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The Effect of Cellulolytic Rumen Bacteria or TheirCell-Free Extracts on the Germination ofEuonymus americanus L.Gary Thomas Howard

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Recommended CitationHoward, Gary Thomas, "The Effect of Cellulolytic Rumen Bacteria or Their Cell-Free Extracts on the Germination of Euonymusamericanus L." (1987). Masters Theses & Specialist Projects. Paper 1431.http://digitalcommons.wku.edu/theses/1431

THE EFFECT OF CELLULOLYTIC RUMEN BACTERIA OR THEIR CELL-FREE

EXTRACTS ON THE GERMINATION OF EUQnymus americanus L.

A Thesis

Presented to

the Faculty of the Department of Biol ogy

western Kentucky University

Bowling Green, Kentucky

In Partial Fulfillment

of the Requirements for the Degree

Master of Science

by

Gary Thomas Howard

July, 1987

•

AUTHORIZATION FOR USE OF THESIS

PerlTlission is hereby

~ granted to the Western Kentucky Unive rsity Library to lTlake. or allow to be lTla e photocopies, lTlicrofilm Or other copies o f this the s is for appropriate research or scholarly purposes.

O reserved to the author fo'r the making of any copies of this thesis e xcept for brief sections for research or scholarly purposes.

Signed )1alll~ 1. iJtWlQAJ

Date !D JI.S L 87

P l e ase place an "X" in the appropriate; box.

Thi s form w ill be filed with the original of the thesis and will control fu t ure u s e of t h e thesi s.

THE EFFECT OF CELLULOLYTIC RUMEN BACTERIA OR THEIR CELL-FREE

EXTRACTS ON SEED GERM INATION OF ~nymua americanua L.

Dean of the Graduate lege

Recommended ((/~ a\) 1~~7 Date)

;

DEDICATION

Dedicated to my mother, Mildred Colling s Howard, and to

my father, Hurrol Thomas Howard, who took an active interest

in my work.

11

j

ACKNOWLEDGE ME NTS

I would like to thank Dr. Larry P. Elliott, chairman of

my adviso r y committee, for his guidance and encouragement in

directing me through this project. Thanks are also extended

to the other members of my committee , Drs . Martin R. Houston

an d Joe E. Winst ead.

Other individuals who also deserv e special thanks for

their assistance are Dr. Delmer Davis for his help in

collecting t he rumen fluid, Dr. Donald Bailey for his help

i n obtaining deer rumen fluid, and Mr. Rodney McCurry for

his help in the photographic aspects and assistance in the

electron microscopy.

i11

TABLE OF CONTENTS

Pa ge

ACKNOWLEDGMENTS • • • • • • • • • • • • • • • • • • • iii

LIST OF TABLES • • • • • • • • • • • • • • • • • • • • v

LIST OF FIGURES • • • • • • • • • • • • • • • • • • • • vii

ABSTRACT • • • • • • • • • • • • • • • • • • • • • • • viii

INTRODUCTION • • • • • • • • • • • • • • • • • • • • • 1

MATERIAL A~D METHODS • • • • • • • • • • • • • • • • • 6

RESULTS • • • • • • • • • • • • • • • • • • • • • • • • 15

DISCUSSION • • • • • • • • • • • • • • • • • • • • • • 37

SUMMARY • • • • • • • • • • • • • • • • • • • • • • • • 45

LITERATURE CITED • • • • • • • • • • • • • • • • • • • 46

iv

LIST OF TA BL ES

Table Page

1. The Effect of Age on the Viability of Euonymus americanus Se eds Maintained at Room Temperature. 16

2. Germination of Unscarifi e d Euonymus americanus Seeds After Treatment with Clostridium cellobioparum Grown in Thioglycollate Broth . •• 11

3 . Germination of Unscarified Euonymus After Treatment with Clostridium cellobioparum for 24 h . ...•.

americanus

• • • • • • •

4. Germination of Unscarified Euonymus americanus Seeds After Treatment with Clostridium

18

cellobioparum for 48 h. . • • . . • . . . . • •. 19

5.

6.

Germination Seeds After Clostridium Broth . . •

Germination Seeds After Clostridium Broth . • .

of Unscarified Euonymus americanus Treatment with Serial Dilutions of cellobioparum Grown in Thioglycollate • • • • • • • • • • • • • • • • • • •

of Unscarified Euonymus americanus Treatment with Cell Concentration of cellobioparum Grown in Thioglycollate • • • • • • • • • • • • • • • • • • •

1. Germination of Un sca ri f ied ~onymus americanus Seeds After Treatment wil h Ruminococcus

21

22

flavefaciens for 24 h . . . . . . . • • • . . . . 23

8. Germination of Unscarified Euonymus americanus Seeds After Trea tment with Buminococ cus flayefaciens for 48 h . . . • • . • • . • • . .. 24

9. Red ucing Sugars Produced from Euonymu s americanus Seeds in the Presence of Rumen Bacteria. • . . . 25

10. Detec tion of Reducing Sugars Produced by Various Cell uloses Hydrolyzing Avicel and Carboxymethyl Cellulose . . . . . . . . . . . . . . . . . . . . 28

11. Germination of Unscarified Euonymus americanus Seeds in the Presence of Enzyme Extracts Prepared from Rumen Bacteria . . . . . . . . . • . . . . . 30

v

Table

12. Reduc ing Sugars Produced from EUQnymus americanus Seeds in the Presence of Enzyme Extracts Prepared from Rumen Bacteria • • .

13. Perc ent Seed Germinat i on of Euonymus americanus Seeds After Treatment with an

• • •

Endocellulase . . . . . . . . . . , • • • • • • •

vi

Page

31

33

LIST OF FIGURES

Figure Page

1. Congo red screening for endo ellulase activity of Clostridium cellob i ogarum • • . • • . . • . • 27

2.

3.

Reduc i ng sugars liberated from Euonymus americanus seeds by 10 U of a commercial endocellulase . . . . . . . . • . . . .

