Microbial Population Changes and Fermentation Characteristics of Ensiled Bovine Manure-Blended Rations

Microbial Population Changes and Fermentation Ensiled B o v i n e M a n u r e - B l e n d e d

Characteristics of

Rations E. F. KNIGHT 1 , T. A. McCASKEY, W. B. ANTHONY, and J, L. WALTERS Department of Animal and Dairy Sciences Agricultural Experiment Station Auburn University Auburn, AL 36830

ABSTRACT

led to extensive research in recycling animal wastes. Animal wastes possess substantial nutritional value and yield satisfactory results when fed to farm animals (2, 3, 9, 11). Ensiling bovine manure with a basal ration is an economical and convenient means of preserving waste-blended rations and insuring the safety of the product (12). In the ensiling process, lactic acid-producing bacteria that occur naturally on plant material ferment water soluble carbohydrates to lactic acid, and the reduction in pH and establishment of anaerobic conditions suppress undesirable activities of aerobic and sporeforming microorganisms (20). The purpose of this study was to investigate the principal microbiological and chemical changes that occur in ensiled bovine manure-blended rations and to characterize the predominant acid-producing bacteria.

Manure from beef steers confined on concrete was blended with a basal ration at 20, 40, and 60% and ensiled at 25 C. The moisture content of the rations increased with manure added. The rations with higher moisture developed a lower pH and higher lactic acid during the 10-day ensiling. Coliform bacteria were not detected in rations with 40% and 60% manure after 5 days ensiling and after 10 days in the 20% manure-blended ration. Coliform count of the basal ration (no manure) remained constant at about 104/g. When pH dropped to 4.4 to 4.7, coliforms died in all manure-blended rations. Salmonellae were isolated twice from 66 samples of the manure-blended rations and ration constituents (basal ration and m~nure) prior to ensiling. Salmonellae were not recovered from the salmonellae positive manure-blended rations after 3-days ensiling. The total bacterial count, numbers of acid-producing bacteria, and numbers of yeasts and molds decreased in the manure-blended rations after 10 days ensiling. Sporeforming bacteria survived but did not proliferate in the ensiled rations. The predominant acid-producing bacterium in the manure-blended rations prior to ensiling was Streptococcus faecalis. After 10 days ensiling Lactobacillus plantarurn was the predominant type.

MATERIALS AND METHODS Collection and Preparation of Bovine Manure-Blended Rations

INTRODUCTION

Pollution abatement plus an increasing demand for more efficient sources of feed have

Received October 20, 1976. 1National Sales, Inc., P. O. Box 1019, Jackson, MS 39205.

Manure from beef steers confined on concrete was scraped daily into a pit and twice weekly blended with a basal feed composed of 81.5% ground corn, 2.0% cottonseed meal, 7.5% dehydrated alfalfa meal, 7.5% alfalfa hay (ground), 1.0% defluorinated phosphate, and .5% trace mineralized salt. Wet bovine manure was blended with the basal feed at 20, 40, and 60%. The bovine manure used in formulating these rations was collected from three separate pens of steers continually receiving a diet of their recycled waste blended at 20, 40, and 60% in their ration. A portion of each mixture was brought to the laboratory and five portions of approximately 600 g of each ration were ensiled in polyethylene bags and incubated at 25 C. The basal feed was treated similarly to serve as a control.

416

FERMENTATION OF MANURE-BLENDED RATIONS Microbial and Chemical Examination of the Ensiled Manure-Blended Rations and Basal Feed

