APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1990, p. 1509-15150099-2240/90/061509-07$02.00/0Copyright C) 1990, American Society for Microbiology

Isolation, Identification, and Characterization of a

Feather-Degrading BacteriumtC. M. WILLIAMS,lt C. S. RICHTER,2 J. M. MACKENZIE, JR.,2 AND JASON C. H. SHIH1*

University Biotechnology Program and Department ofPoultry Science' and Electron Microscopy Center andDepartment of Microbiology,2 North Carolina State University, Raleigh, North Carolina 27695

Received 16 November 1989/Accepted 2 March 1990

A feather-degrading culture was enriched with isolates from a poultry waste digestor and adapted to growwith feathers as its primary source of carbon, sulfur, and energy. Subsequently, a feather-hydrolytic,endospore-forming, motile, rod-shaped bacterium was isolated from the feather-degrading culture. Theorganism was Gram stain variable and catalase positive and demonstrated facultative growth at thermophilictemperatures. The optimum rate of growth in nutrient broth occurred at 45 to 50°C and at pH 7.5. Electronmicroscopy of the isolate showed internal crystals. The microorganism was identified as Bacillus licheniformisPWD-1. Growth on hammer-milled-feather medium of various substrate concentrations was determined byplate colony count. Maxinum growth (approximately 109 cells per ml) at 50°C occurred 5 days postinoculationon 1% feather substrate. Feather hydrolysis was evidenced as free amino acids produced in the medium. Themost efficient conditions for feather fermentation occurred during the incubation of 1 part feathers to 2 partsB. licheniformis PWD-1 culture (107 cells per ml) for 6 days at 50°C. These data indicate a potentialbiotechnique for degradation and utilization of feather keratin.

Feather waste, generated in large quantities as a by-product of commercial poultry processing, is nearly pure

keratin protein (7). Keratin in its native state is not degrad-able by common proteolytic enzymes such as trypsin, pep-

sin, and papain. However, keratin does not accumulate innature, and keratinolytic activity has been reported forspecies of Aspergillus (W. Koh, A. Santro, and R. Messing,Bacteriol. Proc., p. 18, 1958), Ctenomyces (14), and Strep-tomyces (9). Molyneaux reported the digestion of woolkeratin by a Bacillus species (6).

Currently, feather waste is utilized on a limited basis as adietary protein supplement for animal feedstuffs. Prior tobeing used, the feather is steam pressure-cooked or chemi-cally treated to make it more digestible. These treatmentprocesses require significant energy and also destroy certainamino acids (10-12, 17). Biodegradation by microorganismspossessing keratinolytic activity represents an alternativemethod to improve the nutritional value of feather waste.Only limited information regarding such microbial treatmentof feathers is currently available (5, 8).

In this laboratory, a thermophilic poultry waste digestorsystem for the treatment of poultry waste and the productionof biogas has been developed (15). A bacterial culture thatgrows on feathers as the sole organic substrate for supplyingcarbon, sulfur, and energy was enriched from a laboratorypoultry waste digestor (18). This study describes the identi-fication and characterization of an isolate from this culture.Additionally, a comparative study of feather fermentationconditions for the production of free amino acids by themicroorganism is described (U.S. patent 4.908.220, March1990).

* Corresponding author.t Paper no. 12402 of the Journal Series of the North Carolina

Agricultural Research Service, Raleigh.t Present address: ESE Biosciences Group, 3208 Spring Forest

Rd., Raleigh, NC 27604.

MATERIALS AND METHODS

Media and culture conditions. The basal medium used forisolation, maintenance, growth, and fermentation analysis ofthe feather-degrading microorganism contained the follow-ing (in grams per liter): NH4Cl, 0.5; NaCl, 0.5; K2HPO4, 0.3;KH2PO4, 0.4; MgCl- 6H20, 0.1; yeast extract, 0.1; agar, 20(as needed). pH was adjusted to 7.5. Feathers (ball milled,hammer milled, or whole) were added to the basal medium invarious amounts, as will be described below. Feathers werewashed in distilled water prior to being sterilized by auto-claving (120°C at 15 lb/in2 for 20 min). In some stages of thestudy, feathers were autoclaved for less than 20 min, andsterilization was completed by fumigation with formalde-hyde gas (3).