Reducing sugars l ibe r ated from Euon~mus americanus seeds by 8 U of a commercial endocellulase . . . . . . . . . . . . .

v 1 1

• • • • 35

• • • • 36

THE EFFECT OF CELLULOLYTIC RUMEN BACTERIA OR THEIR CELL-FREE

EXTRACTS ON THE GERMINATION OF Euonymus americanus L.

Gary Thomas Howard July, 1987 50 pages

Directed by: L. P. Elliott, M. R. Houston and J. E. Winstead

Department of Biology W stern Kentucky University

In past at t empts, the e xperimental germination of the

seeds of Euonymus americanus L. in vitro has had little

success. Howe ver, treatmen t of seeds with rumen fluid con­

taining viabl e microflora has been succ essful in stimulating

germination. In the presence of the cellulolytic rumen

bacterium, Clostridium cellobiogarum ATCC 15832, seeds of A.

amecicanus were stimulated to germinate. Subsequent studies

were designed to determine whether the bacterium synthesized

a cellulolytic enzyme responsible for initiating germi­

nat i on. The cell-free endocellulase from ~. cellobiogarum

was demonstrated to irlduce germination of the seeds. To

support the hypothesis that endocellulase from ~.

cellQbiogarum was responsible for triggering germination, a

1,4- e -endo-glucanase (EC 3.2.1.4) from Penicillium

fu nic ulosum was used to treat the s eeds. Also, Ruminococcus

fla vefaciens strain FD-1 was found to initiate germination

of A. americanus seeds. Thus, cellulase activity is

indicated in the degradation of the testa of the seed

allowing imbibition and germination.

viii

1

INTRODUCTION

The seeds of most endemic plants of temperate climates

will not germinate immediately after ripening as these seeds

at maturity will enter a state of dormancy; the two most

common mechanisms of dormancy are impermeability of the seed

coat to water and dormancy of the embryo (Wilson et al.,

1971). The impermea bilit y of the seed coat or testa, a

common cause of delayed germination , can be gradually

decreased by freezing, thawing, and the action of micro­

organisms in the soil (Wilson et al., 1971). These methods,

coupled with wind action and blowing the seeds against

finely abrasive surfaces, tend to scratch the testa and

permit the absorption of water. Scratching of the testa is

commonly referred to as scarific 3tion. An artificial method

of scarifying seed s is to abrade them wi th fine sandpaper or

with a file.

To mediate the physiological condition of the embryo

causing dormancy, seeds must undergo a series of complex

enzymatic and chemical changes collecti ve ly referred to as

after-ripening. After-ripening can occ u r with moisture and

low temperatures by a process known as stratification.

Dormancy can also be caused by various chemicals that may be

present in the seeds . The chemicals that are inhibitors are

commonly l ~ached out of the seeds in wet soil under natural

conditions. In addition, seeds vary grea tly in the length

of time during which they will retai n their viability and

2

ability t o germinate. The l ongevity of most seeds may be

increased by dry storage a t low temperatures (Wilson et al.,

1911). Plants such as Juniperus communis and Juniperus

viriiniana rely on birds to disperse their seeds

(L i vingston, 1912). Holt huijzen and Sharik (1984) showed

that passage of ~. viriiniana seeds through the gastro­

intestinal tract of cedar waxw i ngs and yellow-rumped

warblers i ncreased germination 150 to 350 percent. Seeds of

several different plants that have passed through the

digestive t ract of black bears often show greater germi­

nation r ates (Rogers and Applegate, 1983). Janzen (1970)

reported that Acacia tortilis seeds must pass through the

digestive system of animals, i.e. elephants and i mpalas , in

order for the seeds to germinate. The lack of Calvaria

species younger than 300 years is reportedly due t o the

extinction of the dodo bird sinc e germination of the seeds

depended upon passage through the bird's intestinal tract

(Pig l , 1912).

The results of this thesis report the germination

strategy of Euonymus americanus L. This plant is an abun­

dant understory shrub in remnant ha rdwood forests of south

central Kentucky with a reported distributional frequency

exceeding 98~ (Bougher and Winstead, 1914). This shrub is a

member of the Celastraceae family and contains over 120

other species of this genus worldwide (Strausbaugh and Core,

1913). However, t, americanus is found exclusively in the

United States.

3

Considerable resea r ch on the genus EUQnymus has been

focused on seed germi nation and embryo development in the

European species E. europaeus (Alekseeva, 1975a, 1975b;

Beranger-Novat and Monin, 1971a, 1971b; Beranger-Novat et

al ., 1977 ; Monin, 1965, 1967a, 1967b, 1967c, 1967d, 1968;

Nikolaeva, 1959; Nikolaeva et al., 1973; and Nikolaeva et

al., 1974). More specifically, Nikolaev~ et al. (1973)

found that B-indolyl-acetic acid and abscisic acid behaved

as growth i nh i bitors while g i boerellin and kinetin played an

important ro le in removal of dormancy. The results 0f this

study also revealed that B-indolyl-acetic acid decreased in

concentration under the influence of co l d strat i fication.

Bayer (1984) showed that E. americanus primarily

reproduces asexually. Runners, produced from the base of

the stem, grew horizontal l y along the substrate. These

runners often developed roots and frequently produced a new

stem. Eventually, such runners are severed and separate

plants formed.

Rudolf (1974) using long, cold stratification periods

reported a 15J increase in seed germination. Bayer (1984)

demonstrated that exhaustive tests us i ng HCl/HOH solutions,

heat treatments , long stratification periods, and different

combi nations of photo-periods were unsuccessful. Howard

(1986) showed a 90J germination rate using rumen fluid

containing viable microflora. Howard's study also revealed

a decrea~e in germination time from 20 days, with no

stratlfication, to three days, wi th a cold stratification.

Scanning electron microscopy by Hill et al. (1985) verified

Howard's wo rk concerning the germ in t ion of £. americanus

seeds by the rumen fluid treatment.

4

Herrera ( 1981) sugg ested that the qual i ty of seed

d ispersal bears an important relationship to the nutritional

rewa rd offered by the plant in the form of fruit pulp.

