DIGESTION OF THE FIBER COMPONENTS OF MAIZE ...digeset.ucol.mx/tesis_posgrado/Pdf/Daniel Contreras...
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UNIVERSIDAD CONACyT DE COLIMA DIGESTION OF THE FIBER COMPONENTS OF MAIZE STEM AND CELLULOSE BY RUMEN ANAEROBIC FUNGI AS AFFECTED BYDIFFERENTSULPHURSOURCES A dissertation submitted in partial fulfilment of the requeriments for the degree of DOCTOR IN MICROBlA~BIOTECHNOLOGY Asesores PhD. Héctor González Cerezo PhD. Geoffrey 1. R. Gordon l UNIVERSIDAD DE COLIMA Facultad de Ciencias Biológicas y Agropectiarias 1996
Transcript of DIGESTION OF THE FIBER COMPONENTS OF MAIZE ...digeset.ucol.mx/tesis_posgrado/Pdf/Daniel Contreras...
UNIVERSIDAD CONACyTDE COLIMA
DIGESTION OF THE FIBER COMPONENTS OF MAIZE STEM ANDCELLULOSE BY RUMEN ANAEROBIC FUNGI AS AFFECTED
BYDIFFERENTSULPHURSOURCES
A dissertation submitted in partial fulfilment of the requeriments for the degree of
DOCTOR IN MICROBlA~BIOTECHNOLOGY
Asesores
PhD. Héctor González CerezoPhD. Geoffrey 1. R. Gordon
l
UNIVERSIDAD DE COLIMAFacultad de Ciencias Biológicas y Agropectiarias
1 9 9 6
DIGESTION OF THE FIBEW COMPONEN-I% OF MAIZE STEM ANDCELLULOSE BY I2UMEN ANEROBIC FUNGI AS AFFECTED
BY DIFFEKENT SULPHUR SOURCES
BY
.t
LMNIELCONTRE~SL~
A dissertatim submitted in partial fblftient of the requirements for fhe degree of
DOCTOR IN MICROBIAL BIOTECHNOLOGY
Asesores
PhD. Hwl3r Gom&z CemzoPhD. Geot’frey L.R. GiKdon .
UNIVERSIDAD DE COLIMAFacultad de Ciencias Biolbgicas y Agropecuarias
EI presente trabajo fue auspiciado por el CONACYT dentro delproyecto “Digestión de Paredes Celulares por Hongos Anaerobios delRumen estimulando su Crecimiento con Azufre”. Convenio 4754-N9406.
Alumno:Daniel Contreras Lara
Grado:Doctor en Biotecnología Microbiana
Institución:Universidad de Colima
Facultad de Ciencias Biológicas y Agropecuarias
Tutores:Dr. Luis Felipe Bojalil JaberDr. Miguel Arenk Vargas
Dr. Héctor González CerezoDr. Roger E. Calza
Dr. Geoffi-ey L.R. GordonDr. Fausto Sánchez y García Figueroa
Dr. Danny E. Akin
CSIRO Division of Animal Production
Locked brg 1, Delivery Centre, BlacktownNew South Wnles 2148, Australia
b . (02)840-2700Fax (02)840-2940
24 May 1995
Dr Daniel Contreras LaraFacultad de Medicina Veterinaria y ZootecniaUniversidad de ColimaApartado Postal No. 3628 I OO Tecoman, ColimaMEXICO
Dear Daniel,
‘Re: “Digestion of tire fiber components of mrize stem by rumen rnaerobic fungi IISrnected by different sulfur source@
As you requested, 1 have read this ninety-page manuscript and now make the following commentson it.
The introduction section adequately covers the current state of knowledge on anaerobic fungí,particularly In relatjon to fibre degradation and the influente of dietary sulfir on anaerobíc tingalpopulations ín the rumen.
The methods section is generally adequate for the methods actudly given. A good methods sectionis important for the understanding of the meaning of the experimental results. However this sectionneeds some attention in thc following ateas:1.
2 .
3.
4 .
The presente of much of thedetailed information in an annex is a usefirl format but this factshould be stated very early in the methods section so that a reader is aware that the annexexists.The sulfur content of the maize stem substrate and of the diet. This information is given inthe text on page 3 1 and in Table 5. However, there appears to be no mention of theanalytical method used to measure sulfir. The method should be described, since most ofthe experiments deal with the effect of sulf%r on the rumen anaerobic tingi.The methods given for in vitro incubation of strained rumen fluid (SRF) with maize stemshould be explained more carefilly to allow the experiment to be independently repeated.Beginning at the bottom of page 33 the following information is required: the size of flaskused and an alteratjon to the sentence to give “... containing an accurrtely weighedsample of maize stem (approximately 0.5 g) and 50 ml basal media . . ..“.The antibiotics penicillin, streptomycin and chloramphenicol will not inhibit rumen protozoaas stated on page 34. This statement needs correction, both hete and at other places in themanuscript where it is made. The antibiotics used in these experiments affect onlyprocatyotes (bacteria) and have no enèct on eucaryotes (fungi and protozoa).
The presentation of the results is generally good. 1 suggest that the standard deviations for fivereplicate cultures be reported in Table 10 (page 55) since this information has been reported for theresults with maize stem in Tables 8 & 9 (pages 45 & 48) and Table bl (page 60).
The discussion section and the conclusions are reasonably good. In places there is some repetitionof results cited eadier in the section. However any changes in this area is a matter for the writer todecide.
A large number of corrections of the English have been made directly on the manuscript.
Yours sincerely,
G.L.R. GordonPrincipal Research Scientist
CSIRO Division of Animal Production
Lmcked Bng 1, Delivey Cedre, BlncktownNew South Wnles 2148, Aastrnlin
(02) R40-2700
lPn⌧ (02) 040.2940
10 July 1995
.
Lic. Fernando Moreno PeRaRectorUniversidad de ColimaAvenida Universidad No. 333Colima, Col.MEXICO
Dear sir,
1 wrlte to you at the request of Daniel Contreras Lara. Reccntly, 1 have revicwcd themanuscript of his thesis, entitled “Digestion of the tiber components of maize stcm by rumcnanaerobic fungi as affected by different sulfur sources”. A number of comments were madcon the manuscript togcther with scveral suggestions for its improvemcnt. Now thnt DnniclContreras Lara has acted on these comments, 1 recommend that the thesis is in a properstatc for the candidate to formally dcfend it.
Yours sincerely,
G.L.R. GordonPrincipal Research Scientist
ACKNOWLEDGMENTS
I would like first to sincerely thank Lic. Fernando
Moreno Pella, rector of the Universidad de Colima for his
generous support to made it possible to the completion of my
program. Acknowledgments also go to Ing. Lorenzo Hernandez
Arreguin for the participatfon and supporter during my
doctoral formation.
Acknowledgment and appreciatíon ís expressed to the
Universidad de Colima and Consejo Nacional de Ciencia y
Tecnología (CONACYT) for their economic support to complete
this study.
Appreciation is also extended to Drs. Héctor González
Cerezo (UAM-Mt%xico), Geoff L.R. Gordon (CSIRO-Australia),
Roger E. Calza (WSU-USA), Danny E. Akin (USDA-USA) for their
excellent technical advice during this study.
1 would also like to thank Drs. Luis Felipe Bojal i l
Jaber and Miguel Arenas Vargas, for their knowledge, guidance
and encouragement in the research and document preparation.
Sincere appreciation is extended to the members of my
academic and graduate commitee.
To my other special friends, Gustavo Ceballos, Juan
Mesina Alatorre, Carlos Izquierdo, Leonardo Guti(irrez,
Trinidad Ramirez, Edelmira Galindo, Rambn Govea, thank you
for the support and friendship that has been essential to the
completion of my program.
iv
1 w o u l d l i k e t o e x p r e s s m y g r a t e f u l n e s s t o m y p a r e n t s
Ani ta and Jos& and to my twelve b r o t h e r s . F i n a l l y , m y
g r a t i t u d e and love is extended to my w i f e S o n i a , and
daughters Bianca and JuliB, and my son Daniel.
DIGESTION DE LOS COMPONENTES DE LA FIBRA DE TALLOS DE MA12 YDE CELULOSA PURA POR HONGOS ANAEROBIOS DEL RUMEN
AFECTADOS POR DIFERENTES FUENTES DE AZUFRE.
Daniel Contreras LaraUniversidad de Colima, Facultad de Ciencias Biolbgicas y
Agropecuarias. Apdo. Postal. # 36, Tecoman, Col.C.P. 28100 MEXICO. Tel./Fax. 332/ 4-42-37.
RESUMEN
S e utilizo un medio de cultivo convencional con
algunas modificaciones par estudiar el efecto del azufre
sobre la digestion de tallos de maiz y celulosa pura por
hongos anaerobios del rumen. Como fuentes de azufre se
utilizaron sulfato, sulfuro y sulf ito en concentraciones
para suministrar OX, O.l%, 0.2% y 0.3% de azufre. Un
experimento adicinal se realizo, para evaluar la curva de
digestibn de celulosa pura durante 8 dias con 0.2% de azufre
en forma de sulfuro. Se encontrA que las tres fuentes de
azufre aumentaron l a digestiAn d e l o s tallos de maiz y
celulosa pura comaparando con el c o n t r o l . U n incremento
significativo (P<0.0001I en la digestion de materia seca
(MS), fibra detergente neutro (FDN), fibra detergente Acido
(FDAI , celulosa, hemicelulosa, lignina y celulosa pura se
observA con el uso de sulfuro. El nivel Aptimo de azufre
para todas las fuentes usadas fue 0.2% en el medio de
cultivo. Se encontrA un aumento en la actividad enzimAtica de
vi
d e C a r b o x y m e t i l c e l u l a s a (CMCasa), B - g l u c o s i d a s a , y produccion
d e p r o t e i n a , c o r r e l a c i o n a d o c o n l a d i g e s t i o n d e c e l u l o s a
pura. E l n i v e l o p t i m o p a r a l a a c t i v i d a d enzimAtica f u e d e
0 . 2 % d e a z u f r e e n l a s t r e s f u e n t e s e v a l u a d a s . Fue observado
u n i n c r e m e n t o p r o p o r c i o n a l e n l a d i g e s t i o n d e c e l u l o s a p u r a y
a c t i v i d a d e s enzimhticas cada 24 hora s duran te 0 dias. La
mbxima d i g e s t i o n d e c e l u l o s a p u r a y a c t i v i d a d e s enzimAticas
se o b t u v o a l a s 192 hora s . Todas l a s f u e n t e s d e a z u f r e
e v a l u a d a s i n c r e m e n t a r o n l a digestibn d e l o s t a l l o s d e m a i z y
c e l u l o s a p u r a . E l s u l f u r o ( f u e n t e r e d u c i d a d e a z u f r e ) fue
m e j o r u t i l i z a d o p o r l o s h o n g o s d e l r u m e n . E l n i v e l o p t i m o d e
a z u f r e p a r a e l c r e c i m i e n t o d e l o s h o n g o s d e l r u m e n y p o r e n d e
u n a m a y o r a c t i v i d a d enzimAtica fue e l 0 .2%. Los componentes
de l o s forrages i n t a c t o s m u e s t r a n u n a d i g e s t i o n menor que
l o s c o m p u e s t o s p u r o s , como la celulosa.
DIGESTION OF THE FIBER COMPONENTS OF MAIZE STEM ANDCELLULOSE BY RUMEN ANAEROBIC FUNGI AS AFFECTED
BY DIFFERENT SULPHUR SOURCES
D a n i e l C o n t r e r a s L a r aU n i v e r s i d a d d e C o l i m a , F a c u l t a d d e C i e n c i a s Biologicasy Agropecuarias. Apar tado pos t a l No . 36 , Tecomhn, C o l .
C.P. 28100 MEXICO. Phone z (332) 4 - 4 2 - 3 7 .Fax : (332) 4 - 2 2 - 2 9 o r 4 - 4 6 - 4 2 .
SUMMARY
Ruminants c a n u s e plants w i t h a h i g h f i b r e content a s
f e e d s t u f f s , due to the breakdown of t h i s m a t e r i a l b y a
comp 1 ex microbial p o p u l a t i o n in t h e r u m e n . Until the mid
1970s only two g r o u p s o f rumen microorganisms were
recognized; however, t h e e x i s t e n c e o f a n a e r o b i c fungf was
r e p o r t e d b y O r p i n in 1975. T h e s e f u n g i were f o u n d t o b e able
t o g r o w on f i b r o u s p l a n t m a t e r i a l s a n d p r o d u c e h i g h l e v e l s o f
e n z y m e s capable o f d e g r a d i n g p l a n t structural carbohydrates.
T h e n u t r i t i o n a n d b i o c h e m i s t r y o f r u m e n a n a e r o b i c fungi,is a t
p r e s e n t p o o r l y u n d e r s t o o d . T h e y c a n u s e a w i d e r a n g e o f p l a n t
c a r b o h y d r a t e s a s a carbon s o u r c e . A f e w e x p e r i m e n t s provided
evidente t h a t a d e f i c i e n c y o f d i e t a r y s u l p h u r could l i m i t
t h e i r g r o w t h in t h e r u m e n , a n d hence l i m i t t h e i r c o n t r i b u t i o n
to p l a n t t i s s u e d i g e s t i o n . T h e r e s p o n s e in v i t r o o f r u m e n- -
f u n g i to d i f f e r e n t s o u r c e s a n d l e v e l s o f s u l p h u r w a s m e a s u r e d
W the rate o f m a i z e s t e m c o m p o n e n t e s a n d pure c e l l u l o s e
d i g e s t i o n . R u m i n a l digesta o b t a i n e d f r o m a gelded male g o a t
V i i i
f e d a h i g h f i b e r d i e t was used a s i n o c u l a . M a i z e s t e m m i l l e d
to 1 m m a n d pure c e l l u l o s e (Sigmacell) was used a s a carbon
source. S o d i u m s u l f a t e , s o d i u m s u l f i d e , a n d sodium s u l f i t e
w e r e t e s t e d a s s u l f u r s o u r c e a n d a d d e d in a c o n c e n t r a t i o n o f
O X , O.l%, 0.276, and 0 .3%. A conventional m e d i a w a s used with
c e r t a i n m o d i f i c a t i o n s t o e n a b l e t h e s t u d y o f a d d i t i o n s
o f inorganic sulphur t o a medium deficient in s u l p h u r .
Erlenmayer f l a s k s c o n t a i n i n g 0 . 5 g o f maize s t e m a n d 50 ml
media w e r e f l u s h e d w i t h C 0 2 , s t o p p e r e d a n d autoclaved. For
d e t e r m i n a t i o n o f d r y m a t t e r loss f l a s k s w e r e i n o c u l a t e d
separa te l y w i t h 4 m l o f r u m i n a l f l u i d a n d a n t i b i o t i c s mix.
T h e r e s i d u e s r e m a i n i n g a f t e r i n c u b a t i o n a t 3 9 C b y 8 d a y s
were analyzed f o r d r y m a t t e r . Supernatant was recovered and
the pH was measured immediately. The r e s i d u e s after
i n c u b a t i o n w e r e a n a l i z e d f o r n e u t r a l d e t e r g e n t f i b e r (NDF) ,
acid d e t e r g e n t f i b e r (ADF), a n d acid d e t e r g e n t l i g n i n (ADL).
C e l l u l o s e a n d h e m i c e l l u l o s e w e r e c a l c u l a t e d c o n s i d e r i n g the
NDF , ADF, and ADL determinations. The analytical d a t a w e r e
compared w i t h a c o n t r o l w i t h o u t s u l p h u r s o u r c e . F o r in v i t r o- -
d i g e s t i o n o f pure c e l l u l o s e , culture tubes c o n t a i n i n g 0 . 1 g
S i g m a c e l l t y p e 1 0 1 a n d 1 0 m l m e d i a w e r e used. F i ve tubes w e r e
i n o c u l a t e d w i t h 0 . 5 m l o f r u m e n i n o c u l u m an 3 0 u l o f m i x o f
a n t i b i o t i c s s o l u t i o n . T h e r e s i d u e s a f t e r 8 days incubation
were analyzed f o r c e l l u l o s e disappearance, and enzyme
a c t i v i t i e s . An a s s a y w a s d o n e u s i n g culture tubes described
iX
above but using sulphur 0.2% as sodium sulfide. The residues
remaining each 24 hours for 8 days were tested for
cellulose loss and enzyme activities. It has been observed
that three sulphur sources promoted maize stem and pure
ce1 lulose digestion compared to control wi thout sulphur.
The greater increase (P>O.OOOll for dry matter, NDF, ADF,
cellulose, hemicellulose, 1 ignin and pure cellulose
digestion was observed with the addition of sulphur as
sulfide. The optimum leve1 of sulphur in the media
(P<0.0001) was 0.2% for al1 sulphur sources. Increasing
Carboxymethylcellulase (CMCase) and B-glucosidase activity,
and protein production correlated with the cellulose
digestion. The optimum leve1 for enzyme activities was 0.2%
sulphur for al1 sources tested. A proportional increase of
pure cellulose digestion and enzyme activities each 24 hours
for 8 days was observed. The maximal pure cellulose digestion
and enzyme actitvity w a s obtained at 192 hours. In
conclusion, al1 sources tested increased the maize stem and
cellulose digestion. The sulphur as sulfide (reducing source)
w a s better utilized for rumen fungi. The leve1 of 0.2%
sulphur w a s optimum for al1 sulphur sources. Intact
roughages digestion are less than the digestion of their
individual compounds.