The manure-blended rations and basal feed were analyzed for total aerobic bacteria, coilforms, total acid producers, aerobic and anaerobic sporeformers, yeasts and molds, pH, and lactic and volatile acids after 0, 3, 5, 7, and 10 days ensiling. Prior to ensiling, each ration was analyzed for moisture content and for Salmonella. The microbial frequencies in the manure used in preparation of the manure-blended rations were analyzed also. Five separate trials for each of the three manure-blended rations and three trials for the basal ration (control without manure) were conducted. One bag of each ration was removed from the incubator after ensiling for 3, 5, 7, and 10 days. Each bag was opened aseptically, and an l l - g sample was collected from the center of each ration, weighed into a blender cup, and blended with 99 ml of sterile Standard Methods Phosphate Buffer (1). Enumeration of the various groups of indigenous microorganisms was accomplished by the Standard Plate Count Method (1). Two more samples of each ration were collected, one for pH determination and the other for Salmonella detection. The remaining ration in each bag was sealed and frozen for determination of lactic and volatile acid at a later time. The moisture content of the rations was determined by heating a sample at 100 C for 48 h. Loss in sample weight was assumed to be moisture. The pH of each ration after 0, 3, 5, 7, and 10 days ensiling was determined with a pH meter equipped with a glass electrode. In two separate trials, lactic and volatile acids were determined for each of the three manure-blended rations ensiled for 0, 3, 5, 7, and 10 days. Extraction of the acids was accomplished by weighing 5 g of each sample in a 23 × 100 m m test tube with 20 ml of distilled water and capping with a rubber stopper. The sample was held for 6 days at 5 C and then filtered through cheesecloth. The filtrate was centrifuged at 3164 × g (on a Sorvall refrigerated centrifuge, Model RC-3) for 20 rain. Part of the supernatant fluid was passed through a cation exchange resin (AGS0W-X4, 200 to 400 mesh, hydrogen form, washed in water; Bio Rad Laboratories, Richmond, CA), and the

417

eluant fluid was analyzed for volatile acids (acetic, propionic, butyric, and isovaleric) by the gas chromatographic procedure of Carsson (6). The apparatus was a dual-column gas chromatograph (Varian, Model 1200, Palo Alto, CA) equipped with hydrogen flame detectors and a simple recorder. The remainder of the supernatant fluid was analyzed for lactic acid by the colorimetric method of Barker and Summerson (4). The enumeration of the total aerbic and acid-producing bacteria was achieved on Tomato Juice agar (Difco) with .75% calcium carbonate added. Following plating these were incubated at 32 C for 48 h. Acid-producing bacteria were detected by a clearing zone around the colonies. All colonies on the plate were enumerated for the total aerobic count. Coliforms were enumerated on Violet Red Bile agar (Difco) after 24 h incubation at 32 C. Yeasts and molds were enumerated on Potato Dextrose agar (Difco) acidified to pH 3.5 with 10% tartaric acid for 5 days at 25 C. Duplicate 50-g samples of the manureblended rations and the manure used in formulating the rations were analyzed for Salmonella. Each 50-g sample was incubated in 350 ml of Tetrathionate broth (Difco) at 37 C for 24 h. The broth then was streaked on Brilliant Green Sulfa agar (Difco) and XLD agar (Difco). Suspected salmonellae colonies were picked from the agar, and the isolates were confirmed biochemically and serologically by the methods of Edwards and Ewing (8). The spore-heat test was used to select for sporeforming bacteria. The test was accomplished by heating 25 ml of a 1 : 10 dilution (11 g blended in 99 ml of Sorensen's Phosphate Buffer) of the rations at 80 C for 10 rain in 25 × 150 mm screw-cap tubes. Sorensen's Phosphate Buffer (pH 7.4) was prepared by mixing 80.4 ml of .15 M Na2HPO4 with 19.6 ml of .15 M KH2 PO4 (15). Sorensen's Buffer was used to maintain a pH near neutrality whereas Standard Methods Phosphate Buffer ( 1 ) a l l o w e d the pH of the mixtures to drop with the pH of the fermented rations. The samples were subjected to the spore-heat test, cooled in cold water, and the upper portion of the tube that was not below the level of the heated water was flamed with a Bunsen burner until hot. The cooled samples were plated in duplicate in Brain Heart Infusion agar (Difco). The aerobic sporeformers Journal of Dairy Science Vol. 60, No. 3

418

KNIGHT ET AL.

were enumerated by incubating one set of the plates at 32 C for 48 h. The anaerobic sporeformers were enumerated by incubating the other set of plates in a Brewer Anaerobic Jar with Gas Pak Anaerobic System (BBL) at 32 C for 5 days. Predominant Acid-Producing Bacteria in the Manure-Blended Rations

Prior to ensiling and after 10 days ensiling, isolates of acid-producing bacteria were picked at random from Tomato Juice agar plates, streaked for isolation, and transferred to Tomato Juice agar slants. These isolates were identified according to Bergey's Manual of Determinative Bacteriology (5), VPI Anaerobe Laboratory Manual (14) and by the methods of Gibbs and Skinner (10) and Whittenbury (19). RESULTS AND DISCUSSION

Data for the rations at each sampling period (0, 3, 5, 7, and 10 days) represent an average of three trials for the basal ration and five trials for the manure-blended rations. Moisture and pH Values of the Rations