Those pulps associated with h igh s eed d ispersal had a high

lipid andlor protein content. Har greaves (1971) reported

t he presences 0 lipids and sugars in high concentrations in

the arils of Euonymus. The oil s cont ained the usual range

of C16 and C18 fatty acids but were particularly rich in

unsaturated C18 fatty acids. The sugars were a mixture of

glucose, fructose, and rhamnose. Since seeds of Euonymus

are highly nutritional, they should be di s persed easily.

According t o Halls and Alcaniz (1972), the seeds of £.

am e ricanus are an important food source for deer, and

Blakelock (1951) noted bluebirds and robin s foraged on the

fruit. Sinc e deer forage on this pl ant and Howard's (1986)

study showed inc reased germination of the seeds when treated

with rumen fluid, an hypothes i s that r umen microflora play

an i mportant role in inducing ge rmination of t he seeds of £.

americanus is pl aus ibl e. Windham and Ak i n (1984) reported

that rumen bacte r ia were the most act i ve cellulose and fiber

d i gesting populations in the rumen. Clostridium

cellobioparum and Ruminococcus flaveTaciens are two common

cellulolytic bacteria found in the microbial niche of the

rumen. Thus, the objectiv e of thi s thesis was to elucidate

whether the rumen bacterium k. cellobioparum ATCC 15832

acting alone with the seeds will induce germination and, if

5

so, by what mechanism. Also, B. flayefaciens strain FD-l is

being assayed for initiating germi nation of ~. americanus.

6

MATERIAL AND METHODS

Seed Collection

Fruiting capsules of Euonymu5 americanu5 were collected

i n early October of 1983, 1984, 1985, and 1986 from two

sites in south central Kentucky and maintained for testing.

The first collection site was l ocated east of Interstate 65

on Route 68/80 i n Barren County near the village of Bonayer.

The second site was east of Interstate 65 on Route 31E in

Barren County nea r Barren River Lake at the Brigadoon Nature

Preserve of Western Kentucky University.

Seed Pregaration

Seeds were allowed to dry at room temperature before

removal of the arils. Before treatment, seeds were surfaced

sterilized as described by Kremer et al. (1984), whi oh

involved immersing seeds in 3.0S (w/v) sodium hypochlorite

for five minutes followed by immersing in 70S (w/v) ethanol

for two minutes and then rinsing with distilled deionized

water (DI H20). Subsequently, seeds were then subjected to

various treatments to induce germination. After treatment,

the seed~ wer e rinsed with DI H20 and placed on a single,

sterile nine centimeter circular piece of Whatman #1 filter

paper in petri dishes.

~rtitions for Germination

An Environator Corporation Model 3448 growth chamber

was used to provide the conditions for seed germination.

The chamber was programmed for a cont1nuously cycling 12-h

day temp e rature of 29.4oC and a 12-h night temperatu re of

18 . 30 e wit h cyc1 1ng fluorescent and incandescent lighting.

All seeds were kept moist with DI H20 throughout the

experiments.

Seed Viability Test1n&

7

The two chemicals used in the seed viability testing

were a tetrazo11um sal t and indigo carmine ( Bonner, 1974).

The tetrazolium salt used in this assay was 2,3,5-triphenyl­

tetrazolium chloride (Sigma, St . Louis, MO) which may be

used as a vital stain. Indigo carmine (Sigma, St. Louis,

MO) is a non-vital stain. One-hundred and forty seeds from

1983, 1984, 1985, and 1986 were scarified with a dissecting

probe to facilitate entry of the solutions.

One-ha lf of the seeds (70 of 140) were immersed in a 1S

(w/v) aqueous solution of tetrazolium chloride and were eft

in the dark at 300 c for 48 h. The other 70 sceds were

imme rsed in a 1S (w/v) aqueou solution of indigo carmine

and left in the dark at 30 0 C for 48 h. The seeds were then

removed after 48 h and cut in half to observe staining with

a stero binocular dissecting microscope.

Mi croor&anisms and Growth

The cellulolytic rumen bacterium Clostridium

cellobioparum ATCC 15832 was obtained from the American Type

Culture Collection, Rockville, MD. Ruminococcus

flayefaciens FD-1 was a gift from Dr. B. A. White,

University of Illinois, Urbana, IL. The k. cellobiopa~

was grown r outinely at 37°c in Brew er 's Thioglycollate Broth

(Oifco) Ot- Medium 10 (Caldwel l an d Bryant, 1966)

anae r obically in Gas Pak jars (BBL Microbiology Systems,

Cockeysvill e , MO) for 48 h. For seed germination studies,

an inoculum was prepared by pipetting 1.0 mL of culture into

either 250 mL of Brewer's Thioglycollate Broth or Medium 10.

The inoculated broths were incubated anaerobically in Gas

Pak jars at either 39 0 C or 40 0 C for 24 h or 48 h. R.

flayefaciens was grown routinely at 39°C in Gas Pak jars for

24 h or 48 h. In seed germination studies, an inoculum was

prepared by pi petting 1.0 mL of culture into 250 mL of

Medi um 10 (M 10) and incubating anaerobically in Gas Pak

jars for 24 h or 48 h.

Seed Iooculation with Bacterial Cells

One-half of the seeds (1985) in each assay (80 of 160)

were scarif ied by the use of a dissec t ing probe to scratch

the seed coat, and ha lf were unscarified. Each subset of 20

seeds was covered with 20 mL of sterile thioglycollate broth

(control) or one of the following 48-h thioglycollate broth

cultures of k. cellobioparum: (1) incubated anaerobically,

(2) cell-free extra ct cent rifuged at 10 ,000 x g for 5 min

and filtered through a 0.45 ~m membrane, or (3) autoclaved

at 121°C for 15 min. The same assays were repeated as

described above except that the incubation period for growth

of k. cellobioparum was 48 h at 40°C. The same assays were

repeated except that the i ncubation temperature was 39°C for

24 hand 48 h. All experiments were re peated using

~. cellob ~oparum cultures grown in M 10 i nstead of

thioglycol l ate broth.

Seeds of ~. americanus were treated with A.

flavefacien s by those assays described for ~. cellobioparum;

however, only M 10 broth was used for growth , and an

incubation temperature of 39 0 C was used for 24 hand

repeated at 48 h. At the end of the incubation periods, the

seeds were r i nsed with 01 H20 and placed in the

environmental chamber programmed for the proper germination

conditions.