Nutrient broth, nutrient agar, and yeast extract were fromBBL Microbiology Systems (Cockeysville, Md.); other me-dia used in the various tests for identification and character-ization of the microorganisms were from Difco Laboratories(Detroit, Mich.). For growth determination, the microorgan-isms were cultured in 250-ml Erlenmeyer flasks containing50 ml of 1% hammer-milled-feather medium or nutrientbroth. Growth at 25, 37, and 50°C with aeration by rotationof the flasks at 175 rpm was determined by plate count onnutrient agar. To ensure an adequate distribution of the cellswhen plating them on nutrient agar, several 1-mm glassbeads were included in the first dilution tube (K. L. Bott,personal communication).

Light microscopy. Standard microbiological methods wereused to fix the cells to slides for Gram staining and observa-tion. Endospore staining was by the method of Dorner (13).

Electron microscopy. A pellet of stationary-phase cellsfrom nutrient broth was fixed in 3% glutaraldehyde in 0.1 Mphosphate buffer, postfixed in 1% OS04, dehydrated througha graded series of ethanol washes, and embedded in anepoxy resin. Uranyl acetate-lead citrate-stained thin sectionswere viewed with a transmission electron microscope.

Adaptation of bacterium for maximum feather degradation.Whole intact feathers were autoclaved for various periods (1to 20 min). Single feathers were placed in test tubes contain-

1509

Vol. 56, No. 6

on May 19, 2018 by guest

http://aem.asm

.org/D

ownloaded from

1510 WILLIAMS ET AL.

ing 10 ml of basal salt solution and inoculated with the pure,feather-degrading culture. Seven to ten days after inocula-tion at 50°C, bacteria from the tube showing the most featherdegradation were aseptically transferred to fresh whole-feather medium for further selection. After several monthsof this protocol, a strain able to degrade feathers that hadbeen steam treated for a minimum of 2 min was selected forfurther study.

Determination of feather degradation under aerobic andanaerobic growth conditions. Feather-degrading cells weregrown in 500 ml of 1% hammer-milled-feather medium for 3days, filtered through four layers of sterile cheesecloth, andcollected and washed three times in the basal salt solution.These cells were suspended in the basal salt solution andtransferred to culture tubes containing 10 ml of 1% hammer-milled-feather medium. These tubes were maintained underboth aerobic and anaerobic conditions (anaerobic tubes werebubbled with O2-free N2 for 30 min prior to cell transfer, withthe headspaces flushed with the same both during transferand after transfer until sealed by rubber septa and screwcaps). All tubes were incubated at 50°C for 10 days (shakingat 125 rpm). The cultures were sampled daily for cellnumbers by determining CFU on ball-milled-feather agarplates, and soluble sulfhydryl concentrations were deter-mined by the method of Ellman (4), as modified by Shih et al.(16).

Solid-phase fermentation of feathers. The feather-degrad-ing microorganism was grown aerobically to a concentrationof approximately 107 cells per ml in the basal mediumcontaining 1% hammer-milled feathers. This liquid culturewas aseptically added to 200-ml plastic bottles containing 5 gof sterile hammer-milled feathers in order to obtain ratios of1 part feathers (dry weight) to 2, 4, and 8 parts liquid cellculture. The bottles were flushed with oxygen-free nitrogen,sealed with screw caps, and incubated at 50°C. Agitation wasby vigorous manual shaking twice per day. Duplicate sam-ples of the aqueous fermentation products were collected ondays 0, 3, 6, 12, and 24 after incubation. The liquid wascollected by filtration through four layers of cheesecloth toremove nondegraded feather particles. This filtrate waspassed through a 0.45-,um-pore-size filter and frozen untilamino acid determination.Amino acid analysis. Samples (200 j,l) of the filtrate noted

above were methanol extracted (800 ,ul of 40% methanol in0.1 N HCI) and centrifuged at 13,000 x g for 10 min at 4°C.The supernatant was analyzed for free amino acids byhigh-performance liquid chromatography. The analysis wasperformed at the North Carolina State University ProteinSequencing Facility.