The moisture content of the various manure-blended rations and the basal feed is in Table 1. The basal ration contained an average of 12.0% moisture which was increased by addition of wet bovine manure. The initial pH of the manure-blended rations increased with the manure added (Table 1). The pH of the manure ranged from 6.9 to 7.2. The pH values of the 40% and 60% manure-blended rations dropped to pH 4.5 within 3 days ensiling whereas the pH of the 20% ration required 10 days ensiling to drop to 4.7. The basal feed remained at about pH 6.0 throughout the 10 days. Acid production was related directly to the percent of manure blended into the rations. As the manure incorporated in the rations was increased a higher moisture content was achieved. Manure-blended rations with 40% and 60% manure (37.3% and 48.3% moisture) had pH values of 4.5 and 4.4 after 10 days ensiling indicating that about 37% moisture or higher was required for maximum acid production. Caswell et al. (7) reported that 40% moisture was required for maximum acid production during fermentation of broiler litter and wood Journal o f Dairy Science Vol. 60, No. 3

shavings. The final pH range of 4.4 to 4.7 in the manure-blended rations was comparable to the pH range of 4.0 to 4.6 reported for ensiled bovine manure-blended rations by McCaskey and Anthony (17) and pH 4.5 reported by Harpster et al. (13). Our pH values are lower than the values (pH 6.8 to 5.2) reported by Saylot and Long (18) for poultry litter ensiled 60 days with grass hay. Harmon et al. (12) reported pH values of 3.7 to 4.7 of broiler litter ensiled with corn forage for 61 to 71 days. Lactic and Volatile Acids

The manure-blended rations contained .14% lactic acid (DM) or less prior to ensiling (Table 1). After 10 days ensiling the rations containing 20, 40, and 60% manure had 2.37, 4.58, and 7.37% lactic acid on a dry matter basis (DM). Maximum acid production measured as percent lactic acid (DM) increased as manure was increased to 60%. However, acid production measured as pH (wet basis) did not increase appreciably with the addition of manure beyond 40%. Harmon et al. (12) reported lactic acid of 4.19% to 8.82% for poultry litter ensiled with corn forage for 61 to 71 days. Harpster et al. (13) reported 3.89% lactic acid (DM) for ensiled rations containing 60% bovine manure. Volatile acids (acetic, propionic, butyric, and isovaleric) increased with manure in the rations (Table 1). However, change was little during the 10-day ensiling which indicated heterofermentative organisms were relatively inactive during fermentation of the rations. Populations of Indigenous Groups of Microorganisms

Prior to ensiling, the basal feed (no manure) had 106 bacteria/g whereas the manure-blended rations (basal with manure) contained an average of 108 bacteria/g (Fig. 1). During 7 days of ensiling, the total bacteria count of the 20% ration remained at 108/g and dropped to 107/g after 10 days of ensiling. The total bacteria count of the 40% and 60% rations declined faster and to a lower count during 10 days of ensiling than did the count for the 20% ration. The ration containing 60% manure had a total bacteria count after 10 days ensiling similar to the basal feed. The number of total bacteria in the basal feed was relatively unchanged during

TABL E 1. Chemical parameters of ensiled bovine m a n u r e - b l e n d e d rations. Manure level

Days ensiled

Percent moisture

0 3 5 7 10

12.0

0 3 5 7 10

24.5

0 3 5 7 10

37.3

0 3 5 7 10

48.3

pH

Lactic acid

Acetic acid

Propionic acid

Butyric acid

lsovaleric acid

(Percent DM) 0% a

20%

40%

r~

6.0 6.0 6.2 5.9 6.1

Z -q

6.5 5,2 5.0 4.9 4.7

.03 .95 .97 1.50 2.37

.30 .38 .67 .51 .56

.029 .034 ,057 .058 .065

.019 .012 .015 .020 .O21

.008 .002 NDb ND ND

6.8 4.5 4.5 4.5 4,5

.02 4.00 4.02 4.89 4.58

.79 .74 .81 .80 .83

.108 .O10 .116 .087 .097

.089 .O78 .090 .082 .081

.011 .002 .002 .002 .001

6.8 4.6 4.6 4.5 4.5

.14 4.53 4.42 7.05 7.37

1.33 1.23 1.36 1.41 1.46

.132 .110 .132 .143 .126

.I 29 .145 .161 .137 .137

.020 .002 ND ND ND

Q

> 7

t" Z

7~

E

60%

E Z

aBasal ration. t~ <

Z

bNondetectable.