In additi on, ~. americanus seeds were treated with

decimal dilutions of ~. cellobioparum. The dilutions were

made in 90-mL blanks containing 0.1J (w/v) peptone wate in

an anaerobic glove chamber Model AL 12441. One-tenth of a

milliliter of each dilution was then transferred onto

triplicate petri dishes which contained 4J thioglycollate

agar. Glas 3 L-rods were used t o spread the inoculum on the

plates. Plates were incubated anaerobically at 39 0 C for

48 h. After the incubation period, the seeds were

elutriated with 01 H20 and placed in an environmental

chamber programmed as previously descri bed, and the seeds

were check ed for germination daily.

A con~entrated suspension of ~. celloPioparum was also

used to assay for induction of germination in ~. americanus

seeds. An inoculum of 1.0 mL of a 24-h culture of~.

cellobioparum was used to inoculate 250 mL of Brewer's

10

Thioglycollate Broth, and the inocul a ted broth wa s

anaerobic~lly incubated for 48 h at 39 0 C. Afte r the

incubation period, the broth was centrifuged at 10,000 x g

for 5 min. The supernatant was removed, a nd 40 mL of saline

solution (0.9%) were added to the pellet. The cell

suspensi on was pipetted into two petri dishes, 20 mL/plate.

To these petri dishes, 20 seeds of t. americanus were added

per dish. One petri dish was incubated anaerobically at

39 0 C for 24 h, and the other was i ncubated anaerobically at

39 0 C for 48 h. Then the seeds were elutriated with Dr H20

and placed in an environmental chamber programmed as noted

bef ore and checked daily fo r germination.

ScreeninB Cultures for Cellulase Actiyity

~. ~ ellobioparum was assayed for the production of

cellulase by two met hods . The fjrst method consisted of

inoculating 0.1 mL of a 48-h culture from thioglycollate

broth into 10 mL of Omelianski Both which contained a strip

of Whatman #1 f il ter paper as t he sole source of carbon

(Omelianski , 1902). Three cultures at each temperatu re were

incubated anaerobically at 39 0 C and 40 0 C for 21 days.

The second method used was Teather a ll d Wood's (1982)

technique of congo red screening. A 48-h culture of ~.

cellobioparum grown in Brewer's Thioglyc0!la t e Broth was

used to spot inoculate 0.05 mL of inoculum on 45 plates of

Medium 10 Agar. Three sets of tripl i cate ~lates (15

plates/set) were placed at 37 0 C, 380 e, 3S J C, 400 C and 41 0 C

and were incubated anaerobically for 48 P. After 48 h, the

11

plates were overlayed with M 10 which lacked the normal

carbohydrates but contained 0.8~ agar a nd O . l~ carboxymethyl

cellulose (CMC). One set of plates was reincubated for 24 h

at the same tempera tu re while another set was reincubated

for 48 h and the third set reincubated for 72 h. After

these time periods, the plates were flooded with an aqueous

solution of congo red (1 mg/mL) for 15 min. The congo red

was decanted, and the plates we re flooded with 1M NaCl for

15 min . This was fo llowed by decanting the NaCl solution

and flooding the plates with 1M HC1. The clear zones around

the colon i e s were then measured in millimeters.

Isolation of Cell-Free Cellulase

A 48-h culture of ~. cellobiogarum grown in 250 mL of

Brewer' s Th i oglycollate Broth and Medium 10 Broth and a 48-h

culture of R. flavefaciens grown in Medium 10 Broth were

used to prepare cell-fre~ extracts. Cells were sedimented

by cen~rifugation a t 10,000 x g for 5 min . The supernatant

was decanted and proteins concentrated by precipitation with

ammonium sulfate at an 80~ (wt/vol) concentration. The

preCipitate was removed by centrifugation at 22,000 x g for

20 min at 40 C. The protein was resuspended by addition of

5 mL of 0.05 M sodium acetate buf fe r, pH 5.2. The ammonium

sulfate was remov ed from the resulting enzyme extract by

dialysis in 1.0 1 of 0.05 M sodium acetate buffer, pH 5.2,

which was changed twice in a 24-h period. The crude enzyme

extract was stored at _200 C until needed.

2

Enzyme Assays

(a) Endog l ucanase activity

The reaction mixture containing 1% CHC (w/v ) (Sigma, St.

Lou i s, HO) in 4.0 mL of 0 . 05 H sodium acetate buffer, pH

5.2, and 1. 0 mL of enzyme extract was s haken in a New

Brunswick Scientific Incubator shaker at 90 strokes per min

at 37°C for 2 h (Hayashida and Ho, 1986). Reducing sugars

formed were determined by the method of Nelson (1944).

(b) Exocellulase activity

Assays were conducted using a 1% (w/v) Avicel (Baker,

Phillipsburg, NJ) in 4.0 mL of 0.05 H sodium acetate buffer,

pH 5.2, and 1.0 mL of enzyme extract. The reaction mixture

was shaken in a New Br unswick Scientific Incubator shaker at

90 strokes per min at 37 0 C for 2 h. Reducing sugars f ormed

were determined by the method of Nelson (1944).

(c) Cel l obiase act i vity

Assays were conducted using a 1% (w/v) D (+) cellobiose

(Sigma, St. Louis, HO) in 4.0 mL of 0.05 H sodium acetate

buffer pH 5 .2 and 1.0 mL enzym~ extract. The reaction

mix ture was shaken in a New Brunswick Scientific Incubator

shaker at 90 strokes per min at 37°C for 2 h. Reducing

sugars formed were determined by the method of Nelson

(1944) .

Protein Determination

Protein was est i mated by the Bio-Rad protein assay with

bovine serum albumin as a standard (Bradford, 1976).

13

Enzyme Inhibition

Ass a ys were conducted using a 11 CMC (w/v) in 4.0 mL of

0.05 M ~od i um acetate buffer, pH 5.2, and 1.0 mL enzyme

e xtract of ~. cellobiopa rum were incubated a t 37oC, 45 0 C,

and 600 C for 2 h. In addition, an enzyme reaction mixture

was inactivated with 1.0 mL of 0.001 M HgCl 2 and incubated

at 370 C for 2 h. This assay was repeated except that 1.0 mL

of 0.01 M HgC1 2 was used. Reduci ng s ugars were measured by

the method of Nelson (1944).