X
TABLE OF CONTENTS
ACKNOWLEDCMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i i i
RESUMEN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V i i
TABLE OF CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X i i
LIST OF FIQURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X i i i
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
ANTECEDENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Some characteristícs and the classification of rumenfungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Culture media and nutrition of the rumen fungi . . . . . . . . . . 8
Utilization of sulphur by ruminants . . . . . . . . . . . . . . . . . . . . . 9
Requirement of rumen microorganisms for sulphur . . . . . . . . . 14
The effect of sulphur on populations of ruminal fungi . . . 17
Fiber degradation and other digestive activity by rumenfungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Potential applications of ruminal fungi and theirenzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chemical analysis of ce11 wall constituents . . . . . . . . . . . .
MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Source of inocula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preparation of media for @J vitro incubations . . . . . . . . . .
In vitro incubations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 4
31
32
34
34
34
34
36
37
X i
Test to verify the efficacy of culture media withthree sulphur sources........................,....,...
Analysis of NDF, ADF, and ADL.............. .,..........
In v i t r o d f g e s t i o n o f c e l l u l o s e . . . . . . . . . . . . . . . . . . . . . . . .
Enzyme activity from cellulose fermentation............
RESULTS..................... ..,............................*.
Polyester bag technique (PBTI to analysis ofNDF, ADF, and ADL.... . . . . . . . . . . . . . . . . . . . ..i...........
Digestion of dry matter and ce11 wall components fromm a i z e s t e m . . . . . . . . . . ..,................................
Cellulose digestion by rumen fungi... . . . . . . . . . . . . . . . . . .
Enzyme activities for CMCase and B-glucosidase.........
Cellulose digestion by rumen fungi and sulphur assulfide.............. ..,..............*................
Enzyme activities for CMCase and B-glucosidaseinvolved in degrading cellulose by rumen fungi and0 .2% su l phu r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
DISCUSSION.... . . . . . . . . . . . . . . . . . . . . . . . . . . ..*..................
CONCLUS ION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
LITERATURE CITED................. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
38
39
45
45
48
48
50
61
64
66
69
73
88
91
ANNEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * . . . . . . . . . . * . ío5
LIST OF TABLES
TABLE
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
G l a s s i f i e d r u m e n a n a e r o b i c fungí..................... 7
P e r c e n t a g e d i g e s t i o n o f structural c o m p o n e n t s o fwhea t s t raw b y pure cultures o f r u m e n a n a e r o b í cf u n g í f o r 4 d a y s i n c u b a t i o n . . . . , . . . . . . . . . . . . . . . . . . . . 20
Degradation of wheat straw by the rumen fungíand rumen c e l l u l o l y t i c b a c t e r i a , í n m o n o c u l t u r e sand cocul tures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Degradation of maize straw by the rumen fungíand rumen c e l l u l o l y t i c b a c t e r i a , í n m o n o c u l t u r e sand aocul tures . . . . . . . . . . . . . . . . . . . . ..B...............
G o m p o s i t i o n o f t h e d i e t t o f e d t h e g o a t d o n o r o fr u m e n i n o c u l a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
29
30
35
G o m p a r i s o n o f p o l y e s t e r b a g t e c h n i q u e w i t h t h econventional proeedures for NDF, ADF, and ADL....... 49
pH changes and dry matter degradation from maizes t e m b y r u m e n a n a e r o b i c f u n g í w i t h t h r e e l e v e l s o ft h r e e s u l p h u r s o u r c e s f o r 8 days (XI . . . . . . . . . . . . . .
Ce11 w a l l s c o m p o n e n t s d e g r a d a t i o n o f m a i z e s t e mW rumen anaerobic f u n g í w i t h t h r e e l e v e l s o ft h r e e s u l p h u r s o u r c e s f o r 8 d a y s ( X I . . . . . . . . . . . . . . .
G e l l u l o s e d e g r a d a t i o n b y t h e r u m e n fungí wi th3 l e v e l s o f 3 s u l p h u r s o u r c e s a f t e r 8 d a y s ( X I . . . , . .
51
55
62
G a r b o x y m e t h y l c e l l u l a s e (GMGase) a n d B - g l u c o s i d a s ea c t i v i t i e s , a n d p r o t e i n í n an a s s a y u s i n g r u m e nf u n g í a n d t h r e e s u l p h u r s o u r c e s a f t e r 8 d a y si n c u b a t i o n o f c e l l u l o s e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
X i i i
LIST OF FIGURES
FIGURE
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
l l .
12.
13.
14.
S c h e m e o f f i b e r component a n a l y s i s . . . . . . . . . . . . . . . . . . . . . . . 4 1
I n c r e a s e c o m p a r e d t o c o n t r o l w i t h o u t s u l p h u r o f d r ym a t t e r d e g r a d a t i o n f r o m maize stem by rumen fungif o r 8 d a y s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
PH increase compared to no sulphur control fromd i f f e r e n t t r e a t m e n t s m o n i t o r e d a f t e r 8 d a y so f i n c u b a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
I n c r e a s e r e s p e c t t o c o n t r o l o f N D F d e g r a d a t i o n fromm a i z e s t e m b y r u m e n f u n g i a f t e r 8 d a y s o f i n c u b a t i o n . . . . 5 6
I n c r e a s e r e s p e c t t o c o n t r o l o f A D F d e g r a d a t i o n fromm a i z e s t e m b y r u m e n f u n g i a f t e r 8 d a y s o f i n c u b a t i o n . . . . 5 7
I n c r e a s e r e s p e c t t o c o n t r o l o f c e l l u l o s e degradationf r o m m a i z e s t e m b y r u m e n f u n g í a f t e r 8 d a y s o fi n c u b a t i o n . . . . . . . . . . .,..............................*... 58
Increase r e s p e c t t o c o n t r o l o f h e m i c e l l u l o s edegradation from maize stem by rumen fungi after 8d a y s o f i n c u b a t i o n . . . . . . . . . . . . . . . . . . * . . . . . . . . . . . . . . . . . . . 59
I n c r e a s e r e s p e c t t o c o n t r o l o f l i g n i n d e g r a d a t i o nf r o m m a i z e s t e m b y r u m e n f u n g i a f t e r 8 d a y s o finoubation . . . . . . . . . . . . . . . . . . . . . . . . . . ..*.............*.... 60
íncrease r e s p e c t t o c o n t r o l o f pure c e l l u l o s ed e g r a d a t i o n b y r u m e n f u n g í a f t e r 8 d a y s o f i n c u b a t i o n . . . 6 3
C e l l u l o s e d i s a p p e a r a n c e (X) r e c o r d e d b e t w e e n 2 4 a n d192 h in a m e d i u m w i t h 0 . 2 % s u l p h u r a n d Turnen f u n g i . . . . . 6 7
Changes in t h e p H t h r o u g h t h e i n c u b a t i o n p e r i o d w i t hrumen anaerobic fungi, c e l l u l o s e a n d 0 . 2 % s u l p h u r . . . . . . . 6 8
T i m e s c o u r s e s f o r c a r b o x y m e t h y l c e l l u l a s e (CMCase)(IU/mlI b y r u m e n a n a e r o b i c f u n g i g r o w i n g onc e l l u l o s e a n d 0 . 2 % s u l p h u r a s sulfide............., 7 0
T i m e s c o u r s e s f o r B - g l u c o s i d a s e (IU/ml) b y r u m e na n a e r o b i c f u n g i g r o w i n g on c e l l u l o s e a n d 0 . 2 % s u l p h u r . . 7 1
S u p e r n a t a n t p r o t e i n (ug/mlI r e c o r d e d d u r i n g 8 d a y sf o r r u m e n f u n g i i n c u b a t e d in presente o fc e l l u l o s e a n d 0 . 2 % s u l p h u r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2
viii
fed a high f iber d iet was used as inocula . Maize stem mil led
to 1 mm and pure ce l lu lose (Sigmacell) was used as a carbon
source . Sodium sul fate , sodium sul f ide , and sodium s u l f i t e
were tested as sul fur source and added in a concentrat ion o f
O%, O.l%, 0.2%, and 0 .3%. A conventional media was used with
certa in modi f i cat ions t o e n a b l e the study of addi t ions
o f inorganic sulphur t o a medium deficient in s u l p h u r .
Erlenmayer f lasks containing 0 .5 g o f maize stem and 50 ml
media were f lushed with C02, stoppered and àutoclaved. For
determinat ion o f drY matter loss f lasks were inoculated
separate ly with 4 ml o f ruminal f lu id and ant ib iot ics mix.
The res idues remaining a f ter incubat ion at 3 9 C b y 8 d a y s
were analyzed for dry matter . Supernatant was recovered and
the PI-I was measured immediately. The res idues af ter
incubat ion were anal ized for neutral detergent f iber (NDF) ,
acid d e t e r g e n t f i b e r (ADF), and acid detergent l ign in >(ADL).
Ce1 lulose and hemice l lu lose were ca lculated consider ing the
NDF > ADF, and ADL determinations. The analytical data were
compared with a control without sulphur source . F o r in v i t r o-
digest ion o f pure c e l l u l o s e , culture tubes c o n t a i n i n g 0 . 1 g
Sigmacell type 101 and 10 ml media were used. Five tubes were
inoculated with 0.5 ml o f rumen inoculum an 30 u l o f mix o f
ant ib iot ics so lut ion . The res idues a f ter 8 days incubation
were analyzed f o r ce l lu lose disappearance, and enzyme
a c t i v i t i e s . An assay was done using culture tubes described
1
INTRODUCTION
T h e p o s s i b i l i t y o f i n c r e a s i n g f i b e r d i g e s t i b i l i t y o f
high f i b e r c o n t a i n i n g m a t e r i a l b y r u m i n a n t s t o o b t a i n high
p r o t e i n produc ts is o f p a r t i c u l a r in te re s t in d e v e l o p i n g
c o u n t r i e s , w h e r e these f e e d s t u f f s constitute t h e m a i n o r o n l y
d i e t a r y component f o r animals, since f e e d s o f h i g h e r energy
a n d p r o t e i n v a l u e ( c e r e a l s , l e g u m e s , e t c . ) a r e r e s e r v e d for
human n e e d s . I n t h i s respect, i t is i n t e r e s t i n g t o n o t e tha t
the rumen microflora and microfauna can produce protein from
s i m p l e nitrogen sources, such a s u r e a , a n d s u l p h u r , a n d s o
the n u t r i t i v e v a l u e o f some s t r a w s a n d b y - p r o d u c t s , l o w in
p r o t e i n and h i g h in c a r b o h y d r a t e content, c a n b e improved
s i m p l y b y a d d i n g an a m o u n t a d d i t i o n a l o f u r e a a n d s u l p h u r .
P l a n t ce11 wal l s provide t h e m a i n e n e r g y source f o r
m o r e t h a n 1 . 4 b i l l i o n r u m i n a n t l i v e s t o c k u n i t s in the world.
Domestic r u m i n a n t s p r o d u c e 7 0 % o f t h e t o t a l animal p r o t e i n
eaten a n d 1 0 % o f t h e n a t u r a l f i b e r used b y humans (Wilson,
1994). Ce11 wal l s comprise 20 and 80% o f f o rage dry weight
a n d a r e composed m a i n l y b y c e l l u l o s e , h e m i c e l l u l o s e , l i g n i n ,
and pectin. C e 1 1 w a l l s d i g e s t i b i l i t y b y r u m e n microbes can
vary w i th in the range 30 and 60%, a n d t h e d i g e s t i b i l i t i e s o f
i n d i v i d u a l ce11 types can vary f rom 0 to 100% (Susmel and
Stefanon, 1993; Wil son , 1994) .
Rumen microorganisms a r e l a r g e l y r e s p o n s í b l e f o r ce1 1
wa l l s degradatíon. They a r e p r e s e n t in the rumen ín the
2
1 iquid p h a s e o f digesta content, a s s o c i a t e d wi th sol id
fragments, a n d a s a l i n i n g on t h e r u m e n e p i t h e l i u m . B a c t e r i a9 10
in r u m e n f l u i d m a y b e p r e s e n t a t c o n c e n t r a t i o n s o f 1 0 a n d 1 05 6
/ml, whereas p r o t o z o a l p o p u l a t i o n s r a n g e f r o m 1 0 a n d 10
/ml (Hungate, 1 9 6 6 ) . A v a l u e f o r t h e p o p u l a t i o n d e n s i t y o f3 5
rumen f u n g a l zoospores, w i th in the r a n g e o f 1 0 and 10
/ml, has been p r o v i d e d b y O r p i n a n d J o b l i n (1988). Since tha t
O r p i n (1975) r e p o r t e d t h e r u m e n f u n g i a n d t h e i r r o l e on f i b e r
d i g e s t i o n , the s tud i e s on t h e s e m i c r o o r g a n i s m s w e r e i n t e n s e s
b y some laboratories. However , in o u r c o u n t r y none l a b o r a t o r y
s t u d y t h e r u m e n f u n g i a n d f i b e r d e g r a d a t i o n .
T h e k n o w l e d g e o f r u m e n f u n g i in r u m i n a n t d i g e s t i o n is
yet incomplete. A common c h a r a c t e r i s t i c o f the se
microorganisms is t h e i r a b i l i t y to e x t e n s i v e l y colonize the
h i g h l y l i g n i f i e d areas c o n t a i n e d in the ce11 w a l l s o f
forages (Akin and Borneman, 19901, and Carboxymethylcellulase
(CMCase) a n d B - g l u c o s i d a s e a c t i v i t i e s have been d e m o n s t r a t e d
in s u p e r n a t a n t s from culture g r o w n on ce1 lulose ( L i a n d
C a l z a , 1 9 9 1 ; Morgavi j$ al., 1 9 9 4 ) . T h e s e a b i l i t i e s sugges ts
tha t they have a p o t e n t i a l to c o n t r i b u t e s i g n i f i c a n t l y t o
l i g n o c e l l u l o s e d i g e s t i o n in r u m i n a n t s .
T h e n e e d f o r s u l p h u r (SI in rumen microorganísms
has been r e c o g n i z e d since it was observed that they can
syn thes i ze a l1 the needed amino acids from carbohydrates,
non-protein n i t r o g e n , and sulphur (Block et a l . 1951).-
3
Addi tiotl o f e i t h e r s u l f a t e s a l t s o r m e t h i o n i n e t o p u r i f i e d
and s e m i p u r i f i e d d i e t s h a s been s h o w n to have a b e n e f i c i a l
e f f e c t upon c e l l u l o s e d i g e s t i o n b o t h &J vitre a n d jrJ v i v o
(Barton e t a l- -0 1 9 7 1 ) . T h e r e s p o n s e t o d i f f e r e n t s o u r c e s and
c o n c e n t r a t i o n s o f s u l p h u r o f in vitre d i g e s t i o n b y rumen-
b a c t e r i a has been s t u d i e d f o r ce1 lulose d í g e s t i o n . The
optimum c o n c e n t r a t i o n s varied between 0.16% and 0.24%
inorganic sulphur source and 0.32% f o r organic sulphur
s o u r c e (Bu11 and Vander sa l l , 1973) .
However t h e r o l e p l a y e d b y t h e d i f f e r e n t m i c r o b i a l
groups ( b a c t e r i a , p r o t o z o a , a n d f u n g i ) w e r e n o t considered
s e p a r a t e l y . L a t e r , in a study of s u l p h u r - d e f i c i e n t wheat
s t r a w f e d t o s h e e p , G o r d o n et Q. (1983) showed that two
tmes o f anaerobic f u n g i w e r e p r e s e n t in t h e r u m e n , b u t the
s t raw d i e t d i d n o t support normal p o p u l a t i o n s o f the se
fungi. However, methionine supplementation al lowed the
p r o l i f e r a t i o n o f t h e m y c e l i a l t y p e (probably N e o c a l l i m a s t i x
frontalis), b u t n o t the non-mycel ial tme (Sphaeromonas
communis), both of which are monocentric fungi. Gordon and
Ashes (1984) r e p o r t e d very l i t t l e s o l u b i l i z a t i o n o f l i g n i n
b u t much m o r e d i g e s t i o n o f o r g a n i c m a t t e r , NDP , ADF , and
c e l l u l o s e b y b o t h m y c e l i a l a n d n o n - m y c e l i a l t y p e s o f fungi.
S t u d i e s in v i t r o ktablish the- c a p a c i t y o f rumen
microorganisms t o u s i n g sulphur sources wi th d i f f e r e n t
e f f i c i e n c y . T h e n , is p o s s i b l e t h a t t h e r u m e n f u n g i u s i n g the
s u l p h u r f r o m s o u r c e s o f d i f f e r e n t l e v e l s o f o x i d a t i o n .
I n a r e v i e w , O r p i n (19881 r e p o r t e d that s u l p h u r c a n
b e u t i l i z e d b y N e o c a l l i m a s t i x p a t r i c i a r u m w h e n s u p p l i e d a s L-
c y s t e i n e , m e t h i o n i n e or s o d i u m s u l f i d e . S u l f a t e or a d d i t i o n a l
reduced s u l p h u r s o u r c e s w e r e n o t u t i l i z e d , b u t t h e y d i d not
i n t e r f e r e wi th t h e u t i l i z a t i o n o f c y s t e i n e or s u l f i d e .
M a r k e d d i f f e r e n c e s in the p r e v a l e n c e o f sporangia and
z o o s p o r e s o f r u m e n f u n g i b e t w e e n d i e t s a n d f u n g a l t y p e s have
been o b s e r v e d . T h e r e is evidente t h a t r u m e n f u n g i a r e always
f o u n d in s h e e p f e d f o r a g e w i t h S , b u t f e w or n o fungi a r e
f o u n d w i t h f o r a g e w i t h o u t s u l p h u r (Akin e t a l 19831.- - -
On t h e b a s i s o f c u r r e n t evidente, t h e n u t r i t i o n a l
r e q u i r e m e n t o f r u m e n anaerobic f u n g i a r e n o t very demanding.
Therefore, t h e d e t e r m i n a t i o n o f t h e i r p r e c i s e n u t r i t i o n a l
parameters w i l l t a k e f u r t h e r s t u d y (Theodorou et al. 19951.
