, room

temperature; Ol, moisture content; -x-, organic carbon; ---xtotal nitrogen; *, C/N ratio.

fatty acids in the feces were completely assimilated byactinomycetes in 24 h (Fig. 2). The white mycelia of acti-nomycetes penetrated vigorously into feces and maturedspores were fully developed in the inner part of the fecesafter 3 days of incubation in the controlled plastic house. Inlaboratory experiments, however, mycelia grew only on thesurface and were not able to penetrate into feces in an

incubator because of high humidity.Treatment of poultry feces had been very difficult because

of the presence of a large amount of uric acid. However, byusing these five actinomycetes, treatment of poultry feceswas performed properly with respect to pH, moisture con-tent, and cell number of coliform bacteria, as in the treat-ment of swine feces (Fig. 3). In addition, about 50% of theuric acid contained in poultry feces was decomposed duringthe treatment.

Maturity of treated swine feces was judged by (i) 40%reduction of the biochemical oxygen demand of the originalorganic matter, (ii) a carbon-to-nitrogen (C/N) ratio of about10, and (iii) the smell of soil, or rather actinomycetes, in thetreated feces.

Pot experiments. When the series of the plant cultures wasfertilized with ammonium sulfate, the highest yield was

obtained at a nitrogen content of 0.2 g per pot (Fig. 4).Growth of the plants was suppressed by fertilization withlarger amounts of ammonium sulfate-nitrogen. In the case ofrapeseed meal, maximum plant yield was obtained at a

nitrogen content of 0.8 g per pot. The best yield of the plantswas obtained with the treated swine feces at a highest

C

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0 2 4 0 2 4 0 2 4MIN MNI MI,

FIG. 2. Gas chromatogram of volatile fatty acids in swine feces.(A) Volatile fatty acids in fresh swine feces (attenuation, 16 x 102);(B) volatile fatty acids in mixture of fresh swine feces and seedculture at the beginning of treatment (attenuation, 4 x 102); (C)volatile fatty acids in treated swine feces after 48 h (attenuation, 4 x102). Peaks: 1, acetic acid; 2, propionic acid; 3, n-butyric acid; 4,isovaleric acid. Volatile fatty acids were separated by a glass column(2 m by 3 mm) of 2% DEGS plus 0.5% H3P04 on Chromosorb W AWat 80°C with an injection temperature of 150°C and a carrier gas (N2)flow rate of 30 ml/min.

nitrogen content of 1.6 g per pot. Within 4 days, 40% oforganic nitrogen was degraded. Treated swine feces, abun-dant in mycelia, contained 2.4% nitrogen, 5.6% P205, and1.9% K20. Organic nitrogen in mycelia and treated swinefeces was gradually mineralized, and so there was no inhib-itory effect on plant growth, which could often be seen withinorganic nitrogen.

DISCUSSION

A previous paper (14) described a novel use of actinomy-cetes for practical treatment of swine feces. In the presentpaper, the five strains of actinomycetes were selected andidentified. Furthermore, the techniques for growing theactinomycetes fully inside the domestic animal feces weredeveloped. The actinomycetes showed vigorous growth onnonsterilized fresh swine feces without any additives andassimilated the offensive volatile fatty acids as carbonsources. The feces were deodorized very rapidly at an initialmoisture content of 65% of the feces at pH 8.0 to 8.5. Underthese conditions, the growth of anaerobic bacteria wasinhibited.Of the five strains, HIR-50 grew most predominantly on

both swine and poultry feces. In the practical treatments,this thermophilic strain played the principal role in compost-ing, because the mycelial growth phase coincided with theincrease in temperature of the manure, and the dark-greenishspores of this strain were often observed in the treated feces.The composting process was divided into two steps,

covering of feces with mycelia and penetration of myceliainto feces. The surface of the feces was initially covered withthe mycelia in 30 h under the optimal conditions. Althoughthese conditions were quite satisfactory for growth on the

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*c' 10

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FIG. 3. Changes in pH and moisture content during microbialtreatment of poultry feces. Cell numbers (CFU per gram) of acti-nomycetes (0) and coliform bacteria (A) are indicated. Othersymbols: ---A---, pH; O, moisture content; -x-, organic carbon;- x---, total nitrogen; *, C/N ratio; -0-, N as NH4; V, N as

N03-N; V, N as urate-N.

surface, mycelia of these obligately aerobic actinomycetescould not penetrate into feces. Therefore, we tried a novelapproach for allowing the mycelia to penetrate into feces onthe basis of the principle of rice koji-making technique insake brewing (7). During the rice koji-making process, the