4~

420

KNIGHT ET AL. LEVEL OF MANURE 0 ~-Q' =

°.I

0 0% -*, 20% 13 40% = 60%

E = io7 o 1-

I

|

I

I

5

5

7

10

OAYS ENSILED

FIG. 1. Effect of ensiling time on total bacterial count in manure-blended rations.

the 10 days. The number of coliform organisms in the rations prior to ensiling increased with manure blended into the rations (Fig. 2). The basal feed

LEVEL OF MANURE I0 ?

o

,

-"

o%

:. ~0%

E~ 43 4 0 % H 6 0 %

rO6

~

o

10 5

~10 4

o

,0

10 2

i0 ~

I

3

5

7

I0

DAYS ENStLED

FIG. 2. Effect of ensiling time on coliform bacteria in manure-blended rations. Journal of Dairy Science Vol. 60, No. 3

contained 104 coliforms/g throughout the 10 days, whereas the 40% and 60% rations declined to nondetectable after 5 days ensiling. Ten days were required for the coliforms to decrease to nondetectable in the 20% ration. Destruction of coliforms in the ensiled rations was attributed to acid development. Rations with 40% and 60% manure developed acid more rapidly than the 20% ration which resulted in a more rapid die-off of coliforms. The greater acid development of rations with more manure also may be attributed to the higher moisture o f t h e s e rations (Table 1). Coliform bacteria decreased to nondetectable in all the ensiled manure-blended rations when the pH declined to 4.4 to 4.7 whereas the basal feed (pH 6.0) contained viable coliforms throughout the 10 days. Three days of ensiling were required for the 40% and 60% rations to reach pH 4.4 to 4.7, and 10 days for the 20% ration. These results show that manure-blended rations may be rendered safe from coliforms by ensiling for sufficient time to develop acid to a pH of 4.7 or less. These results support the work of Caswell et aL (7) in that complete destruction of coliforms occurred after ensiling broiler litter with wood shavings with 20 to 50% moisture. Litter alone adjusted to 40% moisture completely destroyed all coliforms. McCaskey and A n t h o n y (17) reported decreases in coliform counts from 9.7 × 106 to 1.2 X 103 in bovine manure-blended rations ensiled for 4 days at 25 C. Harmon et al. (12) reported a decline in coliforms to less than 150/g after ensiling poultry litter with corn forage for 61 to 71 days. Salmonellae were isolated from two of 66 samples (6 basal feed, 30 manure, and 30 manure-blended samples). Salmonella was isolated from one sample of fresh manure but was not isolated from the manure-blended ration prepared from the manure. Another Salmonella isolate was in a sample of 60% ration but was not in the manure used to prepare the ration. Samples positive for salmonellae were rechecked following 3 and 5 days of ensiling, but no salmonellae were recovered on either of these days. This further confirms McCaskey and Anthonys' (17) observation that salmonellae do not survive in ensiled bovine manure after fermentation for 3 days. Acid-producing bacteria are responsible for the lactic acid fermentation that is essential for

421

F E R M E N T A T I O N OF MANURE-BLENDED RATIONS

the production of good quality silage. The number of acid-producing organisms in the manure-blended rations was 107]g in the 40% ration and 10S/g in the 60% ration (Fig. 3). The acid producers in the basal feed remained about lOS/g during the 10 days. The number of acid producers in the manure-blended rations prior to ensiling increased with manure incorporated into the basal feed. During ensiling acid producers declined faster in the rations containing the higher manure. There was no appreciable change in numbers of acid producers in the basal feed during the 10 days. The initial number of yeasts and molds in all rations including the basal feed was 106/g (Fig. 4). The yeast and mold count declined to 104/g after 10 days ensiling for the 20% ration, 103/g for the 40% and 60% rations, and the basal feed remained at 106 yeasts and molds/g. Reduction in the population of yeasts and molds in the ensiled rations is important in preventing spoilage of the rations. Yeasts and molds utilize residual carbohydrates and lactic acid resulting in an undesirable silage. The sporeforming bacteria remained fair/y constant in the manure-blended rations throughout the 10 days ensiling (Fig. 5 and 6). These organisms were able to withstand the

0 zL O

o

0 0% z. 20% [] 40% 41 6 0 %

:E

0 O

Ld

0 0% ~ 20% D 40% 60%

,d

104

!