L i t t l e i n f o r m a t i o n h a s been r e p o r t e d on the c h a r a c t e r i s t i c s
o f t h e r u m e n anaerobic f u n g i u t i l i z i n g s u l p h u r s o u r c e s , and
the e f f e c t o f t h i s s u b s t r a t e on f iber d i g e s t i o n and
production of fiber-degrading enzymes.
In t h e p r e s e n t s tudy , the e f f e c t o f sulphur
a d d i t i o n on t h e r o l e o f r u m e n f u n g i in the ma ize stem and
c e l l u l o s e d e g r a d a t i o n was ivest igated. Antibiotics were used
t o select f o r f u n g a l a c t i v i t y , a n d a v a r i e t y o f m e t h o d s were
used to d e t e r m i n e m a i z e s t e m d e g r a d a t i o n a n d enzyme activity.
O u r w o r k w i l l a l l o w c o n s o l i d a t e in Mexico o n e g r o u p that
s tudy the se microorganisms important for the rumen
metabolism.
5
ANTECEDENTS
Some characterist ics and the classi f ication of Turnen fungi .
The rumen has been considered as one of the most capable
ecosystem for the bioconversion of plant ce11 walls and their
components (celllulose, hemicel lu lose, lignin) t o usable
energy. The digest ion of cel lulose in the rumen is undertaken
by a dense microbial p o p u l a t i o n w h i c hinclude bacter ia ,
protozoa and fungi (Heath et a l- --p 1988) . Unti l recent ly the
microbiota of the rumen was considered to be composed only of
bacteria and protozoa. However in 1975 Orpin, and later Bauchop
in 1979, reported chytr idiomycete- l ike fungi in the the rumen.
Recent research by Akin et al- --9 (1988) on anaerobic fungi has
emphasized their zoospore-producing abi l i ty and indicated a
monocentric pattern of growth (i.e., a thal lus radiating from a
single p o i n t a t w h i c h a sporangium is formad). However,
anaerobic ruminal fungi also inc lude types wi th polycentr ic
growth patterns (Akin and Rigsby, 1987) (i.e., a thal lus
radiat ing from many g r o w t h center a t whí ch sporangi a are
formed).
A c l a s s i f i c a t i o n f r o m morphological characterist ics of
anaerobic fungi has been shown by Teunissen and Op den Camp
(1993):
Dl/vision: Eumycota
,Subdivision: Mastigomycotina
Class: Chytridiomycetes
6
Order :
Family:
Genera:
Spize l lomyceta les
Neocal l imasticaceae
Caecomyces (zoospore has one or two flagella)
Neocal l imastix (zoospore h a s f o u r t o twenty
f l a g e l l a )
Piromyces (zoospore has one to four f lagel la )
Orpinomyces (zoospore is multiflagellated)
Anaeromyces (zoospore has one flagellum)
Unti l now up to eleven species of rumen anaerobic fungi
have been described a n d c l a s s i f i e d (table 1). The monocentric
fungi have a s imple asexual l i fe -cycle which occurs both in gut
and in culture. The poster ior ly f lage l lated motile zoospore
contacts a suitable substrate, typ ica l l y a piece of forage, and
sheds i t s f l a g e l l a ; the zoospore encysts developing a rhizoid,
which attaches the fungus to the substrate, and a cell-wall is
synthesized around the original ce1 1-body to form a
sporangium(Orpin, 1988). After a variable degree of ce11 body
enlargement, zoosporogenesis can b e induced (Gold et a l-*t
1988). Zoospores are released from the sporangium by
dissolution of the sporangial wal l and the cycle is completed
(Heath et a l - . , 1983) .
In contrast the polycentric fungi are not dependent on
zoospore production f o r their propagation as their growth
pat tern resembles that of the higher fungi ; fungal g r o w t h is
propagated by hyphae Wo et Q., 1991) . Zoospore are produced
7
TABLE 1. CLASSIFIEB RIMEN ANAEROBIC FUNQI.
Genus Speci es
MONOCENTRIC or POLYCENTRIC:Caeccmyces communis’L/
110s t Ref erence
sheep Gold et al., (1988)
RON~ENTRIC:Piromyces
Neocallimastix
POLYCENTRIC :Anaeromyces
Orpinomyces
communis~/ sheep Gold et al., (1988)minutus deer Ho et al., (1993~)spiral is goat Ho & Q., (1993dI
frontal is sheep Heath et al., (1983)patriciarunQ/ sheep Orpin and Munn (1986)hurleyensis sheep Webb and Theodorou 11991)variabilis cow Ho et al., (1993a)
el egans4/ cow Ho et al., (1993b)mucronatus sheep Breton et @. , (1990)
joyoni iS_/ sheep Breton et aJ., (1989)
Originally called: i/ Sphaeromonas communis (Orpin,l976); 2/ Piromonasconnnuni s (Orpin, 1977bI; 3/ Neocallimastix frontalis (Orpin, 1975) ;$/ Ruminomyces elegans (Ho pJ aJ., 1990); 5/ Orpinomyces bovis tBarr$3& al-., 1989); and Neocallimastix joyonii (Breton et al-., 1989).
i n f r e q u s n t l y o r z o o s p o r o g e n e s i s is even a b s e n t (Phillips,
1 9 8 9 ) . Some p o l y c e n t r i c anaerobic f u n g i f o r m z o o s p o r e s r e a d i l y
and a b u n d a n t l y in c e r t a i n m e d i a , b u t t h e m a j o r i t y o f the
s p o r a n g i a d o n o t d i f f e r e n t i a t e a n d release z o o s p o r e s (Teunissen
and Op den Camp, 1993). The development of sporangia OCCUrS
l a t e r a l l y o r t e r m i n a l l y on r h i z o m y c e l i u m , a n d s p o r a n g i a c a n b e
b r a n c h e d o r u n b r a n c h e d (Barr e t a l . , 1989) .- -
P o r m o n o c e n t r i c f u n g i O r p i n (1977) r e p o r t e d t h a t & vivo
z o o s p o r o g e n e s i s a r e induced b y w a t e r s o l u b l e c o m p o n e n t s in the
d i e t o f t h e h o s t animal. The components r e s p o n s i b l e for
i n d u c t i o n o f z o o s p o r o g e n e s i s in p o l y c e n t r i c f u n g i have n o t been
inves t iga ted ye t .
C u l t u r e m e d i a a n d n u t r f t i o n o f t h e f u m e n fungf.
The culture media most commonly employed is described by
Orpin (1975)) and consist o f centrifuged rumen f l u i d ,
t r y p t o n e , yeas t extract, a c a r b o n s o u r c e , a carbon dfoxide-
bicarbonate b u f f e r at pH 6 . 7 - 6 . 9 , L - c y s t e i n e a s reducing
agent, and v i t a m i n s . I f b a c t e r i a 1 c o n t a m i n a t i o n o f cul tures
occurs, a n t i b i o t i c s m a y b e i n c o r p o r a t e d i n t o t h e m e d i u m .
C u l t u r e m e d i a incorporating p l a n t t i s s u e s a s c a r b o n
source a r e v a l u a b l e f o r e n s u r i n g t h a t t h e o r g a n i s m s d o n o t l o s e
t h e i r a b i l i t y t o f e r m e n t p l a n t structural c a r b o h y d r a t e s in-
vi t ro (Orpin and Le tcher , 1979) . Cu l ture s fn the l i q u i d m e d i a
9
of O r p i n (1975) g a v e h i g h c e 1 1 y i e l d s b u t n e e d e d subculturing
a f t e r 24 -48 hour s t o ma in ta in v i ab i l i t y , wherea s cultures on
p l a n t t i s s u e s m a y b e v i a b l e f o r u p to 7 d a y s , t h u s s i m p l i f y i n g
routine maintenance.
Only orle species, N e o c a l l i m a s t i x p a t r i c i a r u m , has been
grown in a minimal medium (Orpin and Greenwood, 1986b). Minimal
n u t r i t i o n a l requirements f o r g r o w t h o f N, p a t r i c i a r u m on
c e l l o b i o s e w e r e s a t i s f i e d b y t h e p r o v i s i o n o f s o u r c e s o f haem,
b i o t i n , thiamin, ammonium ions, a f o r m o f reduced s u l p h u r and
t r a c e elements. H a e m s a p p e a r t o p l a y a m a j o r r o l e in both the
growth and z o o s p o r o g e n e s i s o f rumen fungi (Orpin, and
Greenwood, 1986aI.
U t i l i z a t i o n o f s u l p h u r b y r u m i n a n t s .
C e l l u l o s e a n d h e m i c e l l u l o s e a r e ma.ior components o f p l a n t
ce11 w a l l s a n d a r e t h e p r i n c i p a l s o u r c e o f f e e d f o r ruminants.
S p e a r s e t a l- -* (19761, w o r k i n g w i t h p u r i f i e d d i e t s low in
sulphur, i n d i c a t e d t h a t s u p p l e m e n t a l s u l p h u r is b e n e f i c i a l in
i n c r e a s i n g celulose d i g e s t i o n a n d nitrogen r e t e n t i o n . Bu11 and
Vander sa l l ( 1973) repor ted increases í n nitrogen r e t e n t i o n and
A D F d i g e s t i o n w h e n organic o r i n o r g a n i c s o u r c e o f sulphur
content in t h e d i e t was increased f rom 0 .20% to 0 .32%. Because
t h e r u m e n microorganisms c a n r e d u c e o x i d i z e d f o r m s o f sulphur
t o f o r m s whi ch can be incorporated in to organic
10
compounds, ruminants have the abi l i ty to obtain their sulphur
supply from inorganic sources of sulphur (Kandylis, 1984) . This
demonstrated that inorganic sul fate can be substituted f o r
organi c sulphur sources such as methionine and vice versa.
However, elemental sulphur has been used as sulphur source, but
it has general ly been shown not to be so readi ly avai lable than
s u l f a t e or organic sources, probably o w i n g t o i ts lower
s o l u b i l i t y in the rumen f luid (Spears et a l_ _. 1 9 7 6 ) . A l b e r t et
a l-* (1956) reported that sulphur from elemental sulphur and
sodium sulfate was 30% and 50% as a v a i l a b l e a s t h a t o f
methionine. However, Johnson et &. (1971) using true retention
data, concluded that sulphur from elemental sulphur and sodium
sulfate were 38 and 80% as available, as sulphur to methionine,
respect ive ly .
McLennan e t a l- -’ (1989) demonstrated t h a t a d d i t i o n o f
s u l p h u r t o urea-based supplements f o r ruminants consuming
mature forages increased N retention and forage digest ib i l i ty .
The reason for the increase in intake and digestibi l i ty of dry
matter and organic matter when s o d i u m s u l f a t e is added to
diets c a n b e explained by the better u t i l i z a t i o n o f the
ni trogen present. However, the e f f e c t o f sulphur
supplementation only is present i f the leve1 of nitrogen is
a d e q u a t e in t h e d i e t (Playne, 1969). Moreover, with the
addit ion of sulphur ta the diet of spear grass and urea, the
concentration of rumen ammonia is reduced and more nitrogen is
íl
retained (Kennedy and Sieber t , 1972) .
Hume and Bird (1970) suggested that when the N:S ratio was
í1:1, each gram of added dietary sulphur c o u l d p o t e n t i a l l y
y i e l d 6 9 g o f b a c t e r i a 1 p r o t e i n in the fumen. L a t e r , McLennan
e t JiJ. (1989) r e p o r t e d a m e a n v a l u e o f 18.5:l r a t i o (N:S) for-
mixed rumen bacteria, r a n g i n g b e t w e e n 8.6:1 a n d 30.8:1, a n d a
mean v a l u e o f 21.6:1 f o r protozoa, r a n g i n g b e t w e e n 14:l a n d
38:l. From t h i s t h e y c o n c l u d e d t h a t a r a t i o o f 2O:l b e t w e e n
a v a i l a b l e nitrogen a n d a v a i l a b l e s u l p h u r s h o u l d b e a d e q u a t e t o
meet the requirements of rumen microbes.
Recent s t u d i e s have i n d i c a t e d t h a t t h e optima1 d i e t a r y
sulphur leve1 f o r m a x i m u m d a i l y g a i n o f goat k i d s was
approximately 0 .22% o f d i e t a ry dry ma t t e r w i th a N :S ra t i o o f
1O:l. Therefore, t h e o p t i m a 1 d i e t a r y s u l p h u r l e v e 1 presumably
improved performance by enhancing bacteria1 protein syn thes i s
in t h e r u m e n a n d i m p r o v i n g t h e amino acid b a l a n c e (Qi et al-.
1993). However, & v i v o d i g e s t i b i l i t y w a s h i g h e s t wi,th 0.11%
of s u l p h u r a s calcium s u l f a t e in the d i e t . Apparent
d i g e s t i b i l i t i e s o f d r y m a t t e r , organic ma ter , ADF, and crude
p r o t e i n w e r e i n c r e a s e d l i n e a r l y b y a d d i n g 0.6 , 0 .28 , and 0 .36%
s u l p h u r a s c a l c i u m s u l f a t e in t h e d i e t (Qi e t a l- -* 1992).
Multani et aJ (1986) de termined t h e a v a i l a b i l i t i e s o f
s e v e r a 1 s o u r c e s o f s u l p h u r a n d f o u n d t h a t a N:S r a t i o o f 1O:l
r e s u l t e d in majar m i c r o b i a l p r o t e i n s y n t h e s i s in v i when- t r o
s u l p h u r w a s s u p p l i e d t h r o u g h s u l f a t e s a l t s o f s o d i u m , p o t a s s i u m ,
12
calcium, íron, and ammonium, as wel l as through methionine.
However, the best leve1 with zinc sul fate or cysteine was
4O:l. Onwuka and Akinsoyinu (19891 using elemental sulphur at
15:l rate in dwarf goats and sheep observed better N retention
and apparent digest ib i l i ty of dry matter compared with 28:1,
7:1, a n d 6:l r a t e s (N:S).
Momont et (1993) f o u n d n o d i f f e r e n c e s in- al. apparen t
d i g e s t i b i l i t i e s o f dry matter NDF and ADF among 1 ambs
supplemented with sodium sulfate or methionine. With a semi-
puri f ied diet contaíning 4% urea and the S-content increasing
from 0.05 to 0.15% of either inorganic sulfate or methionine,
both cel lulose digestion and nitrogen retention were increased
(Bray and Hemsley, 19691. In addit ion t h e o b s e r v a t i o n s b y
B a r t o n et al. (1971) indicated that levels of sulphur as high
as 0 . 3 5 % in sulfate form had no adverse e f f e c t s upon f i b e r
d i g e s t i o n fn v i t r o and 0.18% was optima1 f o r ce l lu lose
digestion. This in vitro work is supported by in vivo work ín- - -
which cel lu lose digest ion was s igni f icant ly depressed in steers
fed with puri f ied diets without adequate l e v e l s o f sulphur
( M a r t i n e t a l- - - 1964) . Clark and Petersen (19881 n o t e d an
increased rate of in situ digestibi l i ty of dry matter of 1 ow-- -
qual i ty grass hay in cows supplemented with urea plus 15 g of
methionine compared with ammonium sulfate. However, McCracken
et gJ. (1993) noted that methionine supplementation did not
i ncrease forage NDF disappearence at 96 h compared with
13
oontrol. Greater f o r a g e d r y m a t t e r disappearence has been
reported by Huisman et alII -* (1988) a s a r e s u l t o f supplementing
m e t h i o n i n e in ei ther l i q u i d o r r u m i n a l l y p r o t e c t e d f orms .
Conversely, Lodman et Q. (1990) n o t e d o n l y increases in the
e x t e n t a n d rate o f d r y m a t t e r a n d N D F d i g e s t i o n a s a r e s u l t o f
methionine a n d u r e a s u p p l e m e n t a t i o n . H a l l e t a l . (1990) f ound- -
t h a t p r o v i d i n g m e t h i o n i n e t o s t e e r s f e d w i t h b e r m u d a g r a s s haY
and g r o u n d corn h a d n o e f f e c t on f e e d i n t a k e o r d i g e s t i o n and
o n l y t e n d e d t o i n c r e a s e t o t a l tract N D F d i g e s t i o n .
T h e r u m e n p o p u l a t i o n in animals f e e d i n g w i t h l o w - q u a l i t y
roughage and supplemented with 0.21% s u l p h u r , c o n t a i n 10%
more b a c t e r i a , 2 2 % m o r e protozoa, and 69% more fungal
zoospores. T h e p r o p o r t i o n a l i n c r e a s e in r u m e n f u n g i p o p u l a t i o n
was c o u p l e d w i t h a 1 6 % i n c r e a s e í n f i b e r d í g e s t i o n (Gulati e t
a l- - 1985).
T h e r a t i o o f nitrogen t o s u l p h u r r e t e n t i o n is determíned
W t h e a m o u n t s o f nitrogen a n d sulphur d e p o s i t e d ‘ in body
t i s s u e . G e n e r a l l y , d i e t s s u p p l y i n g m o r e s u l p h u r t e n d t o resul t
i r1 higher r e t e n t i o n o f b o t h n í t r o g e n a n d s u l p h u r (Kandylis,
1984). Gulati e t a l . , (1989) s u g g e s t e d t h a t an increased
b i o m a s s o f rumen fungi in s h e e p may r e s u l t in increased
q u a n t i t i e s o f e s s e n t i a l a n d s u l p h u r amino a c i d s b e i n g a b s o r b e d
from t h e s m a l l i n t e s t i n e . This would be expected to s t imula te
wool growth and may have important i m p l i c a t i o n s for wool
p r o d u c t i o n in s h e e p g r a z i n g l o w - q u a l i t y r o u g h a g e s .
14
Requirement of rumen microorganisms for sulphur.