RESULTS

Isolation and adaptation of feather-degrading microorgan-ism. It was found that a previously enriched, feather-de-grading culture (18) contained microorganisms exhibiting atleast two distinct colony morphologies when streaked ontonutrient agar plates. One type contained only rod-shapedbacteria; the other was a coccus, which appeared singly andin chains. Although each type displayed clearing zones whenstreaked onto the ball-milled-feather agar plates, the rod-shaped bacterium demonstrated the most pronounced clear-ing zones. This isolate was selected for the purpose ofidentification and for its adaptation to feather degradation.Cells of the isolate were grown on whole feathers andtransferred at frequent intervals to the basal medium, con-taining whole feathers, in which the autoclave treatment

time was systematically reduced. Eventually, after severalmonths of repeated selection, the isolate was able to degradefeathers which had been autoclaved for only 2 min. Attemptsto maintain the growth of the isolate on non-steam-treatedfeathers were not successful.

Identification and characterization of feather-degrading iso-late. Microscopic observation of the isolate showed astraight rod (2 to 3 by 0.5 to 1 ,Lm) (Fig. 1) with endospores(Fig. 2). The bacterium grew aerobically, was stronglycatalase positive, and was Gram stain variable. Transmis-sion electron micrographs showed, in some cells, internalcrystals in a rectangular array with regular spacing (Fig. 1B).Collectively, these characteristics indicated that the isolatewas of the genus Bacillus.The optimum rate of growth in hammer-milled-feather

medium occurred at temperatures near 50°C (Fig. 3). Nogrowth occurred at temperatures in excess of 65°C. Addi-tional morphological and physiological tests conducted onthe isolate in this laboratory were confirmed by the Ameri-can Type Culture Collection, which characterized the isolateas thermophilic Bacillus licheniformis. We propose PWD-1as the strain designation to indicate isolation of the strainfrom a poultry waste digestor.

Degradation of feathers by isolate. It was observed thataerobic growth by the isolate on feathers, with the feathersas its primary source of carbon, energy, and sulfur, resultedin nearly complete degradation of the keratin protein after 7to 10 days of incubation at 50°C. Washed cells from suchaerobically grown cultures were inoculated onto fresh ham-mer-milled-feather medium and incubated at 50°C underboth aerobic and anaerobic conditions. Biodegradation wasmeasured as the appearance of soluble sulfhydryl in thegrowth medium. Growth of the isolate and the concentra-tions of soluble sulfhydryl under these conditions are shownin Fig. 4. The amount of soluble sulfhydryl present wasproportional to the number of viable cells. These results aresimilar to those previously observed for the enriched,feather-degrading culture (15), although the concentration ofsoluble sulfhydryl is higher for the pure culture isolate. Onthe basis of (i) previous evidence that free amino acidconcentrations were higher for the enriched culture underanaerobic conditions and (ii) data showing that neither theenriched culture nor pure isolate actively grows anaerobi-cally (Fig. 4), the effect of anaerobic incubation time and theratio of feather substrate to liquid cell culture on amino acidliberation was examined in greater detail. The results areshown in Fig. 5, and the profiles of the specific amino acidsthat were liberated are shown in Table 1. The profiles of theamino acids that were measured were relatively similar forall conditions of fermentation. Glutamic acid, alanine, andthe branched-chain amino acids isoleucine, leucine, andvaline were consistently the most abundant amino acids. Themole percent of arginine decreased significantly under an-aerobic conditions.

DISCUSSION

A bacterium isolated from a thermophilic poultry wastedigestor has been shown to degrade feather keratin by usingfeathers as a primary source of energy, carbon, and sulfur.The isolate, which grows optimally at thermophilic temper-atures, has been classified as B. licheniformis PWD-1. Thebacterium was isolated from an anaerobic habitat; however,it showed maximum growth under aerobic conditions, aswould be expected of a member of the family Bacillaceae.One explanation for the presence of this species in a poultry

APPL. ENVIRON. MICROBIOL.

on May 19, 2018 by guest

http://aem.asm

.org/D

ownloaded from

S

A `

A~::

FIG. 1. Electron micrograph of B. licheniformis PWD-1. Longitudinal sections of a vegetative cell (A) and of a cell containing a crystalinclusion (B) are shown. Bar, 0.25 jim. Insert: Section of a B. licheniformis PWD-1 crystal. Bar, 0.1 jim.