Seed Assay with the Enzyme Extracts

Enzyme extract s from ~. cellobioparum and B.

flavefaciecs obtained as described earlier were used in thi s

assay. One milliliter of cell-free extract prepared from

each bacte rial species was used to treat ten seeds of ~.

americanus for 24-h and 48-h incubation periods with shaking

at 37oC. After each time period, the amount of reducing

sugar libera ted was measured by t he method of Ne l son (1944).

The seeds were removed, e l utriated with OI H20, placed in

the environmental chamber programmed as previously described

and observed daily for germination.

Seed Assa y with an Endoglucanase

A 1,4- 8-0 glucan glucanohydrol ase (EC 3.2.1.4) from

Penicillium funiculosum was obtained from Si gma Chemical

Co., St. LOUis, MO. This endocellulase was a purified

enzyme extract with a specif i c activity of 11 U/mg of

protein. In this assay, quantities of 10 U and 8 U of the

14

endocellulase was buffered with 2 mL, 1 mL, and 0.5 mL of a

0.05H s odium acet ate buffer, pH 5.2.

The buffe. ed endocellulase was ster ilized by filtrat i on

using a Hillex HA SLHA0250S ul t ra c lean 1ng filter wh i ch was

attached to a s t erile syringe. The filtrate was collected

in sterile test tubes. To seven test tubes (10 x 100 mm),

ten seeds were added and i ncubated with shaking at 370 C for

2 h, 4 h, 6 h, 8 h, 12 h, and 48 h. Af t er e ach time

i nterval, the amount of glucose liberat ed was measured by

the method of Nelson (1944), the seeds removed, elutriated

with 01 H20, placed in the programmed environmental chamber,

and checked dai l y for germination.

See d Assay with an ExoKlucanase

A 1,4-8-0 glucoside glucohydrolase (EC 3.2.1.91) was

obtai ned from Sigma Chemical Co., St. Louis, HO. This

exocellulnse was purified from almonds and had a unit

act i vity of 5 U/mg. In th i s assay, quantities of 5 U and

8 U of the exocellulase was buffered with 1 mL and 0.5 mL of

a 0.05 M sodium acetate buffe r , pH 5.2. The buffered

exoce l lulase was sterilized by filtering through a Hillex HA

SLHA0250S ultracleaning filter into sterile test tubes. To

each test tube, ten seeds were placed and incubated with

shaking at 37°C for 6 h, 8 h, and 12 h. After these time

periods, the amount of glucose liberated was measured by the

method of Nelson ( 1944), the seeds removed, elutriated with

01 H20, placed in a programmed e nvironmental chamber, and

observed daily for germination.

15

RESULTS

Seed Viability Assay

Seeds of Euonymus amerioanus tested with indigo

carmine and tetrazolium showed an average of 20.7S decrease

i n viability in a three-year period when stored at room

temperatu re (Table 1). The method us i ng tetrazolium

reflected a 2.2S higher viability of the seeds per year than

the ind igo carmine method. Seeds three years old had an

average of 79.3S viab i lity as determined by both of the

assays.

Seed Germination Assay with Baoterial Cells

Seeds treated with ~. oellobioparum germinated only i n

the presence of viable cells. When the bacterium was grown

i n Brewer's Thioglycollate Broth, germination was induced at

a 65S rate within 9 days after 48 h of i ncubation with ~.

oellobioparum (Tab le 2). Seeds did not germinate when

treated with Brewer's Thioglycollate Broth. Also, seeds did

not germinate whe n subjected to ~. oellobioparum f or only

24 h nor d id germination occur when they were scarified.

When ~. oellobioparum was grown in M 10 broth, seeds were

induced to germinate at a 20~ rate within 9 days after a

24-h incubat i on period with the bacteria (Table 3). An

i ncrease of germination to 70S was achieved when the seeds

were treated with ~. oellobioparum for 48 h (Table 4).

1

16

Table 1 . The Effect of Age on the Viability of Euonymus

americanus Seeds Maintained at Room Temperature

ASSAY

Indigo Carmine Tetrazoli um

Year of Collection Vi a ble ( J) Nonviable ( J) Vi3ble (S) Nonviabl e( S)

1986 98.0 2.0 100 0

1985 88.89 11. 11 91. 43 8.57

1984 84.29 15.71 88 .57 11.43

1983 78.34 21.66 80 .19 19.81

170 seeds per year were assayed in each method .

• Ii

Table 2. Germination of Unscarified Euonymys ~ricanys

Seeds 1 after Treatment 2 with Clostridiym

cellobioparym Grown in Thioglycollate Broth

17

Trea tment Germination Time (Days)

~ of Germination

Anaerobically Grown Culture 6-9 65

Ce ll-Free Extract

Autoclaved Culture

'20 seeds per assay.

o

o

2Seeds were held anaerobica l ly for 48 h at 40 0 C i n a Brewer's Thioglycollate Broth culture preparation of Clos t ridiym cellobiQparym. The culture preparations were treated as indicated.

o

o

Table 3. Germination 0 ' Unscarified Euonymus americanus

Seeds 1 after Treatment2 with Clostridium

cellobioparum for 24 h

18

Treatment Germination Time (Day s)

J of Germination

Anaerobically Grown Culture

Cell-Free Extract

Autoclaved Culture

6-9

o

o

20

o

o

120 seeds per assay.

2Seeds were held Medium 10 broth cellobillparum.

anaerobically for 24 h at 39 0 C in a culture preparation of Clostridium The culture preparations were treated

as indicated.

Table 4. Germination of Unscarified Euooymus americanus

Seeds 1 after Treatment 2 with Clostridium

cellobioparum for 48 h

19

Treatment Germination Time (Days)

J of Germination

Anaerobically Grown Culture

Cell-Free Extrac t

Autoclaved Culture

120 seeds per assay.