The u t i l i z a t i o n o f inorganic s u l f a t e b y rumen
microorganisms in the synthesis of the sulphur containing amino
acids has been wel l substantiated (Block et a l- -* 1951). Numerous
studies have indicated that methionine is o n e of the amino
acids l imiting rumen microbial growth and substrate
fermentation r a t e s in the rumen (Salter et a l . 1979). For- -
instance, sulphur h a s l o n g been r e c o g n i z e d a s an essentia l
element f o r ruminal microorganisms. Sulfur and ni trogen
metabolism are c losely associated in ruminants. The action of
the Turnen microf lora may completely alter the dietary form of
b o t h o f these elements; for instance, the d e g r a d a t i o n o f
dietary protein to yield ammonia and sulfide or the synthesis
o f microbial protein from dietary urea and inorganic sul fate
(Kandylis, 1984).
Kahlon et a l . (1975) demonstrated that rumen- -
microorganisms can uti l ize inorganic as wel l as organic f orms
o f s u l p h u r t o synthesize sulphur containing amín0 acids.
Al though the de novo bacteria1 synthesis of methionine from- -
sul fate , ammonia, and carbohydrates occurs (McMenimam et a l .- -
19761, the extent to which this pathway supplies adequate
methionine f o r optima1 c e l l u l o l y t i c a c t i v i t y is debatable
(Mathers and Mi l ler , 1980) .
When rumen microorganisms are incubated with radioactive
sul fate , radioactive sulphur can be found in cysteine, cyst ine
and methionine of the bacteria1 proteins (Whanger, í972). But,
in order f o r sulphur amino acids to be s y n t h e s i z e d in the
rumen, sulphur must be present in the diet . In addit ion, the
sulphur containing vi tamins, such as thiamine and biotin are
synthesized by the rumen microbes; moreover, sodium sulfate and
methionine have been shown to stimulate the s y n t h e s i s o f
ribof lavin and vitamin 812 by rumen microorganisms to a greater
degree than when the s o u r c e o f sulphur was c y s t e i n e o r
e lemental sulphur CBriggs et a l . 1964).- -
Data provided by Kahlon et a l . 11975) indicated that the- -
sources of su‘lphur differ greatly wi th respect t o the
avai labi l i ty of sulphur. They determined the avaílabili tie3 Uf
severa1 sources of sulphur and found that sulphur concentration
o f 21.5 ug/ml of inoculum (0.32% sulphur in the substrate dry
matter) resul ted in less than optimum microbial protein
synthesis in an ín vitro s y s t e m which used starch a s substrate,-
while concentrations of 86.7 and 130.0 ug/ml (1.3 and 1,95X in
the substrate dry matter) were apparent ly inhib itory . However,
a sulphur concentration of 43.3 ug/ml in the incubation medium
( 0 . 6 5 % in the substrate dry matter) r e s u l t e d in the most
protein synthesis .
As sources of sulphur sodium sulfate, calcium sulfate, and
methionine were equal at equal sulphur concentration in their
abi l i ty to promote in vitro l ignocel lulose digestion with rumen-
16
bacter ia ; the optimum was 0.16 to 0.24% S as sodium sulfate or
calcium sulfate and 0.32% S as methionine (Bu11 and Vandersall,
1973). The theory which best explains the resul ts of this
study, is the one in which the supplemental sulphur stimulates
bacteria1 activity in the rumen. A l t h o u g h some pure s t r a i n s o f
rumen microorganisms have a preferente for certain f o r m s o f
sulphur, due to the symbiotic relationship of a míxed cul ture
of rumen microorganisms, the form of sulphur does not appear to
have a significant influente on t h e i r a c t i v i t y . However some
strains isolated from the rumen were able to synthesize
cysteine and methionine from inorgaaic sulfate (Whanger, 1972).
The a d d i t i o n o f sulphur compounds to an &I vitro
incubation of rumen microorganisms, has been shown to stimulate
cel lu lose digest ion. When a steer was fed with a diet high in
corn stubble , about lo-20 ppm of sulphur as sodium sulfate was
required for optimum cellulose digestion and about 30 wm of
sulphur inhibited cel lulose digest ion (Trenkle et a l . . 1958).- -
Sul f ide added to an incubation mixture of rumen microbes was
r e p o r t e d t o lower the incorporation of sulfate sulphur into
bacteria1 protein more than the addition of methionine or
cysteine (Halverson et a l . 19681. This suggested that sul fate- -
was reduced t o s u l f i d e before incorporation into the
bacteria1 proteins. Komisarczuk-Bony et a l . (í992), in a study- -
to determine sulphur requirements of rumen microbes, reported
that cel lu lose digestion was of 45.3% with 12 and 22 mg S as
17
sodium sulfate/day in a semi-continuous fermentor. However,
microbial nitrogen content was larger with 12 mg/day than 22
mg/day .
Wi th the inorganic sulphur as supplement in low-qual i ty
diets, more organi c mat ter and acid detergent f i b e r were
d i g e s t e d in the s tomach and in the alimentary tract as a
whole, a greater quantity of non-ammonia was digested ín the
intestines per unit of digestible organic matter intake and
fungal act ivity in the rumen was higher. This indicated that an
inadequate amount of S in the low S diet impaired the
metabolism of the rumen microbiota which in turn affected
variables re lat ing to digestion and metabolísm (Weston et a l- -**
1988).
The effetct of sulphur on populatíons of rumínal fungí.
T h e structural components o f f o r a g e have been considered
to p lay a part in l imit ing forage digest ibi l i ty . In addit ion to
structural components, mineral l e v e l s in forage are often
inadequate f o r e f f i c i e n t microbial activity and f i b e r
degradation. Moreover, the total sulphur content in forages may
not a lways be a re l iab le indicator of sulphur avai labi l i ty to
the rumen microorganisms (Spears et a l- -* 1976; Akin and Hogan,
1983). This suboptimal leve1 of minerals is indicated by
enhanced f i b e r digest ibi l i ty with addit ion of m i n e r a l s . I n
particular sulphur supplement has been reported to enhance the
18
b r e a k d o w n o f ce l lu lose and l i g n o c e l l u l o s e b y Turnen
microorganisms (Bu11 and Vandersal l , 1973) .
The importance of rumen anaerobic fungi in digestion by
h e r b i v o r e s is not clear, but their c e l l u l o l y t i c capaci ty
(Bauchop and Mountfort, 1981) and their abi l i ty to digest up to
45% of the dry weight of plant t issue in culture in vitro (Lowe- -
et al- -* 1987b) sugges t that they have the p o t e n t i a l t o
contribute signi f icant ly t o l i g n o c e l l u l o s e d i g e s t i o n in the
host animal as shown by Gordon and Phillips (1993).
In recent years, the sulphur content of forage diets has
been recognized as a significant factor governing the size of
the rumen fungal populat ion (Akin & al., 1983j; whereby , a
def ic iency of dietary sulphur could l imit their growth in the
rumen, and hence l i m i t t h e i r c o n t r i b u t i o n t o p lant tissue
digestion.
Anaerobic fungi were not detected in sheep fed Dig itar ia
pentzii hay harvested from sulphur deficient plots, whereas the
haY collected from t h e same p l o t s a f t e r an a p p l i c a t i o n o f
sulphur f e r t i l i z e r supported an active population of rumen
fungi , the number of zoospores per ml were 4,000 to 6,000, and
with methionine supplementation (4.5 g/day) the amount was only
of 4,100 per ml (Akin and Hogan, 1983). Morrison et al. (1990)- -
r e p o r t e d an increase in bacteria, protozoa and s p o r a n g i a o f
rumen anaerobic fungi in sheep supplemented with sulfate. This
increase was not m a r k e d in the amount of sporangia.
Simi lar ly , the sulphur content of wheat stubble affects the
number of rumen fungi. Unl ike low sulphur Dig i tar ia pentzi i ,
fungi were found in sheep receiving low sulphur wheat straw
even though they were present only in low numbers (Gordon et-
a l . , 1983). This di f ference between Digitar ia and straw may be
due to varying components of the feeds or, more p r o b a b l y , to
t h e a b i l i t y to detect smal l populat ions of rumen anaerobic
f u n g i , an a b i l i t y the which has been gained wi th wider
experience.
Ruminal anaerobic fungi have been found in the rumen of
sheep fed a t ropica l hay, and they appeared to c o n t r i b u t e t o
digest ion when there was adequate dietary sulphur (Akin et a l .- -
1983). In a study of sulphur deficient wheat straw f e d t o
sheep, Qordon et al- - - (1983) showed that two types of anaerobic
fungi were p r e s e n t in the rumen. I n culture, one f ungus
produced an obvious myeelium, whereas the other produced a
sporangium from spher ica l bodies. The s t r a w d i e t did not
suppor t normal populat ions of these fungi . However, dietary
methionine supplementation al lowed the prol i feration of the
mycel ia l tyw (MT) but not the non-mycelial twe (NMT). I n
contrast Orpin (1981) noted that similar MT fungi canno t use
methionine as a source of sulphur.
In addit ion to cel lu lose digest ion, the fungi apparent ly
degrade other structural elements of plant t issues, such a s
hemicel lulose and pectin. Moreover the data shown in T a b l e 2
2 0
TABLE 2. PERCENTAGE DIGESTIGN OF SIRUCTURAL CXMPONENTS OF WHEATSTRAW BY PURE CULTURES OF RUMEN ANAEROBIC FUNGI AFTER 4DAYS OF INCUBATION (X1.
O@/ NDF ADF Ce11 HCel Lig Pect2/ 96.6 80.7 52.6 44.3 33.9 13.8 8.0
Source: Orpin and Hart (1980):
Neocallimastix frontalis 35.0 ---- --- 58.1 52.3 19.4 20.5
Piromonas communis 37.0 --- --- 50.4 55.0 21.9 47.3
Sphaeromonas communis s/ 30.0 --- --- 39.4 39.6 16.4 16.3
Source: Gordon and Ashes (19841:
MT-1 (N. frontalis14/ 23.3 20.8 18.1 21.2 --- 2.2 ---
MT-2 (& frontalisl 24.2 16.3 23.0 21.3 - 0 ---
NMT-1 (& communis) 2.1 1.9 0 6.0 w--B 0 - -NKT-2 (Z& communisl 7.8 4.7 0 8.6 - 0 ----
&/ oh! = organic matter, NDF = neutral detergent fiber, ADF = aciddetergent fiber, Cell = Cellulose, HCel = Hemicellulose, Lig = Lignin,Pect = Pectin. 2/ Tissue composition (X dry matterl. 3/ Named alsoCaecomyces. 4/ MT = mycelial type, NRT = non-mycelial type.
21
suggest that fungal act ivity could solubi l ize as much as 22% of
the lignin component of wheat straw. As Orpin and Hart (1980)
pointed out, this result should be treated cautiously as there
is no evidente that any of the l ignin can be used to support
fungal growth. McSweeney e t a l (1994)- - r e p o r t e d an apparent
l ignin solubilisation of 33.3% ín sorghum rind and 21.2% when
sorghum was NaOH extracted. The inocula used were
Neocal l imastix patriciarum for 6 d a y s incubation. Bauchop
(1979) indicates that the d iet has a substantial e f f e c t on
fungal populat ions; a f ibrous diet supported more fungi than a
leafy diet. Grenet et a l- _* (1989) showed that rumen fungí are
part icu lar ly abundant wi th l ignocel lu lose-r ích díets, and
se lect ive ly colonize plant t issues, especia l ly those with thick
or l igni f ied ce11 walls. The development of fungi depends not
only on the substrate on which they become attached, also on
the ruminal medium. This fact can explain the results obtaíned
by Millard e t a l- -* (19871, on the change of the sulphur status
o f the feed had no detectable ef fect upon numbers of viable
bacter ia . However , changing the feed from s u l p h u r (+) t o
sulphurq-1 grass increased the number of ce l lu lo lyt ic fungi .
This ef fect probably was caused by the increasing of f íber in
grasses without sulphur fert i l ization.
Orpin (1977) reported that p lant ce11 wa l l s compounds
stimulated zoospore p r o d u c t i o n r e s u l t i n g í n a higher
population, whi le more detailed work by Orpin and Greenwood
88
(1986a) indicated that zoosporogenesis was induced by heme-type
compounds . In Austra l ia sulphur fert i l ized grass (Akin et al.
1983) and methionine supplemented diets (Gordon et al. 1983)
resul ted in increased fungal populat ions . Perhaps r e l a t e d to
these f i n d i n g s is the work by Akin and Windham (1989) who
showed that increased ruminal fungi populations ocurred wi th
a l f a l f a versus bermudagrass and mature versus immature wheat
forage diets.
Sulphur can be utilized by N. patriciarum when- supplied
as L-cyste ine, m e t h i o n i n e or sodium sul f ide . S u l f a t e , or
reduced sulphur sources including dithiothreitol , 2-
mercaptoethanol and thioglycol late were not uti l ized, but did
not interfere with the uti l ization of cysteine S or su l f ide S .
The amino acid methionine acted as a source of sulphur only in
the presente of compounds which generated a low redox potential
in the medium, such as d i t h i o t h r e i t o l or 2-mercaptoethanol
(Orpin and Greenwood (1986b). T h e l o w redox potential in the
rumen would ensure the avai labi l i ty of L-cysteine and sulf ide
from the microbial hydrolysis of dietary proteins and sulphur -
containing amino acids. Sulfur-containing amino acids and
sul f ide can be detected in rumen contents at any time and are
u n l i k e l y t o limit the growth of Neocallimastix spp. in v i v o- -
(Orpin, 1988).
Ph i l l i p s and Gordon (1991) indicated that sulphur is a
l imiting nutrient to Neocal l imast ix sp. LMl, because- varying
the concentration of sulphide both alone and with equal amounts
a3
o f cysteine or methionine gave increased growth of LM1 u p to
l -2 mhí total sulphur, and the growth on sulphide in combination
with cysteine or methionine was better than growth on sulphide
alone. However, no growth is found with a range of compounds
including inorganic oxides of sulphur, sulphonic or su lphid ic
acids, dimethyl sulphide, thiourea, thioacetamide, sulphones,
sulphoxides, or dithiothreitol .
Rees et al. (1978) showed that sulphur f e r t i l i z a t i o n- -
reduced retention time of forage in the rumen and increased
voluntary intake and digest ib i l i ty compared with unfert i l ized
Digitaria decumbens forage. The authors suggest that depressed
.microbial activi ty due to a sulphur def ic iency could have
caused poor animal response to t-S) forage. In s imilar studies
with (+s) and f-S) D i g i t a r i a p e n t z i i , Rees e t a l- -0 (1982)
indicated that S fert i l ization increased voluntary intake and
reduced rumen ammonia levels , indicating enhanced microbial
activity in the rumen. Akin et al.- - (1983) reported that rumen
fungi were absent or in extremely small numbers in sheep fed
sulphur unfert i l ized forage.
In a study, Gulati e t a l . , (1990) indicated that the- -
d igest ib i l i ty of sulphur amino acids in rumen bacteria1 protein
was less t han t h a t o f rumen fungi , this sugges ted that
increasing fungal populations in the rumen at the expense of
bacter ia would be unl ikely to cause deleterious effects on the
q u a l i t y o f p r o t e i n avai lab le f o r absorption at the small
2 4
intestine. This may have important i m p l i c a t i o n s in sheep
grazing low qual i ty forages i f procedures can be developed to
increase the rumen fungal population thereby enhancing the
voluntary feed intake and rate of f iber degradation.
I n many sulphur-def ic ient areas of t h e w o r l d sulphur
f e r t i l i z a t i o n has been used to promote forage production, and
under these condit ions the probabi l ity of a def iciency of this
element ocurring in animals fed forages and grains has become
more remote. However, the total sulphur and others minerals
content in forages may not a lways be a re l iable indicator o f
avai labi l ity to the rumen microorganisms (Spears et a l . 1976).- -
Thereby , wi th poor qual ity forages or with forages g r o w n on
sulphur deficient soi l or with these forages fed non-protein
nitrogen, sulphur supplementation will most likely be needed to
meet the sulphur requirements of the animal (Whanger, 1972).
However, Rees and Minson 11978) demonstrated that voluntary
intake and dry matter digest ibi l i ty of sulphur fert i l ized grass
was lower than t h a t o f the control grass when the
determinations were made with sheep supplemented with sulphur.
.Fibrs d e g r a d a t i o n a n d other digestivs a c t i v i t y b y r u m i a s 1fungi.
Rumen fungi can prol i ferate and survive in an ecosys tem
extensively colonized by f ibrol ityc bacteria and protozoa, and
this may be related to their abi l i ty to penetrate and degrade
plant t issues not normally accessible t o o t h e r rumen
microorganisms. rumen fungi are the primary invaders of plant
f i b e r , part icular ly vascular p lant f ragments (Bauchop, 1981)
and they made the plant fragments more accessible to bacteria
(Theodorou e t a l . 1988). The facts that high-f iber diets are- -
associated wi th the greatest p o p u l a t i o n s o f rumen fungi
indicated that they are more resitant to phenolic monomers than
b a c t e r i a (Borneman e t a l . 19901, this suggests that fungi- - play
a majar role in digest ing l ignocel lu lose.
An examination of plant fragments which were removed
sequent ia l ly from nylon bags suspended in the rumen drew
a t t e n t i o n t o the population densi ty o f p a r t i c l e - a s s o c i a t e d
thal l i in digesta contents and led to the suggestion that rumen
fungi part ic ipate in init ial microbial colonization of p lant
ce11 walls (Bauchop, 1979a, 1979b). Rumen fungi may also assist
in c e l l u l o l y s i s b y increasing the a c c e s s i b i l i t y o f p lant
biomass ta invasion by other rumen microorganisms (Theodorou et-
a l 1988). However, the precise role and overa11- • 9 contribution
o f rumen fungi to the fermentation of plant b i o m a s s in the
rumen has yet to be determined.