t

I

I

3

5

7

IO

DAYS ENSILED

FIG. 4. Effect o f ensiling time on yeasts and molds in manure-blended rations.

conditions imposed on them during the ensiling process; however, they did not proliferate in the rations. The aerobic sporeformers (Fig. 5)

,°71

LEVEL OF MANURE t09

LEVEL OF MANURE

l0? k

LEVEL OF MANURE

0 00% ~--------~ 20 % n D40 % ¢

¢ 60%

II I0 ~

Q

~o

:~ I0 ( 0

105

I

I

I

•

3

5

7

I0

DAYS ENSILED

FIG. 3. Effect o f ensiling time on acid-producing bacteria in manure-blended rations.

I

|

I

I

3

5

7

I0

DAYS ENSILED

FIG. 5. Effect o f ensiling time on aerobic sporeforming bacteria in manure-blended rations. Journal of Dairy Science Vol. 60, No. 3

422

KNIGHT ET AL. TABLE 2. Profile of acid-producing bacteria in manure-blended rations.

LEVEL OF MANURE C 0 0% ~~ 20% r't rt 40% ¢ ¢ 60%

,d
Level of manure

o:

Prior to ensiling

20%

40%

60%

t9

Prior to ensiling No, of isolates Fecal streptococci Pediococcia Lactobacilli Gram. neg.b Other

63 27 19 7 0 10

47 22 18 6 0 1

59 33 9 12 1 4

o

After 10 days ensiling No. of isolates

53 38 15

45 45 0

33 32 1

a o: I0E

z

L, plantarum Lactobacillus spp. d

I

I

I

3

5 DAYS

1

I

7

I0

ENSILED

FIG. 6. Effect of ensiling time on anaerobic sporeforming bacteria in manure-blended rations.

remained at 106/g whereas in the basal feed they increased from 104 to 10S/g. The anaerobic sporeformers (Fig. 6) were constant at 10S/g in the manure-blended rations whereas in the basal feed they increased from 104 to 10S/g during the ensiling period. Since these organisms are responsible for undesirable butyric acid fermentation in silage and since they represent a potential health hazard if certain pathogenic members become established in silage, it is imperative that their proliferation be prevented. Manure from cattle fed the manure-blended rations was tested to determine if any change in the microbial population occurred as the result of recycling. In the population of microbes tested (total bacteria, coliforms, acid producers, yeasts and molds, and aerobic and anaerobic sporeformers) there appeared no significant change in their concentration in the manure after 3 mo of recycling. Predominant Acid-Producing Bacteria

The predominant acid producers prior to ensiling were fecal streptococci which were approximately one-half of the day-0 isolates (Table 2). As manure was increased in the rations there was an increase in the proportion Journal of Daffy Science Vol. 60, No. 3

apediococcus urinae-equi. bproteus inconstans. CCatalase + Gram + Coryneform-like rods. dLong thin rods that require anaerobic conditions for growth.

of fecal streptococci. Pediococcus urinae-equi, Lactobacillus spp., one isolate of Proteus inconstans (Gram-negative rod) and coryneformlike Gram-positive rods constituted the remainder of the day-O acid-producing bacteria. Approximately half of the fecal streptococci were S. faecium and S. faecalis. The remainder were streptococci exhibiting fecal streptococci characteristics but different from S. faecium and S. faecalis. The various lactobacilli isolated prior to ensiling were all homofermentative except for one heterofermentative L. fermentum isolated from the 60% ration. The homofermentative lactobacilli isolated were L. plantarum, L.

xylosus, L. leicbmannii, L. delbrueckii, L. lactis and L. belveticus. Laetobacillus plantarurn was the predominant acid producer after 10 days ensiling (Table 2). All isolates from the manure-blended rations ensiled for 10 days were identified as L. plantarum except for 15 of 53 isolates from the 20% ration and 1 of 33 in the 60% ration. These 16 organisms were Lactobacillus spp. that required anaerobic conditions for growth. According to Woolford (20) Lactobacillus plantarum is the most suitable organism for silage fermentation. The results of this study