6-9 70

o o

o o

2Seeds were held anaerobically for 48 h at 390 C in a Medium 10 broth culture preparation of Clostridium ~_llobioparum. The culture preparations were treated as indicated.

20

Seed3 did not erminate when they were only treated with

M 10 broth.

To determine if concentrated cells were needed for

germ i nation, seeds were treate with serial dilutions of ,.

cellobioparum. Germination was induced with 10- 1 to 10-3

dilutions of the culture. In addition, lOS germination of

the seeds occurred within 10 days with the 10-8 dilution

(Table 5) . When cells of ,. cellobioparum were

concentrated, increased ge r mination of E. americanus seeds

occurred. A 24-h treatment of seeds with concentrated cells

resulted in 75S germination in 9 days; however, a 48-h

treatment resulted in an 85S germination rate in 7 days

(Table 6).

The question arises whether other ruminant bacteria

would stimulate germination. Seeds treated with E .

flavefaciens germinated only in the presence of viable

cells. When li. flavefaci€ns was grown in M 10 broth and

used to treat seeds for 24 h, 70S of the seeds germinated

within 9 days (Table 7). An increase of 80% germination was

achieved when the seeds were treated with E. flayefaciens

for 48 h (Table 8). Again, seeds treated with M 10 did not

germinate.

RedUCing sugars liberated f r offi E . americanus seeds by

the rumen bacteri a were detected by the method of Nelson

(Table 9). There was an increase in the glucose

concentration released from the seeds as time of treatment

was increased f rom 24 h to 48 h indicating the hydrolysis of

Table 5. Germination of Unscarified Euonymus americanus

Seeds' after Treatment2 with Serial Dilutions

Dilut ion

of Cl ostridium cellobiQparum grown in

Thioglycollate Broth3

Germination S of

21

of Culture Time (Days) Germination

100 7-9 70

10-1 6-9 30

'0-2 6-9 40

10-3 6-9 '0

'0-4 0 0

'0-5 0 0

10-6 0 0

10-7 0 0

'0-8 10 10

'20 seeds pel" assay.

2Seeds were held anaerobically for 48 h at 390 C in Brewer' s Thioglycollate Broth prepa r at i on of Clostridium cellobioparum. The culture was d i l u~ ed as indicated.

3An average count of 49 x 108CFU/mL was obtained on three plates of thioglycollate agar.

i

22

Table 6. Germination of Unscarified Euonymus americanus

Seeds' after Treatment 2 with Cell Concentration

of Clostridium cellobioparum grown in

Thioglycollate Broth

Incubation Time (h)

24

48

'20 seeds per assay.

Germina t ion Time (Days)

6-9

5-7

~ of Germination

75

85

2Seeds were held anaerobically at 39 0 C in a concentrated cell suspension of Clostridium cellobioparum. The culture preparations were incubated as indicated.

Table 7. Germ i nation 0 Unscar i fied EUQnymus americanus

Seeds' after Treatment 2 with Ruminococcus

f layefaciens for 24 h

23

Treatment Germination Time (Days)

S of Germination

Anaerobically Grown Culture

Cell-F r ee Extra ct

Autoclaved Culture

'20 seeds per assay.

6-9

o

o

2Seeds were held anaerobically for 24 h at 390 C in a Medium '0 broth culture preparation of Ruminococcus flayefaciens. The culture preparations were treated as indi cated.

70

o

o

Table 8. Germination of Unscarified Euonymus americanus

Seeds' after Treatment 2 with Ruminococcus

flavefaciens for 48 h

24

Treatment Germination Time (Days)

S of Germination

Anaerobically Grown Culture

Cell-Free Extract

Autoclaved Culture

'20 seed s per assay.

6-9

o

o

2Seeds were held anaerobically for 48 h at 39 0 C in a Medium '0 broth culture preparation of Ruminococcus flavefaciens. The culture preparations were treated as indicated.

80

o

o

25

Table 9. Reducing Sugars Produced from Euonymus americanus

Seeds 1 in the Presence of Rumen Bacteria

Rumen Bact eri a

Clostridium cellobioparum

Clostridium cellobioparum

Clostridium c ellob ioparum

Clostridium cellobioparum

Ruminncoccus flayefaciens

Ruminococcus flayefaciens

Exposure Time (h)

24

48

24

48

24

48

120 seeds per assay

Culture Broth

Thioglycollate

Thioglycollate

Medium 10

Medium 10

Medium 10

Medium 10

Glucose2 Liberated

8.4

11. 1

14 .3

14.3

17 • 1

2Glucose concentration measured in vg/mL by the method of Nelson.

cel l ulose by cel lulase. In addition, more glucose was

liberated from the seed s that were subjected to

26

~. cellobioparum grown in M 10 ~roth as opposed to growth in

Brewer's Thiogl ycollate Broth.

Screenini Cultures for Cellulase Activity

Initially the ~. cellobioparum bacterium was screened

for cellulase activ i ty in Omelianski Broth. The organism

was found t o produce cellulase both at 39 0 C and 40 0 C as

indicated by the paper strips being decomposed.

Subsequently, the congo red method was used to detect

endocellulase activity. The halo around colonies of ~.

cellobioparum by the congo red staining procedure indicated

that the bacterium was positive for endocellulase synthesis.

The zone diameter of the cellulase enzyme increased with

time; however, ther e was a slight decrease i n zone diameter

when the temperature was increased or decreased f rom 39 0 C

(Figure 1).

Comparison of Enzyme Activi t ies

To prove that cellulase synthesis by the rumen bacteria

i s involved in triggering seed germinat i on, enzyme extracts

from the bacteria must be assayed. The extent of hydrolysis

of cellulose from concentrated enzyme extracts of ~.

cellobioparym and R. flavefaciens was measured by the amount

of reducing sugar released from CMC and Avicel (Table 10).