Rumen fungi produce a wide range of hydrolyt ic enzymes
and u t i l i z e carbon sources ranging from simple s u g a r s t o
comp1 ex polymers. Ce l lu lo lyt ic , xy lanolyt ic , g lyco lyt ic ,
amylolyt ic and proteolyt ic enzyme a c t i v i t i e s have been
demonstrated against both model and natural substrates (Pearce
and Bauchop, 1985; Mountfort and Asher, 1989; Wallace and
26
Joblin, 1985; Wood et a l . , 1986; Lowe et a l . , 1987c, Williams- - - -
and Orpin 198713).
To accomplish hydrolysis of ce l lu lose and xylan into
monosaccharides, two enzyme complexes, cel lu lases or xylanases,
respectively are essentia l (L i and Heath, 1993) . The act ivit ies
of al1 these enzymes against cel lulose or hemicel lulose (mainly
xylanl have been found in ruminal fungi (Akin and Rigsby, 1987;
Akin et al. Hebraud and- - 1990; Barichievich and Calza, 1990;
Fevre, 1990). Some of these enzymes are constitutive whereas
others are apparent ly inducible (Qordon and Phi l l ips , 1989).
It appears that the enzymes necessary for cel lu lose digestion
from large complexes are associated with the fungal ce11 Wall,
an associat ion that apparently is essential for their a b i l i t y
to d i g e s t crysta l l ine cel lu lose (Wi l son and Wood, 1992).
Digest ive enzyme a c t i v i t i e s c o u l d b e detected in zoospores,
vegetative thalli, and culture supernatant and were regulated
by the growth substrate (Williams and Orpin, 1987; Morrison et-
a l-* 1990). The enzymatic a c t i v i t i e s are l o w e r in media
containing mono- or di -saccharides than in media containing
plant cell-wall polymers.
Cel lulases of rumen fungi have pH and temperature optima
o f 5 . 0 - 6 . 0 a n d 45-55 c , respect ive ly , and t h e a c t i v i t i e s
described include carboxymethylcel lu lase, ce l lobiase , and
avice lase . Cel lu lase production in batch c u l ture is always
accompanied by the production o f h e m i c e l l u l a s e a n d o t h e r
27
glycosidase enzymes (Lowe et al., 1987c; Williams and Orpin,- -
1987a, 1987b).
Xylanases (hemicellulasesl o f rumen fungi have been
described b y a number of workers IOrpin and Letcher, 1979;
Pearce and Bauchop, 1985; Williams and Orpin, 1987a; Lowe et-
a l . , 1987c: Mountfort and Asher, 1989) . In general , xylanase
activities have pH and temperature optima of 5.5-6.0 and 50 C,
respect ive ly .
Lignocel lu lose digestion is undoubtedly substantia l ly
aided by the fungal production of esterases that cleave lignin
from the hemicel luloses (Borneman et a l . 19921. Furthermore, i t- -
was shown tha t d e g r a d a t i o n o f p l a n t f i b e r b y rumen fungi
caused up to 40-70% losses in plant t issue dry matter (Lowe et-
a l-* 1987a; Akin et al- -* 1990). Akin and Rigsby (19871 suggested
that fungi a lone were able to digest virtual ly a l1 of the ce1 1
wa l l and were more able than cel lulolyt ic bacteria to degrade
sclerenchyma and thus weaken plant structures.
The ef f ic iency in degrading the p lant polymers varies
according to the fungal species and strain, and the substrate
used as carbon source (Gordon and Phi l l ips , 1989) ; the
cel lu lo lyt ic act ivity of Caecomyces being general ly lower that
of Neocallimastix and Piromyces. Not al1 ingredients a l low the
development of anaerobic fungi ín the rumen, so hígh amount of
zoospores per ml ruminal fluid was observed in rumen of cows
fed wi th grass s i l a g e and straw in contrast w i t h cows
28
f e d w i t h m a i z e silage o r lucerne h a y (Grenet et al. 1989).
Roger et al. (1993) showed a comparison between fungal species
and their interactions with ruminal bacteria (tables 3 and 4).
The whea t straw was less degraded than maize s t e m ; but
wha tever the substrate, the maximum amount of dry mat ter
degraded by N2 frontal is was reached after 4 days of culture.
The two bacteria degraded maize stem with the same ef f ic iency
as the two fungi . However, the substrate was more rap id ly
d e g r a d e d b y FA succinogenes after two days of incubation.
Coculture & frontal is p lus F. succinogenes was more efecctive
than others cocultures. Morgavi e t a l (1994)- - reported
interferente in f i l ter paper ce l lu lose digest ion by protozoal
population ín vitro using Piromyces sp. as inoculum.-
Besides ce l lu lo lyt ic act iv i ty , at least Neocal l imastix
frontal i . shows proteolytic activity that is probably due to a
metal loprotease. Although the proteolytic activity is not very
high compared wi t,h t h a t o f some aerobic f u n g i , í t is much
higher than that of many rumen bacteria (Wallace and Joblin,
1 9 8 5 ) . It is noteworthy that the common rumen cel lu lo lyt ic
bacteria are not actively proteolytic , whi le rumen fungi have
both activities.
It was also found that rumen fungi are able to produce
alpha-amylase and hence are able to digest statich (Mountfort
and Asher, 1988). However, l i t t le pect inase act iv ity has been
found (Pearce and Bauchop, 1985).
TABLE 3. DEGRADATION OF WBEAT STRAW BY THE RUMEN FUNQI AND RUMENCELLULOLYTIC BACTERIA, IN MONOCULTURES AND CXXXJLTURJZS. A/.
X of dry matter disappearence after (days):
2 4 6 0
Neocallimastix frontalis ---- 34.1 35.1 37.1
Orpinomyces joyonii 15.5 27.0 33.5 41.4
Ruminococus flavefaciens 22.7 24.5 26.5 28.0
Fibrobacter succinogenes 22.3 36.6 34.7 38.1
& fronatlis plus R. flavefaciens-
N. frontal& plus-& succinogenes
---- 23.1 23.5 24.2
---- 32.0 59.5 34.2
02 joyonii plus R.- flavefaciens 24.8 26.2 29.4 36.4
2 0joyonii plus F.- succinogenes 26.1 37.4 38.0 39.2
'/ Adapted from Roger et al. (1993).
TABLE 4. DEGRADATION OF MAIZE STRAW BY THE RUMEN FUNQI AND RIJMENCELLUUILYTIC BAClXRIA, IN MONOCULTURES AND COCULTURES. '/.
% of dry matter disappearence after (days):
2 4 6 8
Neocallimastix frontalis 54.7 61.0 59.8 59.5
Orpinomyces joyonii 39.1 52.0 58.7 58.5
Ruminococus flavefaciens 52.6 58.5 58.2 57.8
Fibrobacter succinogenes 60.1 61.0 62.2 62.2
& fronatlis plus & flavefaciens 46.1 49.5 52.3 52.5
& frontalis plus F. succinogenes 53.8 59.5 61.5 60.4
0. joyonii plus R. flavefaciens 39.5 48.0 48.5 49.5
0. Joyonii plus F. succinogenes 42.6 53.8 55.2 56.6
'/ Adapted from Roger et al. (1993).
31
Potential applications of ruminal fungi and their enzymes.
Lignocel lu losic mater ia ls whether primary S0UTC8S or
w a s t e s o f agricultural, domestic or i n d u s t r i a l origin, are
,mainly composed o f c e l l u l o s e , hemicel lulose and l ignin and are
potentially huge storehouses of energy and chemical feedstocks
(Coughlan, 1985).
Anaerobic fungi may be used f o r t h e conversion o f
lignocellulosic wastes and residues to enzymes and fermentation
products. Furthermore, they could be used for making si lage as
feed for catt le or to improve feed for mono-gastric animals by
c o n v e r t i n g a par t of the indigest ib le f i b e r into s ugars ,
fermentation products and fungal biomass (Teunissen and Op den
Camp, 1993).
Ce11 wall hydrolyzing enzymes could be used for part ia l
hydrolysis of ce11 wal ls of oi l containing seed to improve cold
extraction procedures, c l a r i f i c a t i o n o f juices (Biely e t al.,-
19851, the preparation of dextrans as food thickeners, and the
production of fluids and juices from plant materials (Woodward,
19841. The nutrit ional value of oil seed cakes and other high-
f i b e r f eeds c o u l d b e enlarged b y part ia l hydrolysis ;
furthermore, ce11 wal l hydrolyzing enzymes could improve
r e h y d r a t i o n o f dr ied vegetables (Mandels, 1 9 8 6 ) or f i b e r
properties of cotton for the manufacturing or clothes (Mora et-
a l . , 1986).-
The monomeric sugars which retain the chemical energy of
l ignocel lu lose are easi ly separated from the digest , and more
readi ly useable for animal or human food or for the production
o f chemicals (Wang et a l . , 1988) . The xylanases could be used- -
as b leaching reagents for Kraft pulps (Paice et al., 1988).
Chemical analyses of ce11 wal ls constituents.
F r o m an analyt ica l point of v iew, scanning electron
microscopy, transmission electron microscopy, histochemical
techniques, pyro lys is mass spectrometry, and near infra-red
ref lectance are promising techniques to qual itat ively describe
the degradative process of cell wal ls constituents (Akin, 1982;
C h e n g et al., 1983/84; Reid & al., 19881, a l t h o u g h , at
present, they are l ike ly to be app l ied on ly to l imited numbers
o f closely defined samples rather than the necessar i ly large
number of samples required in extension work.
An easy routine method to describe carbohydrates and
!. ignin in plant tissue was proposed by Goering and Van Soest
(1970)) and it can determine d i g e s t i o n o f ce1 1 wall
constituents. T h e p r o c e d u r e is b a s e d on the a b i l i t y o f
detergent solution to solubi l ize non-f ibrous components of the
feed sample and separate b y f i l t r a t i o n the f i b e r , a s
part ículate mater ia l .
The detergent analys is s y s t e m consists o f an acid
detergent f iber fraction, resulting from a extraction with 2X
33
hexadecyltrimethyl ammonium bromide ín l.ON sulphuric acid hot
(Van Soest, 1963). This fraction can be used to determine
l i g n i n b y the 72% sulphuric acíd method, in which case the
c a r b o h y d r a t e is dissolved, or a permanganate oxidation which
leaves a ce1 lulose preparat ion. A t o t a l f i b e r fraction,
referred to as neutral detergent f iber, results from t h e h o t
extraction of a forage with a buffered 2% sodium lauryl sul fate
solution (Van Soest and Wine, 19671. In this way it is poss ib le
t o distingufsh between soluble cellular components and ce11
wall constituents - ce l lu lose , hemicel lu lose , l ignin, cutin and
minerals. These analyses have become very wide ly accepted in
the last 15-20 years.
The above method has the advantage of general
app l icat ion because it is comparatively easy to perform and
gíves an adequate descríption of the compounds of nutrít íonal
re levance. Al though t h e above m e t h o d is useful f o r the
c h a r a c t e r i z a t i o n o f the rumen d i g e s t i o n , i t g ives l i t t l e
ínformation about the extent and nutrit ive v a l u e o f the
digested f iber .
34
MATERIALS AND METHODS
Chemicals.
Al1 chemicals were purchased from the Sigma Chemical Co.
(St. Lous, MO) , the Aldr ich Chemical Co. (Mi lwaukee, WI ) , or
Difco l a b . ( D e t r o i t , MI) and were reagent grade.
Subs t ra to s .
Maize stem (Zea maiz L.f were grown in a f i e l d í n the
municipal i ty of Ixtlahuachn, Colima, MBxico. Stems were
collected in the period of fall to winter, 1993 and were
maintained at room temperature ín s toppered f lasks unti l
used. Stems were mil led tolmmin a Wiley mill. The
composition of maize stems was: dv matter 94.91%, NDF
76.80%, ADF 54.39, l ignin ll .73%, Hemicel lulose 22.41%,
Cel lu lose 40.89%, crude protein í.68%, and sulphur 206 ppm.
Cel lu lose (Sigmacell type 101) was purchased from Sigma
Chemical Co.
So.urce o f i n o c u l a .
Ruminal digesta were obtained through a cannula from a
ge lded male goat cross-bred about three years old (30 kg body
weight) as described by Hungate (1969). The animal was fed a
high f i b e r d i e t (Table 5). Ruminal digesta were strained at
the animal pen through cheesecloth into a vacuum bottle at
39 C and transported to the laboratory, where it was again
strained through eight layers of sterí le cheesecloth before
use.
Preparatfon of media for in vitro incubstíons.-
The media used in this work is described by Akin (1980)
as quoted by Heath (1988) and Barichievich and Calza (1990).
with certain modif ications to enable the study of addition of
inorganic sulphur source. The medía containing the
fol lowing: (wt/vol) K2HPO4, 0 .045%; Na2C03, 1%; Trypt icase
peptone, 0.05%; yeast extract, 0.05%; the fol lowing values
are expresed in (vol/vol): mineral solution without sulphur,
15%; haemin, 0.2%; resazurin, 0.2%; v o l a t i l e f a t t y acids
(VFA) , 1%; vitamin mixture, 0.5%; and sodium ascorbate
solution, 1%. The composition of these solutions is shown in
the annex.
The reducing agent used in this study was sod i um
ascorbate (1.2% solution wt/vol) which replaced the sodium
sul f ide and cysteine mixture normally used in this media.
Ammoni um sul fate was replaced by ammonium chloride in an
amoun t to supp ly the same leve1 of nitrogen. The amount of
t rypt icase peptone was reduced to 5 mg per 100 ml and yeas t
e x t r a c t a t 10 mg per 100 ml; these c o n c e n t r a t i o n s provide
essentia l nutrients and vi tamins f o r the fungí wi thout
acting as a sulphur source (Phillips and Gordon, 1991).
Magnesium sulfate was replaced by magnesium chloride in an
37
amount calculated to supply the same leve1 of avai lable Mg
as that from the sulfate form. The carbon source (cellobiose)
wa,s replaced by maize stem. The pH was controlled between 6.7
and 6.9 in the media. The content of sulphur was measured in
the media after of the addit ion sulphur sources (Bird a n d
Fountain, 1970).
Media were prepared in a flask adding all compounds
plus dist i l ler water . A gas flame to slowly heat the medium
t o b o i l i n g w a s used. During the heating observe the color
change of the medium. After the f lame is removed , a vert ical
Co2 cannula is inserted into the f lask. The oxygen in the
medium was completely removed by the CO2 bubbl ing f o r 3 0
minutes. For anaerobical ly transferring the medium from the
f l a s k to t h e f l a s k s (125 m l ) o r tubes, i t is n e c e s s a r y t o
insert a curved cannula with CO2 into the f lasks or tubes for
at least 20 seconds to remove the oxygen, then use pipet (10
ml) connected with a plastic pipe (about 50 cm) to transfer
medium. The medium is used after autoclaving.
In v i t ro incubat ions .-
Na2S04, Na2S, and Na2S03 were tested as sulphur s o u r c e ’
and a d d e d in an amount of 074, 0.1x, 0.2x, and 0.3% as
sulphur.
Erlenmayer f lasks (125 ml) c o n t a i n i n g an accurate ly
weighed sample of maize stem (approximately 0.5 g) and 50 ml
o f the basal media l isted above were f lushed with CO2 and
stoppered with butyl rubber stoppers and autoclaved at 115 C
/ 15 min. For determination of dry matter loss í n each
sulphur leve1 ( including control without addit ion of sulphur
source) , f ive repl icates were inoculated separate ly with 4
ml of ruminal f l u i d a n d an a n t i b i o t i c s m i x t o inhibit
bacter ia . The antibiotic mix composition is s h o w n in the
annex, and was prepared such that 0.1 ml of solution was
added per ml of broth to give t h e d e s i r e d concentrations
(Akin and Benner, 1988).
Media were prepared, stored and inoculated using the
aseptic and anaerobic techniques.
The residues remaining after incubation at 39 C for 8
days were analyzed with f ive repl icates f o r dry matter.
Supernatant was recovered and the pH was measured immediately
u s i n g a digita l pH meter (Corning Mexicana , S .A . MBxico,
D.F. 1,
Test t o v e r i f y the effioacy o f culture m e d i a w i t h t h r e es u l p h u r sources.
A s s a y s t o veri fy the presente of rumen fungi in the
cul ture media f o r in v i t r o d i g e s t í o n o f maize stem and-
cel lu lose were realized. The incubations were made as point
in earlier paragraphs using only maize stem as carbon source.
During 8 days incubation each 24 hours was removed from the
39
f lasks the supernatant and transfed into the rol1 tubes
containing agar medium with antibiotics mix (Joblin, 1981) l
Qreater fungal growth was observed after 72 hours incubation.
The presente of bacteria1 and protozoal contamination
were determined using supernatant from incubations l isted
above and an e v a l u a t i o n o f culture a f t e r g r o w t h in 1 iquid
media wi th glucose as carbon source without a d d i t i o n o f
antibiotics mix was made (Warner, 1962). N o t g r o w t h f o r
bacteria1 and protozoal population was observed.
Analysis of NDF, ADF, and ADL.
The residues after incubation were analyzed with 15
replicates for NDF, ADF, ADL according to a modif ication of
the aystem of Cloering and Van Soes t (1970) and Komarek
(1993). Cel lu lose and hemicel lulose were ca lculated
considering the NDF, ADF and ADL determinations. The
analyt ica l data were compared with a control without sulphur
source.
The method used herein after extensive refinement
was compared wi th the resul ts from l a b o r a t o r i e s o f
Universidad Autonoma de Chihuahua and campo experimental
“ C l a v e l l i n a s ” o f I n s t i t u t o N a c i o n a l d e Investigaciones
F o r e s t a l e s y Agropecuarias (INIFAP) o f Jal isco. The
substrate (maize stem) used in b o t h l a b o r a t o r i e s was the
40
same used in this experiment, and they used the Goering and
Van Soest (1970) methods wi thout modifications. Other
fibrous forages were tested with analysis procedures used
herein (data not shown).