1511

on May 19, 2018 by guest

http://aem.asm

.org/D

ownloaded from

1512 WILLIAMS ET AL.

FIG. 2. Electron micrographs ofB. licheniformis PWD-1. (A) Longitudinal section of a cell with central endospore. Bar, 0.5 ,um. (B) Crosssection of endospore. Bar, 0.25 ,um.

waste digestor may be that the bacterium is indigenous to thechicken gut. However, it is more likely that, prior to itsaddition to the waste digestor, it was indigenous to theenvironment in which poultry excreta are collected. Thisenvironment also contains feathers, and the isolate may haveadapted to utilize this substrate. Because of its endospore-forming capabilities, its survival in a thermophilic digestorwould be expected. However, it is possible that this isolateis an active component of the hydrolytic microflora presentin the anaerobic digestor. It has been established thatfacultative microorganisms present in anaerobic digestorsare useful in reestablishing the low redox potential requiredby the strict anaerobes during times when oxygen is intro-duced with the substrate (2).When cells of B. licheniformis PWD-1 were grown aero-

bically on feather medium, they yielded appreciable degra-dation products as measured by the soluble sulfhydryl con-centration in the fermentation broth. Such an increase insoluble sulfhydryl did not occur when the cells were sub-jected to anaerobic growth conditions (Fig. 4). This is inaccord with an earlier study in which these parameters weremeasured in the feather-degrading mixed culture from whichthis bacterium was isolated. It is noteworthy that the in-crease in sulfhydryl concentration initially lags behind thegrowth of the bacterium. This may be due to enzymesliberated by the bacterium following a period of logarithmicgrowth. Such enzymes have been documented in strains ofB. licheniformis (1). These enzymes appear in the extracel-lular medium only during the postlogarithmic phase ofgrowth. The increase of soluble sulfhydryl compounds dur-

APPL. ENVIRON. MICROBIOL.

on May 19, 2018 by guest

http://aem.asm

.org/D

ownloaded from

FEATHER-DEGRADING BACTERIUM 1513

FIG. 3. Growth of B. licheniformis PWD-1 at different tempera-tures in feather medium.

ing the degradation of feathers by this strain of B. licheni-formis is evidence that this bacterium possesses a pro-

tease(s) capable of reducing the disulfide bonds of keratin.Although there was not much growth of the bacterium

during anaerobic incubation (Fig. 4), the amount of freeamino acids accumulated in the medium was greater duringanaerobic incubation than during aerobic growth. It is pos-sible that rapid catabolism of the newly hydrolyzed aminoacids occurred during aerobic growth. We used a broth

AerobicCFU

-SH o

Anaerobic 4

CFUU

3

E

06 (0i0 1 3 5 7 10

DAYS

FIG. 4. Growth of B. licheniformis PWD-1 and soluble sulfhy-dryl (-SH) concentrations in the growth medium under aerobic andanaerobic conditions at 50°C.

120 _ w I 'o I/4* 118

100

0

~600zi40wwM 20

0 3 6 12 24DAYS

FIG. 5. Total free-amino-acid concentrations in the feather fer-mentation medium at different ratios of feathers and liquid culture.

culture containing the bacterium, which had been grownaerobically on a feather medium to approximately 107 CFU/ml, to inoculate fresh hammer-milled feathers under condi-tions made anaerobic for fermentation. Under these condi-tions, feather degradation continued, and because there waslittle or no cell growth, a free product in the form of aminoacids accumulated in the medium (Table 1). The mostefficient production of free amino acids was with a semisolidfermentation medium of 1 part feathers to 4 parts liquidculture, incubated for 6 days. Under these conditions, totalfree amino acids in the fermentation liquid were measured ata concentration in excess of 60 mM. Although higher con-centrations of free amino acids were measured at day 24, theexcess incubation time is not practical, especially consider-ing the energy required for thermophilic incubation. Thetreatment of feathers by B. licheniformis in semisolid fer-mentation medium seems to be a useful method for generat-ing partially hydrolyzed feathers, which in turn can be usedas a digestible feed protein (C. M. Williams, C. G. Lee, J. D.Garlich, and J. C. H. Shih, submitted for publication).The degradation of feathers by a Bacillus sp. has not been

reported previously; however, in 1959 Molyneaux reportedthe isolation, from the dermoid cyst of a sheep, of a Bacillussp. which digested wool keratin (6). He noted that althoughthe growth of various bacilli on wool had been previouslyreported, his isolate was the first that could degrade non-steam-sterilized or native wool keratin. Molyneaux con-ducted an extensive classification of his isolate but did notassign a species name. Many of the results of biochemicaltests he conducted match those we observed for B. licheni-formis PWD-1. However, the bacillus isolated by Molyneauxgrew better under mesophilic temperatures and was alsounable to reduce nitrate to nitrite. Additionally, electronmicroscopy of PWD-1 showed some unique properties.Many cells contained internal crystals, and some demon-strated multiple septa. On the basis of our knowledge ofBacillus thuringiensis and other crystal-forming bacilli, itwould be logical to assume that the crystal is protein.