Commercial purified cellulase enzymes were also tested. The

commercial exocellulase showed activity on Avicel; but no

activity was obtained on the CHC. The commercial

Figure 1. Congo Red Screening for Endocellulase Activity

of Clostridium cellobioparum

27

17 16 15

72 • ~ 14 E 13 E ~

12 .... 41

11 - -i 41 - . E 10 48 3 c

tD .-• 0 9

~

::r 41 , c: 8 ~

0 N 7 "0 6 41 .... c 5 • 41 • • ----- 24 -u •

4 3

2 1

37 38 39 40 41 Temperature (OC)

28

Table 10. Detection of Reduc i ng Sugars Produced by Various

Cellulases Hydrolyzing Avicel and Carboxymethyl

Cellulose

Reduced Sugars Produced (\lg/mL)

Enzyme Avicel Carboxymethyl

cellulose

Exocellulase 1

EC 3.2.1.91

Endocellulase2

EC 3.2. 1 .4

Extract from 3 Ruminococcus flavefaciens

Extract from 3 Clostridium cellobiQparum

3.0

0.8

9.5

4.2

1preparation from Almonds.

2preparation from Penicillium funiculosum.

15

20

15.5

3Ex~ract from cells grown anaerobically 48 h in H 10 at 39 C prepared by centrifugation, membrane filtration and ammonium sulfate precipitation.

29

endocellulase showed more activity on CHC than on the

Av icel. The enzyme extract fro m l . flav efaci ens had

activity on both Av i cel and CHC; however, the activity on

the Avicel was one-half that of the activity on the CHC.

The enzyme extract from C. cellobigparum showed activity on

CMC and slight activity on Avicel (Table 10). The enzyme

extracts, prepared from cultures of C. cellobioparum and l.

flavefaciens grown in H 10 broth, revealed no cellobiase

activ i ty.

Seed Germination Assay with Enzyme Extracts

Seeds treated 24 h with an enzyme extracts from l.

flayefaciens enhanced germination compared to seeds treated

for 48 h. Seeds treated with an enzyme extract from C.

cellobigparum germinated optimumly when the cells were grown

in H 10 broth and when treated for 48 h as indicated in

Table 11.

The reducing sugars liberated from the seeds by the

enzyme extracts were quantitated as indicated in Table 12.

An increase in reducing sugars was obtained by an increase

in contact time between seeds and enzyme extract. Also,

more reducing sugars were liberated from the act i vity of the

enzyme extract from C. cellobioparum grown in H 10 broth

than from the enzyme extract prepared from cells grown in

th10gl ycollate broth. The enzyme extract prepared from l.

flayefaciens liberated more reducing sugars from the seeds

than the enzyme extract prepared from C. cellobioparum.

Table ". Germination of Unscarified Euonymus americanus

Seeds' in the Presence of Enzyme Extracts

Prepared from Rumen Bacteria

Species

Clostridium cellobioparum

Clostridium c ellobioparum

Rumi nococcus flavefaciens

Ruminococcus flavefaciens

Time Held in Extract (h)

24

48

48

Culture Broth

Thioglycollate M '0

S of Germination

40 70

60 80

-- 80

20

' Qc curring within 4-8 days in an environmental chamber.

2'0 seeds per assay.

30

31

Table 12. Reducing Sugars Produced from Euonymus americanus

Seeds 1 in the Presence of Enzyme Extracts

Prepared from Rumen Bacter ia

Clostridium cellobioparum

Clostrid i um cellobioparum

Ruminococcus flayefaciens

Ruminococcus flavefaciens

Medium Cells Grown In

Thiogl ycollate Medium 10

Th ioglycollate Medium 10

Medium 10

Medium 10

1'0 seeds per assay.

Exposure Time (It)

24 24

48 48

24

48

Glucose2

Liberated

2.8 9.4

10.2 11.1

12.6

15.3

2Glucose concentration measured in ~g/mL by the method of Nelson.

32

Protein Determination from Enzyme Ex tracts

When ~. cellQbiogarum was grown 24 h an a erob ically in

250 mL of M 10 broth, the cell-free extract was fo und to

contain a concentration of 122 ~g of protein per milliliter.

However, if the incubation period was increased to 48 h,

the pro tein concentration of a cell-free extract from ~.

flavefaciens grown i n M 10 broth for 48 h was 380 ~g/mL.

Enzyme Inhibition

The amount of red ucing sug~r liberated from CMC by the

cellulase activity of the enzyme extract was decreased as

the incubation temperature was increased. The amount of

reducing sugar released at the 45 0 C incubation temperature

was one-half of that released at the 37°C incubation

temperature and the amount of reducing sugar released a t

600 C was one-tenth the amount released at 37°C. In

addition, 0 . 001 M Hg C1 2 and 0.01 M HgC1 2 inhibited all

oe l l ulase activity of the enzyme extract. No reducing

sugars were l i be r nted from the CMC when these concentrations

of HgC1 2 were present.

Seed Germination Assay with Commercial Endocellulase

The total germination percent of seeds exposed to a

purified endocellu l ase from Penicillium f uoiculosum depended

on time of exposure to the enzyme and concentration of the

enzyme (Table 13). Generally, there was an increase in

pe rcent seed germination with more contact time between

seeds and enzyme and when a more concentrated enzyme

suspension was used. However, the percent seed germination

33

Tab l ~ 13 . Percent Seed Germination 1 of Euonymus americanus

Seeds after Treatment with an Endocellulase2

Exposure Time (h)

2

4

6

8

10

12

24

48

10ccurring

2 mL

0

0

0

30

30

30

30

40

within 4-6

2Purified enzyme from

Units of Enzym e

10 U 8U

1 mL 0.5 mL 2 mL 1 mL 0.5 mL

0 0 0 0 0

40 60 0 0 20

60 70 0 40 60

80 90 0 60 80

70 50 0 60 10

60 0 0 50 0

60 0 20 50 0

20 0 20 30 0

days in an environmental chamber.

Penicillium funiculosum.

34

did not increase after 8 h of exposure nd decreas ed

the r eafter with all concentrations of t he enzyme . The more

concentrated enzyme preparation ca sed the greate s t percent

seed germination in the shortest amount of exposure

time, while the les s concentrated enzyme preparation

stimulated less seed germination.

Reducing sugars liberated from the seeds during various

ex posure times with commercial enzym s are indicated in

Figures 2 and 3. There was an increase in the amount of

reducing sugar liberated from the seeds by the different

concentrations up to 8 h of exposure. After 8 h exposure

the amount of reducing sugars leveled off and remained at

the same level throughout the assay.