The methods are based on enclosing a sample of forage
in a polyester bag so that f i l tration and special equipment
were not necessary. Fiber fractions were determined in
dif ferent solvents ( f igure 1): the f irst , neutral detergent
solution, producing a res idue composed of p lant ce11 wa l l s
(NDF) ; and the second, acid detergent solution produced a
fraction corresponding to a l ignocel lulose comp1 ex (ADF) .
L i g n i n is then measured after strong acid hydrolysis . The
data were calculated from the weight losses of the various
samples.
41
SAMPLE
ND ex t rac t i on
INEUTRALDETERGENTRESIDUE
AD ex t rac t i on
ACIDDETERGENTRES 1 DIJE
7 2 % H2SO4 h y d r o l y s i s
LIGNINMINERALSAS RESIDUE
Ash at 550 C
MINERALSAS RESIDUE
SOLUBLECELLULARCOMPONENTS
HEMICELLULOSEMEASURED ASWEIGHT LOSS
CELLULOSEMEASURED ASWEIGHT LOSS
LIGNINMEASURED ASWEIGHT LOSS
F i g u r e 1. Scheme o f f i b e r component ana l y s i s .ND = Neu t ra l d e t e r g en t , AD = Acid d e t e r g e n t .
42
Reagents.
Detergent solutions were prepared according wi th the
procedures detailed by Qoering and Van Soest (1970).
Neutral -detergent solution. To 1 1 dist i l led water add
30 45 sodium lauryl sulfate; 18.61 g disodium EDTA; 6.81 g
sodium borate decahydrate; 4.56 g disodium hydrogen phosphate
anhydrous and 10 ml ethylene glycol. Weigh disodium EDTA and
sodium borate decahydrate then add distilled water and place
on s t i r p l a t e t o f a c i l i t a t e mixing. Mix sodium laury l
sulfate and ethylene glycol then add mix anterior. Dissolve
disodium hydrogen phosphate anhydrous in water. After mixing
with anterior solution. If the solution pH is not between 6.9
and 7.1 adjust with HCl or NaOH.
Acid-detergent solution. Add 20 g of cetyltrimethyí-
ammonium bromide (CTAB) to 1 1 íN H2S04 previous ly
standardízed. Agítate to fací l í tate solutíon.
72% sul fur ic acid.
Decalin: decahydronaphthalene.
Acetone.
Polyclrtsr bala
Synthetic f i b e r bag measuring 5 cm X 6 cm were
fabricated by seal ing three sides wíth thread of the same
materia l . This material was polyester with a u n i f o r m pore
size of 50 um and 1,600 pore/cm2. One side of the bag was not
43
sealed and was used to introduce the sample. Before assays 15
bags were tested for their abi l i ty to withstand the effects
of temperature, detergent and acid solution. The weight loss
by these ef fects represented a b o u t 0.37% and this percentage
was considered in the computation of the data.
Procedure.
Air-dry samples should be ground to pass a l-mm screen
prior to analysis .
Neutrrl-detetraent fiber l nd raid-drtrr#rnt tibor,
1. Weigh p o l y e s t e r b a g at constant w e i g h t (AI.
F)BI Fil1 1x1~; with s a m p l e t o O,iS g s a m p l e o f d r y m a t t e r (BI.
3. Close bag by heat seal ing in a low f lame of a Bunsen
burner (heat seaíing of b a g does not alter total weight) .
4. P lace bags with sample into the f lasks (1000 ml) wi th
detergent solution (ten bag for 200 ml). Add decal in for foam
control led (2 ml for 100 ml).
5. Set flasks in universal supporter and use Bunsen burner
to provide heat.
6. Control the temperature at 95-100 C.
7. After 70 min. for NDF and 60 min. for ADF, remove the bags
from solution and immediately wash the bags containing sample
using b o i l i n g water unti l free of any detergent solution.
Then rinse with acetone. Repeat this rinse 3 or 4 times,
8. Dry overnight or by 8 h. at 100 C and weigh the bags (CI.
44
9. Calculate the percentage of NDF or ADF using data of bag
weight tA), sample weight (BI, and bag with f iber (CI.
C - A% FIBER FRACTION = --------- X 100
B
10. Calculate hemicel lulose b y dif ference between NDF and
ADF resul ts.
Acid-detergent l i g n i n .
This method continues after computer ADF.
1. The b a g s containing acid-detergent extracted fraction
(wi thout d r y i n g a t 100 C) are placed in a f lask adding
s u f f i c i e n t 72% sul fur ic acid to al low that bag c a n b e
submerged.
2. A f t e r 3 h. remove bags from the acid and rinse wi th
boi l fng water unti l the b a g and sample are free from acid.
3. Dry the bag at 100 C overnight or by 8 h. and w e i g h
(calculate the cel lu lose content).
4. Remove sample from the bag and place into the crucible and
ash at 550 C for 3 h. co01 at 100 C and reweight. The loss of
weight on ashing ís the l ignin expressed as a proport ion of
the sample dry matter.
4s
In v i t r o bigsrtion o f cslluloss.
Culture tubes containing 1% (wt/vol) Sigmacell type 101
cellulose and 10 ml basal media as above were autoclaved f o r
15 minutes at 115 C. For each sulphur source and sulphur
l e v e 1 (OX, O.í%, 0.2%, 0.3%) f ive repl icates were inoculated
separately with 0.5 ml of rumen inoculum and 30 ul o f t r ip le
mix antibiotic solution. The residues remaining after 8 days
incubation at 39 C were analyzed by weight loss d r y i n g in a
oven at 70 C overnight.
An assay was done using culture tubes described above
but using sulphur 0.2% as sodium sul f ide . The residues
remaining at time 24, 48, 72, 96, 120, 144, 168, and 192
hours were analyzed (five repl icates per t ime) .
I n b o t h assays supernatant recovered was used t o
measured pH.
Enzyme aotivity from celluloee fermentation.
Enzyme activity of both carboxymethylcel lulase (CMCase)
and B-glucosidase from both cel lu lose fermentation, wi th
dif ferent levels of sulphur sources, and with 0.2% sulphur as
sulfide were measured ín the supernatant.
Enzyme assays were p e r f o r m e d in quintupl icate and
treatments were compared wi th the control . CMCase (EC
46
3.2.1.4) act ivity was measured in a volume containing 100 pl
carboxymethylcellulose (CMC) 1% (wt/vol) in 1X PIPES, (pH 6.8
10 mM P I P E S , 5 mhi NaCl, 0 .01% Triton X-1001, adding 1 0 pl
sample (supernatant). Tubes were incubated at 39 C for 15
min. The reaction was halted by adding 1 ml tetrazolium blue
chloride (TZ) and p lacing the tubes in boi l ing water for 5
min. CMCase was spectrophotometrical ly quanti f ied ( h ~660
nm) as the generation of reducing sugars from CMC by the
method of Jue and Lipke (1985). One international unit (IU)
o f enzyme activity has been def ined as a lpmol of reducing
sugars produced per min. and is e x p r e s s e d in uni ts per
mil l i l i ter of supernatant. f3lucose was used as the standard.
B-g lucosidase (EC 3.2.1.21) activity was determined by
measuring the p-nitrophanol (pNP) released from p-nitrophenol
B-D-g lucoside (10 mM pNPQ3) d i s s o l v e d in 1X P I P E S . The
reaction was stopped after 60 min. by the addit ion of a 1.0
ml 1.0 M Na2CO3. T h e pNP l iberated was measured
spectrophotometrical ly at h ~400 nm. One IU of enzyme
activity has been defined as the amount of enzyme which
produced 1 umol of pNP per minute, and is expressed in IU per
m i l l i l i t e r . pNP was used as the standard.
Protein c o n c e n t r a t i o n in cul ture supernatants was
measured spectrophotometrical ly at h =595 nm by the method
of Bradford (1976). One hundredpl sample plus 1 ml Bradford
47
rengent (100 mg Coomassie Bri l l iant Blue (3 d issolved in 50 ml
95% ethanol plus 100 ml 85% phosphoric acid, bring up to 1
liter) wi th bovine serum albumin (BSA) as a standard were
used.
48
RESULTS
Polyester bag technique (PBT) for analysis of NDF, ADF, andADL.
l t w a s observed that when the bags were p l a c e d in
boi l ing solution they expanded and filled with vapor. The
vapor formation and subsequent bal looning were el iminated by
reducing the temperature of the solution to between 98-100 C.
This re lat ive ly smal l decrease in temperature stopped the bag
from bal looning, and increased t h e precision o f the
procedure.
The NDF, ADF, and ADL analysis comparison of the PBT
and the conventional procedure are presented in the table 6.
The values determined b y the PBT showed 1 ower standard
desviations t han the values analyzed by the conventional
procedure. The means were generally within the range commonly
a c c e p t e d in multi laboratory comparison. Other f ibrous
forages were tested by the analysis procedure used herein
(data not shown).
The improvement of the f i ltration and handling portions
of the detergent analysis system yields major benef its . Fiber
determinations done using PBT are more precise and easier to
preform. Also, savings in time, labor and equipment are
rea l ized . Using the f lasks (1000 ml), thirty f iber analyses
were preformed in 90 minutes. By quantitat ively isolat ing the
49
TABLE 6.COMPARISON OF POLYESTER BAO TECHNIQUE WITH T H E
CONVENTIONAL PROCEDURES FOR NDF, ADF, AND ADL.
LaboratoriesDetermination
U de C UACH INIFAPColima Chihuahua Ja l i sco
NDF % 76.99(1.47) 76.24f3.18) 77.62tl.64)
ADF X 54.39(0.53) 58.00(2.39) 55.98fO.97)
Lignin % 11.73fO.85) 10.56(0.99) 12.23(1.04)
n= U de C 15, UACH 6, INIFAP 3.Maize stem was used as sample.Standard deviation in parenthesis .
50
sample in the f i l ter bag loss of sample due to manipulation
cannot occur. The potential standardization of the NDF, ADF ,
and ADL analysis using the PBT offers a opportunity to reduce
inter- íab variation and permit valid comparisons.
Digestion of dry matter and ce11 wall componentes from maizestem.
The results on dry matter digestion of maize stem by 8
mix of rumen anaerobic fungi are summarized in the table
7. A significant i n c r e a s e (P>O.OOOl) was observed wi th
sulphur addit ion in the incubation media. A lower pH was
observed in the supernatant without adding sulphur source. The
greater increase (16.91%) was obtaining adding 0 . 2 % o f
s u l p h u r a s s u l f i d e ( f i g u r e 2). T h e a n a l y s i s o f variance
(P>0.0001~ indicated that the best sulphur source was
sul f ide and t h a t n o d i f f e r e n c e s w e r e observed be tween
sul fate and sul f i te .
An increase in pH was observed with sulphur sources
addit ion ( f igure 3) . The greater increase of 0.84 units wi th
respect to the control was observed adding 0.3% as s u l f i t e .
However, the values recorded with al1 sulphur sources at 0.1,
0 .2 , and 0.3% are considered optimum for g r o w t h o f rumen
fungi and f iber degradation potentia l .
TABLE 7.pH CHANQES AND DRY MA’lTER DEORADATION FROM MAIZE STEM BY RUMEN ANAEROBIC
FUNQI WITB THREE LEVELS OF ‘lHREE SULFUR SOURCES AFTER 8 DAYS W.
sodiumSu1 fate
Sodium SodiumSulf ide Sulfite
1tem SEN0% 0.1% 0.2% 0.3% 0.1% 0.2% 0.3% 0.1% 0.2% 0.3%
PHab ab b ab ab
6C34 7:03 6.96 7803 7.01 6.75 6.92 6.85 7ao4 7:18 0.21
d bc bDM 21.40 32.02 31:24 3153 34.39 38:09
b bc bc35.02 30:76 33.38 33.66 0.60
DM= dry matter.Each value represents the mean of quintuplicate cultures.Means with the same letter for each line are not significantly different atP<0.0001.SEY = standard error of the mean.
52
20
16
10
6
00.1 % 0.2 %
Sulphur level0.3 %
- SULFATE m SULFIDE I::::::::1 SULFITE
Flg. 2. Increaee compared to controlwithout eulphur of DM degradatlon frommala, etem by rumen fungl for 8 daye.
1
0 .8
0 .6
0.4
0.2
00.1 % 0.2 %
Sulphur level0.3 %
- SULFATE m SULFIDE t::::::::l SULFITE
Flg. 3. pH increaee oompared to nosuphur control from dlferent treatmentemonltored after 8 dayo of Inaubatlon.
Digestion of ce11 wall components from maize stem are
shown in the table 8. Significant increases (P>O.OOOl) ín NDF,
ADF , ce l lu lose , hemicel lulose, and l ignin digestion were
observed adding sulphur sources ( f igure 4, 5 , 6 , 7 , 8 ) . Only
ADF and cel lu lose digestion were 1 ower wi th r e s p e c t t o
control when sulphur was added as s u l f a t e or s u l f i t e .
However, sul f ide was e f f i c i e n t l y u t i l i z e d b y rumen
anaerobic fungi . Increases of 8.37% and 8.77% were observed
in ADF and ce l lu lose d igest ion, respect ive ly at 0 .2% sulphur
( f igures 5, 6) .
The resul ts s h o w e d a little increase in ce11 wa l l s
component digestion of maize stem (NDF, hemicel lulose and
l ignin) adding s u l f i t e versus s u l f a t e .
The preceeding result is the r e f l e c t i o n that rumen
fungi show a tendency to a better u t i l i z a t i o n o f sulphur
present as reduced compounds, since sodium sulfate has one
more oxygen atom than sulf ite. In is r e s p e c t , a significant
dif ference (P>O.OOOl) was observed in the sulphur ut i l izat ion
by fumen fungi between sulf ide (reduced source) , and sul fate
and sul f i te (oxidized sources).
The rumen fungi showed the best growth with the
a d d i t i o n o f 0.2% sulphur as e ither sul fate , s u l f i d e , o r
s u l f i t e . T h i s is shown in the previous figures. Greater
increases (P>0.0001) in the maize stem digestíon by rumen
fungi respect to the control and sulphur at levels of 0.1%
TABLE 8.CELL WALLS CGMPGNENTS DEGRADATION OF MAIZE STEB BY RUMEN ANAEROBIC FUNGI
WXTH THREE LEVELS OF TBREE SULFUI? SOURCES FOR 8 DAYS (X1.
Sodium Sodium SodiumSulfate Sulf ide Sulfite
1tem SEM- -0% 0.1% 0 . 2 % 0 . 3 % 0.1% 0 . 2 % 0 . 3 % 0.1% 0 . 2 % 0 . 3 %
8:04cd cd b b d bc cd
N D F 1 5 . 4 7 16:20 1 5 . 6 2 2 0 . 6 6 25:08 2 0 . 3 1 1 3 . 8 4 1 8 . 9 0 1 5 . 8 3 0 . 5 7
8742d
8:88d bc
16:79b d
8:78d
A D F 5.89 6 . 0 2 9 . 6 3 1 0 . 5 2 5 . 8 5 6 . 7 9 0 . 4 6
12:52cd b cd b cd cd
Ce11 1 0 . 0 5 18.38 10.52 16.95 21:29 13:79 6 8 9 0 l l . 4 6 10.90 0.61
6:99cd bcd d abc ab d a b d
HCell 40.18 41.96 38.93 43.43 45.21 45:96 39.05 43.45 37.99 0 . 7 5
d b d cd b dLig 5:75 1 0 . 3 2 1 6 . 2 4 1 0 . 2 5 13:36 18:41 l l . 3 4 13:07 1 6 . 7 9 9.83 0 . 5 7
NDF= neutral detergent fiber, ADF= acid detergent fiber,Cell= cellulose, Bcell- hemicellulose, Lig= lignin.Each value represents the mean of quintuplicate cultures.Means with the same Ietter for each line are not significantly different atP<O.OOOl.SEY = Standard error of the mean.
56
20
16
1 0
6
00.1 % 0.2 %
Sulphur level0.3 %
m SULFATE k&ii SULFIDE m SULFITE
1
Flg. 4. Increaee reepect to control ofNDF degradatlon from malze etem by rumen
fungl after 8 days of IncubatlonJ
57
10
6
6
4
2
0
- 2
,A I
0.1 % 0.2 %
Sulphur level0.3 %
m SULFATE i&!d SULFIDE m SULFITE
Flg. 6. lncreaee reepect to control ofADF degradatlon from malze etem by rumen
fungl after 8 days of Incubatlon.
1 0
0
6
4
2
0
- 2
- 4
- 60.2 98
Sulphur level
m SULFATE m SULFIDE 1:::::111 SULFITE
Fig. 6. Increaee respect to control ofcellulose degradatlon from malze etem by
rumen fungl after 8 daye of Inoubatlon.
59
60
40
30
20
10
00.1 % 0.2 %
Sulphur level0.3 %
- SULFATE m SULFIDE I:::::‘:rl SULFITE
Flg. 7. Increaee reepect to control ofhemlcellulore degradatlon from malze
etem by rumen fungl after 8 days.
6 0
14
12
10
8
6
4
2
00.1 % 0.2 ‘16
Sulphur level0.3 %
- SULFATE m SULFIDE I::::::::1 SULFITE
.
Flg. 8. Increase reepect to oontrol ofllgnln degradatlon from malze etem byrumen fungl after 8 days of lncubatlon.
61
and 0.3% were abserved. No significant di f ference ~P>0.0001)
was observed between leve l s 0.1% and 0.3% in maize
digestion within the same sulphur source as either sul fate ,
su l f ide , or s u l f i t e .
Cel lulose digestion by rumen fungi .