FERMENTATION OF MANURE-BLENDED RATIONS agree w i t h H r u b a n t ' s r e p o r t (16) t h a t L. plantarum was t h e p r e d o m i n a n t m i c r o o r g a n i s m a f t e r f e e d l o t waste was f e r m e n t e d w i t h corn. T h e p r e s e n c e o f t h e fecal s t r e p t o c o c c i , und o u b t e d l y a d d e d w i t h t h e m a n u r e , p l a y e d an i m p o r t a n t p a r t in t h e early f e r m e n t a t i o n of t h e m a n u r e - b l e n d e d r a t i o n s . W o o l f o r d (20) suggested t h a t a n o r g a n i s m such as Streptococcus faecalis w h i c h p r o d u c e s lactic acid in t h e p H range of 5.0 t o 6.5, w o u l d aid greatly t h e f e r m e n t a t i o n since L. plantarum is slow in lactic acid p r o d u c t i o n u n t i l t h e p H fails b e l o w 5. T h e s t r e p t o c o c c i e v e n t u a l l y w o u l d give way to t h e lactobacilli as t h e p H declined. A p p a r e n t ly t h i s " i d e a l " c o m b i n a t i o n o c c u r r e d in t h e f e r m e n t a t i o n o f t h e ensiled m a n u r e - b l e n d e d r a t i o n s in this study.

REFERENCES

1 American Public Health Association. 1972. Standard methods for the examination of dairy products. 13th ed. APHA, New York. 2 Anthony, W. B. 1970. Feeding value of cattle manure for cattle. J. Anita. Sci. 30:274. 3 Bandel, L. S., and W. B. Anthony. 1969. Wastelage-digestibility and feeding value. J. Anita. Sci. 28:152. (Abstt.) 4 Barker, S. B., and W. H. Summerson. 1941. The colorimetric determination of lactic acid in biological material. J. Biol. Chem. 138: 535. 5 Buchanan, R. E., and N. E. Gibbons. 1974. Bergey's manual of determinative bacteriology. Williams and Wilkins Co., Baltimore. 6 Carlsson, J. 1973. Simplified gas chromatographic procedure for identification of bacterial metabolic products. Appl. Microbiol. 26:287. 7 Caswell, L. F., J. P. Fontenot, and K. E. Webb, Jr.

423

1975. Ensiled broiler litter with different moisture levels. J. Anim. Sci. 40:200. (Abstr.) 8 Edwards, P. R., and W. H. Ewing. 1972. Identification of enterobacteriaceae. Burgess Publ. Co., Minneapolis, MN. 9 Fontenot, J. P, A. N. Bhattacharya, C. L. Drake, and W. H. McClure. 1966. Value of broiler litter as feed for ruminants. Proc. Nat. Syrup. on Anim. Waste Manage. ASAE Pub. SP-0366:105. 10 Gibbs, B. M., and F. A. Skinner. 1966. Identification methods for microbiologists. Academic Press, New York. 11 Harmon, B. G., D. L. Day, A. H. Jensen, and D. H. Baker. 1972. Nutritive value of aerobically sustained swine excrement. J. Anita. Sci. 34:403. 12 Harmon, B. W., J. P. Fontenot, and K. E. Webb. 1975. Ensiled broiler litter and corn forage. 1. Fermentation characteristics. J. Anita. Sci. 40:144. 13 Harpster, H. W., T. A. Long, C. M. Lalonde, and W. W. Saylor. 1975. Nutritive value of ensiled cattle waste. J. Anita. Sci. 41:240. 14 Holdman, L. V., and W. E. C. Moore. 1975. Anaerobe laboratory manual. Southern Printing Co., Blacksburg, VA. 15 Koch, F. C., and M. E. Hanke. 1948. Practical methods in biochemistry. Williams and Wilkins Co., Baltimore. 16 Hrubant, G. R. 1975. Changes in microbial population during fermentation of feedlot waste with corn. Appl. Microbiol. 30:113. 17 McCaskey, T. A., and W. B. Anthony. 1975. Health aspects of feeding animal wastes conserved in silage. Proc. 3nd Intern. Syrup. on Livestock Waste, University of Illinois, Urbana. 18 Saylor, W. W., and T. A. Long. 1974. Laboratory evaluation of ensiled poultry waste. J. Anita. Sci. 39:139. (Abstr.) 19 Whittenbury, R. 1965. A study of some pediococci and their relationship to Aerococcus viridans and the enterococci. J. Gen. Microbiol. 40: 97. 20 Wolford, M. K. 1972. Some aspects of the microbiology and biochemistry of silage making. Herbage Abstr. 42:105.

Journal of Dairy Science Vol. 60, No. 3