100

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NITROGEN (MB/POT, 102)FIG. 4. Fresh-weight yields of Brassica rapa var. perviridis

fertilized by treated swine feces (0), dried swine feces (0), rapeseedmeal (A), or ammonium sulfate (A). Each point represents the meanof eight independent experiments. In Japan, the standard amount ofnitrogen supplied to soil as fertilizer is about 100 mg/600 g of soil.Therefore, 1 unit of soil nitrogen is expressed as 100 mg ofN per 600g of soil.

temperature of steamed rice rises from 35 to 42°C and themoisture content is reduced from 35 to 25% in 40 to 45 h, andthen the mold mycelia penetrates into the steamed rice. In asimilar way, the dehydration rate of feces was controlled soas to be proportional to the mycelial growth rate. In theoptimally moist and therefore aerobic microenvironment,mycelial growth occurred initially on the limited part of thefeces surface and gradually developed on and into the feceswith the gradual dehydration. The heterogeneous structureof feces as a solid mass should not be broken by excessivemechanical mixing to prevent destruction of the aerobicmicroenvironment.

In the pot experiment with Brassica rapa var. perviridis,the growth of plants fertilized with treated feces was betterthan that of plants fertilized with air-dried feces. Sinceorganic nitrogen derived from mycelia in treated swine feceswas gradually mineralized, supplementation with such largeamounts of treated swine feces as 1.6 g ofN per 600 g of soilper pot did not show any inhibitory effect on plant growth.On the contrary, in the case of plants fertilized with ammo-nium sulfate or rapeseed meal, the highest yields wereobtained in the plants cultivated at the lower nitrogencontents of 0.2 or 0.8 g of N per pot, respectively. Additionof larger amounts of ammonium sulfate or rapeseed mealinhibited the growth of plants. These data suggested thatlarge amounts of treated feces (C/N ratio, 10) could be usedas an excellent actinomycete biofertilizer and soil-improvingagent. The treatment of poultry feces with actinomycetes forbiofertilizer will be described in later papers.

ACKNOWLEDGMENT

We are very gratefully indebted to Motoo Shibata at KumamotoUniversity for his valuable advice in the identification of actinomy-cetes.

LITERATURE CITED

1. Aomine, S., and T. Harada. 1977. Dojyo Hiryo-gaku JikkenNote. Yoken-Do Co., Tokyo.

2. Bergmeyer, H. U. 1974. Methods of enzymatic analysis, vol. 4.Academic Press, Inc., New York.

3. Black, C. A., D. D. Evans, L. E. Ensminger, J. L. White, andF. E. Clark. 1965. Methods of soil analysis. American Societyof Agronomy, Inc., Madison, Wisconsin.

4. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergey'smanual of determinative bacteriology, 8th ed. The Williams &Wilkins Co., Baltimore.

5. Greenberg, A. E., J. J. Connors, and D. Jenkins. 1980. Oxygendemand (biochemical), p. 483-489. In Standard methods for theexamination of water and wastewater, 15th ed. American PublicHealth Association, Washington, D.C.

6. Hayashida, S., and Y. Tanaka. 1980. Biseibutsu niyoru Kachi-kufun no Shori to Riyo, p. 243-251. In K. Arima and G. Tamura(ed.), Seibutsu niyoru Kankyo Joka. Gakkai Shuppan Center,Tokyo.

7. Kodama, K. 1970. Sake yeast, p. 225-282 In A. H. Rose andJ. S. Harrison (ed.), The yeasts, vol. 3. Academic Press, Inc.,New York.

8. Kuster, E. 1974. Micromonosporaceae, p. 846-865. In R. E.Buchanan and N. E. Gibbons (ed.), Bergey's manual of deter-minative bacteriology, 8th ed. The Williams & Wilkins Co.,Baltimore.

9. Okami, Y., and A. Seino., 1975. Hosen-kin, p. 1-92. In T.Hasegawa (ed.), Biseibutsu no Bunruito Dotei. Gakkai ShuppanCenter, Tokyo.

10. Pridham, T. G., and D. Gottlieb. 1948. The utilization of carboncompounds by some actinomycetales as an aid for speciesdetermination. J. Bacteriol. 56:107-114.

g %W

m 4-- ,X I,I F 2X~ *

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11. Pridham, T. G., and H. D. Tresner. 1974. Streptomycetaceae, p.747-845. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey'smanual of determinative bacteriology, 8th ed. The Williams &Wilkins Co., Baltimore.

12. Sasagawa, T. 1954. Chapter 5. Application. J. Jpn. Chem. 13:133-137.

13. Shirling, E. B., and D. Gottlieb. 1966. Methods for characteri-

zation of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340.

14. Tanaka, Y., T. Tanaka, N. Nanri, and S. Hayashida. 1978.Treatments of domestic animal feces with actinomycetes. Hak-kokogaku 56:786-793.

15. Waksman, S. A. 1961. The Actinomycetes. The Williams &Wilkins Co., Baltimore.

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