Agains t to the observed in the cel lulose digestion of
maize stem, t h e pure c e l l u l o s e d i g e s t i o n shown dif ferent
result . An increase s i g n i f i c a n t l y fP>O.OOOl) with sulphur
addit ion was observed (table 9). The greatest digestion was
observed a f t e r adding sodium sul f ide. A lower value was
recorded with 0.1% sulphur as sul f i te , whi le with 0.2% and
0.3% S as sul f i te the cel lulose digest ion was greater than
for sul fate.
High amount of pure cel lu lose disappearance was
observed adding 0.2% sulphur as sul fate , sul f ide or s u l f f t e ,
recording increases with respect to the c o n t r o l o f 27.9,
45.9, and 35.7% to sul fate , su l f ide , and s u l f i t e ,
respect ive ly ( f i g u r e 9). Cel lulose digest ion was greatest
with 0.1% sulphur from sulfate and sulf ide sources than with
0 . 3 % f o r the three sulphur sources. However, wi th 0.3%
sulphur as sul f i te the cel lulose digestion was greater than
with sul fate or su l f ide .
62
TABLE 9.CELLULOSE DECRADATION BY THE RUMEN FUNGI WITH
3 LEVELS OF 3 SULFUR SOURCESAFTER 8 DAYS (X1.
Sulfur sourceS u l f u r (%)
0.1 0.2 0.3
Sodium sulfate 54.26a 56.07a 52.66a(5.01) (2.28) (2.92)
Sodium sulf ide 54.90a 74.12b(2.50) (3.00)
50.94a4.23)
Sodium sulf ite 41.99b 63.89c 53.89a(4.65) (4.22) (5.05)
Each value represents the mean of quintupl icatecul tures. Control ( 0 % SI= 28.15% cel lu losedigestion. Leve1 0.2% S was s igni f icantly greaterrespect 0.1% and 0.3% (P>O.OOOl). Means for eachco 1 umn f o l l o w e d b y the same l e t t e r are nots i g n i f i c a n t l y d i f f e r e n t at P<O.OOOl.Standard deviation in parenthesis .
63
1
60
40
30
20
1 0
00.1 Qo 0.2 %
Sulphur level0.3 %
- SULFATE 6\rm SULFIDE r:::::,.rl SULFITE
Flg. 9. Increaee reepect to control ofpure celluloee degradatlon by rumen
fungi after 8 days of incubatlon
64
Enzyme activit ies for CMCase and B-g lucosidase.
The effects of levels of three sulphur sources on the
a c t i v i t i e s o f CMCase and B-g lucosidase, and protein
production are shown in table 10. The activit ies for CMCase
and B-glucosidase were increased with addition of al1 sulphur
sources for three levels. Sulphur sources increased CMCase
activity respect to control (P<O.OOOl). CMCase a c t i v i t y in
cul tures grown without sulphur was 0.056 (IU/ml), against
values since 0.131 (IU/ml) up to 0.314 (IU/ml) f o r 0.1%
sulphur as su l fate and 0 .2% sulphur as su l f ide , respect ive ly .
The act ivity of B-g lucosidase after 8 days of cel lu lose
fermentation was higher respect to control (P<0.0001). One
majar and one minor B-glucosidase activity was found for 0.2%- 3
s u l p h u r a s s u l f i d e (30.6~10 IU/ml) and 0.1% sulphur as- 3
s u l f a t e (12.2~10 IU/ml) , r e s p e c t i v e l y (table 10).
A significant di f ference (P<O.OOOl) between control and
sulphur treatments was observed for supernatant protein. As
observed f o r CMCase and B-g lucosidase, the majar
concentration was observed for 0.2% sulphur as sulfide.
Between sulphur sources, the addit ion of sul f ide shown
higher results, whi le with the a d d i t i o n o f s u l f i t e the
results are minor than sulf ide and sulfate. The better leve1
for al1 sulphur sources was 0.2% sulphur.
TABLE 10CARBOXYMElHYLCELLULASE (CMCase) AND B-GLUCGSIDASE ACIIVITIES, AND PRGTEIN
IN AN ASSAY USING RUMEN FUNGI AND SULPBUR SGURCES AFTER 8 DAYSINCUBATIGN OF CELLULGSE
Sodium Sodium SodiumSulfate Sulfide Sulfite
Item SEM0% 0.1% 0.2% 0.3% 0.1% 0.2% 0.3% 0.1% 0.2% 0.3%
d bCMCase .&6 ,131 .200 .lSS .1;2 .3;4 .150 .lGO .1;5 .lL 0.040
d b d cdB-Glu&/ 6:23 12.21 19.54 16:S8 16:,9 3OaS9 15.26 16:80 17:Ol 16.15 0.005
f b d cds. P . 11.13 18:41 30.54 24:91 26:14 47a28 22.75 26:78 27:54 23.74 0.730
ChiCase = Carboximethylcellulase (IU/mll. B-Glu = B-Glucosidase (IU/ml).S,P.= supernstant protain (~g/ml).'/ The values for B-glucosidase are shown with exponential (E-4) in e, and (E-31 fora, b, c, d, and f.Each value represents the mean of quintuplícate cultures.Means with the same letter for each line are not significantly different atP<O.OOOl.SEu1pI Standard error of tha mean.
66
Cellulose digestíon by rumen fungi and sulphur as sulfide.
Figure 10 shows the amount of cellulose disappearance
using ruminal f luid as inoculum and antíbiotics to select
rumen fungi wi th c e l l u l o l y t i c a c t i v i t y . I n the last
experíment, the greatest rate of dígestíon was observed by
.adding 0.2% sulphur as sul f ide. Therefore, ín thís experíment
the cellulose dígestion was measured each 24 hours for 8 days
using 0.2% of sulphur as sul f ide. A proportional increase to
the incubation time was observed. The greatest increse in
cel lu lose digestion was recorded after 48 hours, and the
maximum cellulose dígestíon was reached at 168 hours. At 192
hours 74.64% cel lulose dígest ion was observed, only 0.14%
more than at 24 hours before. If this tendency fs maintained
by a long incubation time, then cel lulose digestion can reach
100% digestion. Therefore ít ís necessary to determine the
quickest best t ime to total cel lulose digestion.
No signíf icant di f ference ocurred between incubation
times f o r p H ( f i g u r e ll), therefore the rumen fungí
manif iested their ce l lu lose degradation p o t e n t i a l . An
unimportant increase in pH was recorded at 48 hours. However
the cel lul lose dígest ion was greatest in 24 hours before.
67
100.100.
00 ' II II 1I II II II 1l II 12424 4848 7272 9696 120120 144144 168168 192192
Incubation time U-mur)Incubation time U-mur)
Fig. 10. Cellulose dfsappearance (73 mcorded between 24 and 192 h ina medium with 0.2% sulphur and rurnen fungl. Each value, isthe mean of five replicabas. The vertical bars representstandard dsviation.
68
4 _,
0 I I I I l l I I2 4 48 7 2 88 120 144 198 1 9 2
1ncubation tifns (hour)
Fig. l l . Changes in t h e pti thraqh t h s incubation period wíth tunenanaerobic fúngi, cellulose a n d 0.2% su1ptn.w. Each v a l u ela the mean of five replicates. The vertical bars representstandard dsviatlon.
69
Enzyme activit ies for CMCase and B-g lucosfdase i n v o l v e d indegrading cellulose by rumen fungi and 0.2% sulphur.
Enzyme activities were detected since 24 h incubation.
A proportional increase at incubation t ime was observed for
both CMCase and B-glucosidase activit ies ( f igures 12 and 13).
Maximal enzyme activit ies were detected at 192 h. CMCase
s h o w n an activity of 0.33 (IU/ml) and B-glucosidase shown- 3
26.7x10 (IU/mlI. Since 72 h incubation high increase was
observed each 24 h. Supernatant protein increased
simultaneously wi th enzyme production ( f i g u r e 14). The
maximum protein concentration was 5 7 . 4 pg/ml a t 192 h
incubation.
?Q
0 124 4 8
I I I r I7 2 9 8 120 144 188 192
Incubation time WMJLW)
Fig. 12. Times acuses -For carboxymethylcellulase KIPlCase) (IU/ml) byr-unen anaerobic fungi grcwing on cellulose and 0.2% sulphur.Each value is the mean of five replicates.
71
lE-430 -7
0 I I l l 1 I2 4 43 7 2 9 6 120 144 168 192
Incubation time hour)
Fig. 13. Times axwses for B--glucwidase (IUIml) by rumen anaerobi cfungí gvowing on cellulcma and 0.2!4 mulphuv. Each value 16th6 mean of five repllcates.
1 0 -
0 1 I I I I I34 48 96
Incubation time hour)
Fig. 14. Spernatant protein pg/ml) recorded during 8 days fbr unen+üngi incubated in presente o-f cellulose and 0.2% sulphur.Each value la the man CrF five replicates.
73
DISCUSSION
An advantage of the rumen anaerobic fungi is that they
can grow in a culture medium wi thout ruminal f lu id ,
provîded that cofactors and nutrients necessary f o r their
growth are inc luded. The point above was observed by Orpin
and Creenwood (1986) wi th Neocal l imastix patriciarum,
which mult ip l ied in the presente of only two vi tamins and
added carbon, nitrogen and sulphur sources. However, since
this group o f microorganisms is o f recent discovery, the
nutrit ive requirements f o r best growth has not yet been
def ined. This has been demonstrated by d i f f e r e n c e s in
l ignocel lu los ic substrates degradation observed by some
researchers (Orpin and Hart, 1980; Oordon and Ashes, 1984;
Qordon, 1985; Lowe et al- -.? 1987; Roger et al- ,-9 1993).
The effect observed in this study showed that mixed
rumen anaerobic fungi from goats were capable of u t i l i z i n g
s u l p h u r a s sul fate , sulf ide and s u l f i t e . This was
demonstrated by the high weigh loss of dry matter of maize
stem. Increases from 9.36 to 16.7 percentage uni ts wi th
respect to control were observed. This effect confirmed the
abi l i ty of the fungi to degrade l ignocel lulosic substrated as
maize stem used in this work. Both amount and s o u r c e o f
sulphur were determined f o r maize stem degradation.
Therefore, differences between treatments were expected.
Previous research has indicated significant di f ferences
in dry matter and organic matter digestion between species of
rumen fungi . híoreover, t h e s u b s t r a t e c o m p o s i t i o n is
determinat to rumen fungi manifiest i t s growth and
degradation potentia l . As Roger et a l (1993) reported, up to- -
59.5% maize stem digestion at 8 days of incubation with N.-
f r o n t a l i s MCH3 a n d 5 8 . 5 % w i t h 0 . joyonnii TP90-9, these-
resul ts are greater than those obtained fn this work which
used similar substrate at 8 days incubation, It is important
to consider that the inocula used in this study were of 8 mix
of rumen fungí from a goat. In another study, the protozoal
populat ion inter fered with f i l ter paper ce l lu lose digestion
.by rumen fungi (Morgavi et a l . , 1994) . However, interrelat ion- -
be tween dif ferent Turnen fungi spec ies that can give
discrepances in the substrates digestion as wel l as ruminal
fungi-bacterium interactions has not been studied.
T h e e f f i c i e n c y o f fermentation by anaerobic f u n g i
d e p e n d s on t h e s t r a i n s used a n d t h e o r i g i n o f t h e plant
substrate, and may be less than, equal to, or greater than
that of total microbial activity in rumen f luid (Windham and
Akin, 1984; Akin and Rigsby, 1987; Akin et al., 1989).- -
Orpin and Hart (1980) found substantial di f ferences in
the dry matter digestion between substrates (wheat s traw
leaves t haY, and perennial rye-grass). Also between three
rumen anaerobic fungi species (N2 frontalis, $L- communis, and
P. communi s 1 they found a signif icant di f ference. Gordon- -
(1985) reported a difference up to 22.4% in organi c matter
d i g e s t i o n o f wheat straw among two pure c u l ture o f rumen
fungí obtained from sheep. However, Gordon and Ashes (1984)
and Roger et al (1993) found no signif icant díf ferences- -0)
among isolated species (MT-1 and MT-21 and (N frontal isA
‘MCH3 a n d 0. J o y o n í i TPVO-9) , respect ive ly . Gordon and
Ph í l l í p s (1989) reported l i t t l e d i f f e r e n c e s on in vi tro- -
dígestion of dry matter between strain of a same genus and
among strains of Píromonas the dif ference is hígher.
In the present experíment it is possíb le that sources
and concentrations of utí l ized sulphur were not suitable to
fil1 requírements of sulphur for rumen anaerobic fung i , and a
second effect is constítuted b y the substrate used. There ís
evídence that índicates a minor u t í l i z a t i o n o f inorganic
sulphur sources wi th respect t o the organí c sulphur
sources. Thís is supported ín works ín vi tro with ’ rumen-
fungi (Orpin and Greenwood, 1986; Mi l l a rd e t a l 1987;
Phi l l ips and Gordon, 1991), and ín works in vitro using whole-
rumínal f l u i d a s inocula (Barton & al- 1971; Bu11 and
Vandersa l l , 1973; Khalon et a 1975; Speras e t a l 1976;- -
Spears et al 1978; Komisarczuk-Bondy et al- 1992). Also in- -
works & vivo observíng the effect on rumen fungi (Gordon e t
a l 1983; Gordon- e t a l 19841, a n d on- - al1 rumen microbial
groups (Spears et a l 1978; Guardio la et a l 1980; Guardiola- - - -
76
et al 1983; Buttrey et al 1986; Weston et al 1988: Onwuka and--._- _-- - - - -
Akinsoyinu 1989; Murray et al 1990; Qi et al 1992; McCracken- - - -
et a l- - 1993; Qi et & 19931.
Greater dry matter and ce11 wall components digestion
are reported in vivo versus in vitro ín works- - - - - quoted above.
This e f f e c t could be explained by the interactions among
microbial g r o u p s in the ruminal ecosystem. The inoculum
origin is another important factor in the in vitro digestion-
experiments, espec ia l ly the species of ruminant and its feed.
A k i n e t a l (1983) reported higher dry matter- - d i g e s t i o n in-
v i t r o when using ruminal fluid from sheep supplemented wi th
sulphur.
Evidente indicated that reducing sulphur source is used
better by rumen fungi compared with oxidized sources. This
fact m a y b e explained by the absence in rumen f u n g i o f
enzymes capable o f reducing oxidized sulphur compounds
(Phillips and Gordon, 1991). Orpin and Greenwood !1986)
reported no uti l izat ion of sul fate as sulphur source by N.-
patriciarum. F u r t h e r m o r e , i t is poss ib le that oxidized
sulphur sources and other compounds compete f o r avai lab le
hydrogen within the culture media and this could be a
l imfting factor in the reduction of sulfate and s u l f i t e b y
rumen anaerobic fungi and therefore mw influente sulphur
a v a i l a b í l i t y b y decreasing the g r o w t h o f these micro-
organisms. Spears et a l (1977) postulated that a def ic iency- -
77
o f electrons limited the r e d u c t i o n o f sulphate since
supplementation wi th sul f ide produced a higher í n v i t r o-__L
cellulose digest ion.
An e f f e c t on increase of fungal biomass í s o b s e r v e d
wi th sulphur addit ion into of culture media , as wel l as an
increase ín the l ignocel lulosic substrates d isappearance in-
vi tro (Akin et al- -‘? 1989; G o r d o n a n d P h i l l i p s , 1989;
Sijtsma and Tan, 1993) and in vivo (Gordon etI_- - al . 9 1983;
A k i n e t Q.,- 1983; Gordon, 1985; Gulati et aJ., 1985;
Morrison et a l- - - 9 1990; Gordon and Phíllips, 1993; Morrison et-
a l-* f 1994).
The increases í n N D F d i g e s t i o n o f m a i z e stem vs.
control demonstrate that sulphur addit ion as sul fate , sul f ide
and s u l f i t e , develops the fungal growth, r e s u l t i n g in a
greater rate of digestion. The ADF digestion only was better
by adding sulphur as sul f ide. Sul fate and s u l f i t e addit ion
did not increase ADF digestion with respect to the control.
The better e f f e c t was observed adding 0.2% sulphur as
sul f ide , sul fate and sul f i te . A report utilizing rumen fungi
in v i t r o- as inocula was done by Gordon (19851, and our
results could be compared. He reported mínimum NDF digestion
o f whea t s traw up to 24.7% and maximum of 50.1%. In the
present work we reported NDF digestion minimum of 13.8%
adding 0.1% sulphur as sulfite and maximum 25.0% with 0.2% S
as sul f ide . In general the oxidized sulphur sources showed
78
NDF digest ion values s igni f icant ly less u p to 24.7%.
ADF digestion observed with three sulphur sources used
in this experiment are signi f icant ly less than those
reported by Gordon (1985). However, inconsistent NDF and ADF
digestion values are reported by Gordon and Ashes (1984).
They reported between 1.9 and 20.8% in NDF digestion and 0
and 23% in ADF digestion. More substantial ADF digest ion
values are reported by Gordon and Phillips (1989). The ADF
digestion of wheat straw values is between 32.8 and 47% using
pure strain of three genera of rumen anaerobic fungi .
Al though the research quoted uti l ized pure strains of
rumen fungi , insuff icient results are avai lable to show the
real NDF and ADF digestion rates from different substrates.
Moreover, it is still unknown which strains are stimulated by
the sulphur addit ion as sul fate, sul f ide and s u l f i t e . The
dif ferences between data reported by others and our resul ts
suggest that it is necessary to apply more sensitive methods
to future studies, spec ía l l y when using intact fibrous
substrate.