VOL. 56, 1990

on May 19, 2018 by guest

http://aem.asm

.org/D

ownloaded from

1514 WILLIAMS FT AL. APPL. ENVIRON. MICROBIOL.

cn 'I r- cn " ON r- Co W) a

CN 06 6 tl~C-4 0 \

00 - r- C1 00 11 't n r- 'ttoEN r- Nr Cf P- io I'l 't 'It t

nooooe c>~

~~c4~0\O00~~-4O%

0) c*W0 NccO' 00

M "t Qu 0CD 00N 0O

"-4

soooo oooooe

Noo o M-o C oo o-4 OIn ", r- r- 1.

O4 r-

00

Ot0t0 n "lo C4 n00"4'It It I0 N0 M"0O CN 0 1.

"4NO"4"CC r4 -4cl l

In (C n ON - Q r-o- 00o

00;06 00;C t~14 .) _1 _4

"-4

O- tf r- (2 4 -4 Cf C4 t )00 W) W) Ntl0 00 0 C>00 £

cr0aoOOO"4 I' V"4 rC~ l1c

-00r-00 O 00000rQ

.~.~.~ . . . M. . W.

NO00H~ t H 00

ercfO4' 004 N-Rt 0- "4~~~~~~~~~-

MO8oN0eaom.4 In (.O.. .o 0 M .in 0f%0 01 04001e

C vOutn½n W) W)

ON oot (7 mtn r- oo oo o

WIoo4o2oo 0o en - ON

Cf ooo;1- W; ofi0 o4 4 Co

cn ON crOI OC "o C1 trCDWT-mo It oD O r- 00 tt 4T400 0r el 06dr_4 cl 06

r-ChO tN4

a)co 0

CZ Eu*0," *-4

aL)0

0

r-,3 =o .

CA ob Cu

ON 0 Rt'I O 100-

Q0C

cnCf t &.0 00~o 00t C

c rCwIe oo- r-

N000 C1I)tF0-f" "4r-4C

0)C10-IC~ 'nht 0000--CC0 "141*t )

0'W-o 0r-=c

00I0O4 C1 1 W 0r

r- "

r00 ~ r-r-q o

'IOWc f) \0 C00e0r- "Rt

rO-4 r0000O

C4r- C4 f) e'4"40)00 04r'4

- "4

(2~c r- Nt0000 10O

- orio dr-

Of f)rN0f)r00"4 Cft00IRt W-(7, O-- *4'I

oo C r~ r-:ooor-~

0\ IRt 0 N"4N4 00W

~~r~e~ar1~~o e~-4Lr

00000%lf 00\0Cf)0 W0 \r-00N00r4 0Ne7- -40 lo

1.4CiC~~ C; ~d 4 ~a)0-

a0C4) r 1 -

ll 090 >t -0

V)C4 o ' ;C 4W

A.;01

In..-

00_.

C 4

uB

coI..ce

I..

=I

n. -

4)-4)

-S

Co

00oo

00. .

ra

0

4-

co

co

0co

ra

co

.0

Eu

a)

0Cu

'0

Cu

a)

ca)

'0

a)C)a)-Cu

CuCu'0

a)0

Cu0o

mu

0

C."

aN

C."

C."

0

~r)

80

a,-)

'I)

oo

C.."0

0

C."

,)

C."

'I-)0

n

r-4

00a,'

0%

C1

Q

Ci

Cu V:

0

.0

0

0C)CoC1

00

Cu

a)

a)4.

O

C0c

c.

CuC'0

Fa)0".'

C)

C u~

00 Cu

.W

00.

'l

0

(11:

on May 19, 2018 by guest

http://aem.asm

.org/D

ownloaded from

FEATHER-DEGRADING BACTERIUM 1515

However, the origin, composition, and significance of thisultrastructure in B. licheniformis is not known.