The NDF and ADF in vivo digestion rates are- - greater
compared wi th r e s u l t s in v i t r o . G u a r d i o l a e t a l- - -09 (1983)
reported NDF and ADF in vivo digestion of 51% and 40% in- -
NDF and ADF, respectively. They used f istulated sheep fed
with good qual ity fescue and tropical star grass hays and
offered a supplement of 0.15% sulphur as sodium s u l f a t e o r
methionine. Buttrey et al- (1986) reported in vivo digestion- -
79
of 66.5% and 62.8% for NDF and ADF, respectively in sheep fed
wi th corn si lage and sodium sul fate. Qulati et a l (1985)- -
reported 69% ADF digestion in sheep supplemented with sulphur
as methionine (4.5 g per day) and Qordon and Phillips (1993)
reported 57.3% ADF digest ion after t h e e s t a b l i s h m e n t o f
fungi population in sheep fed with barley straw (Hordeum
vulgare) and lucerne (Medicago sativa).
Tha extent of in vitro ce11 wall degradation by rumen- - -
anaerobic f u n g i in t h i s s t u d y (Table 8 , 9) is s igni f icant ly
less when compared with degradation of other, intact, plant
materials, such as Bermudagrass stems (45%) or l e a f b lades
(70%) (Akin et al- -*v 19901, Italian ryegrass haY (SOY.1
(Theodorou et a l- -.f 19891, milled wheat straw (45%) (Qordon
and P h i l l i p s , 19891, barley s t r a w (37-50X) (Joblin et al-.,
19891, or lucerne stems (31%) (Joblin and Williams, 1991).
These dif ferences may be accounted for by d i f f e r e n c e s in
strains, substrate o r i g i n o r substrate particle size,
al though Lowe et a l (1987a) reported that mil l ing of wheat- -
s t r a w d i d n o t enhance t h e rate o r e x t e n d o f substrate
degradation.
The cel lu lose digestion of maize stem was higher
respect control only adding sodium sulfide and with 0.2% S as
sul fate . These results are compared with those reported by
aordon and Ashes tl984) who used used a strain of rumen fungi
tmycelial twe) as inocula t o t r i a l d i g e s t i o n on wheat
80
straw. The results herein with sodium sulfate and sodium
sulf ite are greater that those reported by the same authors
but they used strain of rumen fungi (non-mycelial type).
The solubi l izat ion of the cel lulose component of straw
a f t e r incubation f o r 4 or 5 days was either 39 - 58%
(Gordon, 1 9 8 5 ) or 18 - 5 6 % (Mountfort and Asher, 19891,
depending on the strain of anaerobic fungus being tes ted.
However, Orpin (1983/84) r e p o r t e d a rate o f d i g e s t i o n o f
ce1 lulose from wheat straw leaves to be greater than those
reported herein. The lower value (39.7%) was observed with
the fungus Caecomyces communis and the high (58.1%) with the
fungus Neocal l imastix frontal is . Bernal ier et a l (1988) found- -
that the fungus Neocal l imastix could digest more cel lu lose
alone t h a t in combination wi th c e l l u l o l y t i c bacter ia .
C o m b i n i n g & flavefaciens wi th Neocal l imastix reduced
cel lu lose digestion, whi le essentia l ly no dif ference was
found when FL succinogenes and the fungus were combined. An
example of synergism through end-product u t i l i z a t i o n is
reported by Bauchop and Mountfort (1981) ; Fonty et al (1988) ;I-
Joblin et a l (1989) in some studies on digestion of cel lulose- -
and solubi l ization of straw by rumen fungi ín co-culture with
methanogens.
The di f ferences in cel lu lose digest ion in this study
and other works could be due to the dif ferent f iber sources
or the dif ferents analytical methods used. Even t o the
a n t e r i o r w e may postulate that the addition of sulphur as
sul f ide develops the cel lu lose digest ion.
Although the results shown by some authors indicated
that hemicel lulose is used and l ignin is digested by rumen
fungi (Orpin, 1981; Akin et al., 1983; Gordon and Ashes,- -
1984, Gordon, 19851, the true effects of rumen fungi on these
substrates, especia l ly l ignin, are yet uncertain. I n i t i a l l y ,
l ignin was considered “ indigest ib le” in ruminants, and f o r
this reason i t has been used a s an interna1 m a r k e r to
calculate the d i g e s t i b i l i t y o f feedstuffs . However,
experimental data has shown that lignin, determined ei ther
with sulphuric acid or potassium permanganate, is apparently
digested to a variable extent with values ranging from 2% to
53% (Susmel and Stefanon, 1993).
The present work contributes to elucidate these
e f f e c t s . The results showed that the sulphur sources used
increased signif icantly the digestion of hemicel lulose and
1 ignin of maize stem. However, these values are lower than
those reported by Orpin (1983/84). Up t o 55% loss
of hemicellulose and 21.9% loss of lignin from wheat straw by
Piromyces communis are r e p o r t e d . McSweeney et al- (1994)
reported a l ignin solubi l isat ion by N patriciarum of 33.3%2
and 2 1 . 2 % in sorghum rind a n d NaOH - extracted sorghum
respect ive ly . The results on l ignin d i g e s t i o n w a s g r e a t e r
than reported by Gordon (1985).
82
The capabi l i ty o f the rumen anaerobic fungi to dígest
both puri f ied xylan and hemicel lulose from intact forages has
been demonstrated by Phillips and Qordon (1989) and Theodorou
et al (1989). Windham and Akin (1984)- - reported that the
l ignin determined by the 72% sulfuric acid method was not
digested in the incubations. Akin and Benner (1988) reported
a in vitro l ignin solubi l isat ion by polycentr ic fungi . The-
loss of l ignin probably represents solubi l isation rather than
digestion, for rumen anaerobic fungi connot ferment simple
phenol i c acids (Orpin, 1983/84), and f r e e phenol ic acids
inhibit f iber digestion in vitro (Akin and Rigsby, 1985) .-
Theodorou et al (1989) suggest that the cell-wall- -
hydrolyzing activity of rumen fungi may release substrates
from plant ce11 walls which can be utilized by other members
o f the microbial populat ion in the rumen, t h e r e f o r e an
i n c r e a s e o f f i b e r d i g e s t i o n is observed. However, the
previous studies establ ished that rumen anaerobic fungi
digested and fermented f iber components from intact forages.
Al though cell-wall degrading activity and the
production of cell-wall degrading enzymes have been wel l
documented in the rumen fungi , l i t t l e information is
avai lab le concerning the manner in which they degrade the
structural polysacchar ides (ce l lu lose and hemicellulose) and
l ignin in p lant ce11 wal ls .
1 Il the present study the pH recorded is greater than
83
control although it did not have a determining effect on the
d i g e s t i o n o f maize stem components. T h e p H r e c o r d e d in
control (6.34) and the maximum (7.18) recorded in treatment
wi th 0.3% sulphur as sodium sulfite are considered best f o r
adequate rumen ecosystem function.
The role of sulphur on cel lu lose digest ion is clear in
the table 9. A signif icant d i f f e r e n c e w i t h respect to
the control is observed. An e f f i c i e n t u t i l i s a t i o n b y a m i x
of rumen fungi of the oxidized sulphur sources was observed.
A signif icant increase (46% more relative to the control ) in
cel lu lose digestion adding 0.2% sulphur as su1 f ide was
remorded at 8 days incubation.
The sulphur sources effectiveness could be explained by
the effect that they can produce on the fungal biomass. Then
the increase in cel lu lose digest ion is ín relation with the
increase in fungal populat ion and the consequent increàse in
ezymatic act iv ity . This re lat ionship has been r e p o r t e d in
w o r k s b y (Akin et al-., 1989; Qordon and Phi l l ips , 1989;
Sijtsma and Tan, 1993).
The ce1 lulose and hemicel lulose are digested in the
rumen by dif ferent microbial groups. However, t h e s t u d i e s
have been indicated that the digestion rate change according
the substrate. Ce11 w a l l content o f forage can v a r y in
d i g e s t i b i l i t y within the range of 30-60%, and in individual
84
ce11 types from 0 to 100% (Susmel and Stefanon 1993; Wilson,
1994). It ís this low and v a r i a b l e d i g e s t i o n , and the
prospect o f i t s improvement, that has p r o m p t e d s o much
,research into ce11 wal l characterist ics.
The cel lu lose from intact forages may be hydrol ized
completely by rumen micro-organisms if the incubation time is
extended. However, i t í s poss ib le that rumen fungi d o n o t
possess some mechanism to make disposable the c e l l u l o s e in
intact forages. In the present study the cellulose from maize
stem was probably not disposable completely and therefore
lower digestion was observed.
This p o s t u l a t i o n is supported by Akin and Benner
(19881, who treated ruminal f l u i d wi th antibiotics and
measured the hydrolysis power from different rumen microbial
g r o u p s on Bermudagrass. The bacteria1 a c t i v i t y was
responsible from the greater f iber digestion (as loss weight
o f substrate) and the digestion by fungi was signi f icant ly
less. However, a greater fungal colonization on Bermudagrass
was observed when Turnen fungi were selected with antibiotics
than when the whole rumen fluid without antibiotics was used
(Akin and Rigsby, 1987).
The cellular content in the maize stem is 1 ower than
t h a t o f ce1 1 wal ls ; therefore in the cul ture media the
cellular content ís the f i rst substrate uti l ized, then the
structural pol isaccharides dígest ion ís inhíbited. I n t h i s
respect Morrison et a l (1990) reported a greater ce l lu lo lyt ic- -
activity by rumen fungi when glucose, cel lobiose or starch
were added. They suggest that some f ungal ce l lu lases are
susceptible to catabol ite regulatory mechanisms.
The rumen fungi use cel lu lose as carbon source and
sodium sul f ide as S source for growth. The production and
characterist ics of cel lulases from a number of rumen fungi
have been described (Lowe e t a l 1 9 8 7 ; Hebraud and Fevre ,- -
1988). Cel lu lases of rumen fungi have p H and temperature
optima of 5 - 6 and 45 - 55 c, respect ive ly . Ce1 lulase
p r o d u c t i o n in batch culture is a l w a y s accompanied by the
production of hemicel lulases and other g lycosidase enzymes.
In general xylanase activities have pH and temperature optima
o f 5 . 5 - 6.0 and 50 C, repectively (Mountfort and Asher,
1989). In the present study the pH was increased up to
7.0; however, increases in cel lulose digestion after 24 hours
incubation were observed, to reach the maximum at 168 hours,
which demonstrated that the a b i l i t y t o d e g r a d e pur i f ied
cellulose is a variable character among rumen fungi and among
carbon source. Moreover, some pur i f ied cel lu loses are
fermented more s lowly , but rumen fungi rapidly adapted to
u t i l i s a t i o n of cel luloses with altered unit ce11 structures
(Weimer & jal- 1990).
The CMCase and B-glucosidase activities reached maximum
levels in 8 days-old cultures using 0.2% sulphur as sul f ide ,
86
which is approximate to the resul ts obtained wi th
Neocal l imast ix sp . (Lowe e t a l . 1987b) and Neocal l imastix- -
f r o n t a l i s (Mountfort and asher, 1985) . For other levels and
sulphur sources, the activit ies were less t h a n in 0.2%
sulphur as su l f ide , but major than for control . This resul ts
iS depending of the effectiveness of rumen fungi f o r using
sulphur sources. This is support by Pearce and Bauchop
(1985); Gordon and Phi l l ips (1989). They reported t h a t an
increase in fungal biomass is in relation with i n c r e a s e in
enzyme act iv ity . The f ungal b i o m a s s is increasing wi th
sulphur supplementation (Orpin and Greenwood, 1986; Ph i l l i p s
and Gordon, 1991).
Degradation of cellulose power was determined herein by
measuring the amount of cellulose disappearance. The increase
was observed a f t e r 144 h. This result agrees wi th that
reported by Qordon and Phillips (19891, who observed the
maximum growth and greatest ce l lu lose desappearance a f ter 4
days incubation. Bernal ier et a l (1989)- - reported greatest
cellulose disappearance after 6 days by Piromyces communis.
The enzymic a c t i v i t i e s f o r CMCase and B-glucosidase
general ly reached plateau levels after 6 or 7 days of growth
(Pearce and Bauchop, 19851. Our results are in agreement with
this asseveration. The increases af ter 72 h indicated
poss ib le extracel lu lar enzymic act ivit ies , because the high
rate of growth for rumen fungi is reached at 72 h (Morrison
et a l . , 1994) .- -
The carbon source occasional ly could reach 100%
digestion, but a f t e r growth fungal stops, the c e l l u l o s e
s o l u b i l i s a t í o n is very s l o w , indicating that the ce l lu lo lyt ic
enzymes produced by rumen anaerobic fungi were capable o f
hydrolyzing t h e c e l l u l o s e t o s o l u b l e products detectable in
supernatant of the incubations.
88
CONCLUSION
Our work is the f irst step to stabl ish a investigation
line to study the rumen anaerobic fungi. Although the results
are pre l iminary , we can concluded the fol lowing. It is
evident from the results of this study that sulphur plays a
role in the growth of rumen anaerobic fungi and therefore an
increase in the maize stem and pure cel lu lose digest ion was
observed.
Not al1 sulphur sources can be used by the fungi wi th
t h e same ef f ic iency. The sulphur as sul f ide (reducing
source) was better utilized by rumen fungi . Sul fate and
s u l f i t e (oxidized sources) were used by mixed rumen fungi ,
al though greater r e s u l t s in maize stem and cel lu lose
degradation, and enzyme activities was observed when sul f ide
was added in culture medía for in vitro digestion.-
The results demonstrated that leve1 0.2% of sulphur as
sul fate , sul f ide or sulf ite was the best for growth of rumen
fungi , since greater rate digestion of both maize stem and
pure cel lulose was observed at this concentration.
Digestion of the component of intact roughages are less
than the digestion of their individual compounds. This ef fect
was demonstrated by the extent of digestion of pure ce l lu lose
(Sigmacell type 101) using 0.2% sulphur as sulfide compared
with cel lulose digestion from maize stem.
This work shows a way to increase t h e n u m b e r o f
89
anaerobic f u n g i in the rumen) w h i c h is p o s s i b l e w i t h
appropriate dietary conditions, f o r examp le sulphur
supplmentation. The use of sulphur sources most influenced
the rate of rumen microbial fermentation of f ibrous f orages
by ruminants. Increased rate o f ruminal f e r m e n t a t i o n is
part icu lar ly relevant to obtain the maximum benefit of aw
increaes rumen turnover.
It seems reasonable that the increased growth of rumen
fungi could result in an improved f iber degradation, a lthough
the mechanism by which the rumen fungi use the sulphur
s o u r c e s ( s u l f a t e , s u l f i d e , or sulfite) is n o t clear.
Although our findings support the suggestion that rumen
f ungi m a y b e important in f i b e r degradation, addi tional
research under rumen conditions has to be done to e lucidate
the role of sulphur on rumen fungi growth and their digestion
potent ia l .
Attention needs to be directed towards other
potent ia l ly rate l imiting nutrients other than s u l p h u r , i f
the e x t e n t o f f iber degradation by rumen fungi is to be
further increased.
Techniques are now avai lable for the i s o l a t i o n o f
anaerobic fungi . Therefore the use of sulphur sources is a
method for manipulate specific rumen fungal populat ions to
improve f iber digestion.
The molecular biological approach seems ta be a s trong
too1 t o study ce l lu lo lyt ic and xylanolyt ic enzymes. These
techniques can be applied fox- studying the genes of anaerobic
fungi to obtain information on activity and relat ionships of
these enzymes.
91
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A N N E X
MINERAL SOLUTION WITHOUT SULPHUR
105
3.0 g KII2PO46.0 g NH4CI6.0 g NaCl0.5 g MgC . 6If200.6 g CaC . 2H20
Bring volume up to 1 liter with water.
VOLATILE FA’M’Y ACIDS (VFAI
700 ml6.85 q l3.0 ml1.84 ml0.47 ml0.55 mí0.55 ml0.55 ml
0.2M NaOHacetic acid, glacialpropionic acidn-butiric acidisobutiric acid(+)-2-methylbutyric acidnkleric acidisovaleric acid
The pH of the VFA mixture was adjusted to 7.5 with 1M NaOHand its volume was adjusted to 1 litre with water.
ANTIBIOTIC SOLUTION
Dissolve 100 mg chloramphenicol in 1 ml 95% ethanol .Dissolve 2 g penicillin G plus 800 mg streptomycin sulfate in9 ml water. Slowly combine 2 volumes together, filter andautoclaved, and store at -20 C.
HAEMIN SOLUTION
Dissolve 0.1 g haemin in 10 ml 95% ethanol and adjusting thevolume to 1 litre with O.OSM NaOH and store at 4 C.
RESAZURIN SOLUTION (0.1%)
Dissolve 1 g resazurin in 1 liter water.
SODIUM ASCORBATE SOLUTION (1.2%)
Dissolve 12 g in 1 liter water.
106
VITAhiIN MIX
100 mg100 mg100 mg100 mg100 mg100 mg
5 mg5 w5 mg5 mg
lipoic D,L 6-8 thiotic acidD-pantothenate hemicalciumniacinamideriboflavinpyridoxine HClthiamine HClP-aminobenzoic acidbiotinfolic acidvitamin B12
Bring up to 500 ml with water.
BASAL MEDIUM FCR in vitro INCIJBATIONS- -
0.0451
0.050.05
150.20.2
10.5
1
ggLmlIn1mlmlmlml
K2HPO4Na2CO3trypticase peptoneyeast extractmineral sol. without Shaemin sol.resazurin sol (0.1% wt/volIVFA sol.vitamin mix.sodium ascorbate sol. (1.m wt/volI
Bring volume up to 100 ml with water.
Sulfur source in 100 ml basal media (gI
Sulfur leve1Compound - - - - - - - - - - - - - - - - - -
.lO% .20x .30x
Na2SCM 0.44 0.88 1.32Na2S 0.75 1.50 2.25Na2SC3 0.40 0.80 1.20