Extensive efforts were made to adapt PWD-1 to degradenative feather keratin, but attempts to maintain a viableculture which could consistently degrade non-steam-treatedfeathers were unsuccessful. A minimum autoclave time of 2min was needed for repeated feather degradation activity.Apparently, some steam treatment is required to rendernative feather keratin accessible for hydrolysis by this bac-terium. We have recently conducted studies which indicatethat the application of this bacterium to feathers subjected toa brief steam treatment followed by controlled biodegrada-tion is energy efficient and produces a digestible protein(Williams et al., submitted). These data indicate a potentialnew biotechnique for the degradation and utilization offeather keratin.

ACKNOWLEDGMENT

Grant support from the Southeastern Poultry and Egg Associationis appreciated.

LITERATURE CITED1. Bernlohr, R. W. 1964. Postlogarithmic phase metabolism of

sporulating microorganisms. I. Protease of Bacillus licheni-formis. J. Biol. Chem. 239:538-543.

2. Bryant, M. P. 1977. Microbiology of the rumen, p. 287-304. InM. J. Svensson (ed.), Duke's physiology of domestic animals.Cornell University Press, Ithaca, N.Y.

3. Card, L. E., and M. C. Nesheim. 1972. Controlling diseases andparasites, p. 261. In Poultry production, 11th ed. Lea & Febiger,Philadelphia.

4. Eliman, G. L. 1959. Tissue sulfhydryl groups. Arch. Biochem.Biophys. 82:70-77.

5. Elmayergi, H. H., and R. E. Smith. 1971. Influence of growth ofStreptomyces fradiae on pepsin-HCl digestibility and methio-nine content of feather meal. Can. J. Microbiol. 17:1067-1072.

6. Molyneaux, G. S. 1959. The digestion of wool by a keratinolyticBacillus. Aust. J. Biol. Sci. 12:274-278.

7. Moran, E. T., Jr., J. D. Summers, and S. J. Slinger. 1966.Keratin as a source of protein for the growing chick. 1. Aminoacid imbalance as the cause for inferior performance of feathermeal and the implication of disulfide bonding in raw feathers asthe reason for poor digestibility. Poult. Sci. 45:1257-1266.

8. Nickerson, W. J., J. J. Noval, and R. S. Robinson. 1963.Keratinase. I. Properties of the enzyme conjugate elaborated byStreptomyces fradiae. Biochim. Biophys. Acta 77:73-86.

9. Noval, J., and W. J. Nickerson. 1959. Decomposition of nativekeratin by Streptomyces fradiae. J. Bacteriol. 77:251-263.

10. Papadopoulos, M. C. 1985. Processed chicken feathers as feed-stuff for poultry and swine. A review. Agricultural Wastes14:275-290.

11. Papadopoulos, M. C., A. R. El Boushy, and A. E. Roodbeen.1985. The effect of varying autoclaving conditions and addedsodium hydroxide on amino acid content and nitrogen charac-teristics of feather meal. J. Sci. Food Agric. Abstr. 36:1219-1226.

12. Papadopoulos, M. C., A. R. El Boushy, A. E. Roodbeen, andE. H. Ketelaars. 1986. Effects of processing time and moisturecontent on amino acid composition and nitrogen characteristicsof feather meal. Anim. Feed Sci. Technol. 14:279-290.

13. Seeley, H. W., Jr., and P. J. Vandemark. 1962. The endosporestain, p. 47-48. In R. Emerson, D. Kennedy, R. B. Park, G. W.Beadle, and D. M. Whitaker (ed.), Microbes in action. W. H.Freeman & Co., San Francisco.

14. Sen Gupta, S. R., S. S. Nigam, and R. N. Tandan. 1950. A newwool degrading fungus-Ctenomyces species. Text. Res. J.20:671-675.

15. Shih, J. C. H. 1987. Ecological benefits of anaerobic digestion.Poult. Sci. 66:946-950.

16. Shih, J. C. H., R. H. Jonas, and M. L. Scott. 1977. Oxidativedeterioration of the muscle proteins during nutritional musculardystrophy in chicks. J. Nutr. 107:1786-1791.

17. Steiner, R. J., R. 0. Keilems, and D. C. Church. 1983. Featherand hair meals for ruminants. IV. Effects of chemical treatmentsof feather and processing time on digestibility. J. Anim. Sci.57:495-502.

18. Williams, C. M., and J. C. H. Shih. 1989. Enumeration of somemicrobial groups in thermophilic poultry waste digesters andenrichment of a feather-degrading culture. J. Appl. Bacteriol.67:25-35.

VOL. 56, 1990

on May 19, 2018 by guest

http://aem.asm

.org/D

ownloaded from