Seed Germination Assay with Commercial Exocellulase

No germination was obtained from seeds treated with the

commercial exocellulase enzyme. In addition, no redu ing

s ugars were liberated from the seeds treated with the

commerc i al exocellulase enzyme preparation.

Figure 2. Reducing Sugars Liberated from Euonymus

americanus Seeds by 10 U of a Commercial

Endocellulase

35

16 It = 10u/ 2ml • 15 0-10u/l ml

14 , · 10u/0.5ml

13

12

1 1

10

9 ~

E 8 • "-co 7 ::a. ~

It

'Ii' 6

"' 0 5 <) ::s

,I:>. 4

3

2

1 ; , I ;; y , I

2 3 4 5 6 7 8 9 10 11 12 24 48

Hours of Exposure

Fi gure 3. Reducing Sugars Liberated from Euonymus

americanus Seeds by 8 U of a Commercial

Endocel lulase

36

13 x = Bu/2ml

• 12 o=Bu/lml

11 • = Bu/0.5ml

10

~ 9

E B "-CJI ::1 7 -'V

6 III 0 U X ::l 5 X -,l!),

4 x

3

2 x

1

I , I I , , y, 2 3 4 5 6 7 B 9 10 11 12 24 4B

Hours of Exposure

37

DISCUSSION

Seed Viabi l ity Testini

The seed viability of ~. americanus tested by the

indigo c a rmi ne and tetrazolium methods indicated

approximately the same results and provided confidence that

the seeds used were consistent for testing germination.

Seeds from 986 had a 99J viability wh i le three year old

seeds had an 80J viability. The percent of the seeds that

were viable after three years shows an ample longevity of

the seeds which ensures a greater chance of survival of the

plant.

Seed Germination with Bacterial Cells

Howard (1986) indicated that germination of seeds of ~.

americanus is improved by passage through the rumen of

animals. Once seeds have been ingested by a r uminant

animal, the microflora component of the rumen fluid is the

key facto r i n breaking down th c dormancy and stimulating

germination. The microbial population of the rumen is

composed of a complex mixture of bacteria, protozoa, molds

and yea s ts. Each of these groups comprises a collection of

species whose carbohydrate substrate utilization spectra

overlap ( Leedle et al., 1982). The digestive secretions of

animals are unable to hydrolyze major plant cell wall

components i.e., cellulose and hemicellulose (Russell,

1985), but rumen bacteria are the mo s t active cellulose and

38

fiber digesting populations in the r umen (Windham and Akin,

1984). Thus , the rumen bacteria could degrade the testa of

s eed s al lowing imbibition to occur leading to germination.

The r esults of this study indicate that rumen bacteria

acting alone could induce germination of seeds of S.

americanua by degradation of the seed coat.

Unacarified seeds treated anaerobically for 48 h with

~ . cellobioparum grown in thioglycollate broth resulted in

ge r mi nation while treatment for 24 h i nduced no germination.

Also, seeds did not germinate if scarified and treated with

the bacterium. In addition , unscarified seeds treated

anaerobically for 24 h with ~. cellobioparum grown in M 10

broth resulted in a low percent germination while a 48 h

contact time enhanced the ge r mination of the seeds.

Apparently, a 24-h contact time with the seeds i s not

sufficient time for the bacterium to effectively degrade the

seed coat and a l low germination to occur. A 48-h contact

time allowed the bacterium to sufficiently degrade the testa

resulting in seed germination; however, scarification of the

seeds inhibited germination by allowing the bacteria a

direct pathway to the inside of the seed which could result

in degradation of the embryo.

Seeds treated with ~. cellobiQP·a rUJII grown in M 10 broth

had a higher germination rate than those treated with ~ .

cellobioparum grown in thioglycollate broth. The M 10

broth, which contains cellobiose, enhanced cellulase

production whereas thioglycollate broth did not stimulate

cellulase production to the same extent. Thus, bacterial

cells grown in H 10 would have a higher concentration of

cellulase resulting in Quicker degradation of the seed's

testa leading to more germination.

The congo red screening of ~. cellobioparum for

39

endocellulase i ndicated maximum synthesi~ of cellulase at

39 0 C with the production decreasing as the temperature

inc r eased or decreased. This somewha t correlates with the

fact that ~. cellobioparum does not grow at either 25 0 C or

45 0 C and its optimum temperature is 39 0 C (Gibson and Gordon,

1911). It is i nteresting that maximum endocellulase

synthesis occurred at the temperature of the rumen (39 0 C)

(Hungate, 1966).

A decrease in the number of bacterial cells should

result in a decrease in the amount of cellulase which woul d

lead to a decrease in the amount of cellulase which would

lead to a decrease in seed germination. This hypothesis was

tested when seeds were treated with serial logarithmic

d i lutions of ~. cellobioparu~ . The results obtained from

-8 the study supported the hypothesis; however, the 10

dilution resulted in one seed germinating. The reason why

the seed germinated is unknown. The results demonst rated

that an inc r ease in cell concentration of the bacter i um

results in an increase i percent seed germina tion.

A parallel study was done using ~. flavefaciens grown

in H 10 broth to treat seeds for 24 hand 48 h. This •

bacterium also triggered ger minat on of £. amerlcanus seeds

40

as did the treatment of seeds with k. c ellobioparum.

Although a 24 h contact time was adequa te for seed

germination , a 48 h contact time im roved the germination

rate. The bacterium H. flavefaciens is one of the most

important cellulose decomposers found in the rumen (Fox et

RI., 1980). Since H. flayefaciens is a more efficient

cellulase producer than k. cellobioparum, using H.

flayefaciens to treat ~. americanus seeds should decrease

the a mount of contact time needed for triggering germination

of the seeds. The results of this study support this

conclusion.

One of th e most common ways to measure cellulase

activity is to measure the amount of reducing sugar or

glucose liberated from the cellulose polymer. The method of

Nelson (1944) is a colorimetric method using a copper

reag e nt to measure the concentration of glucose liberated

spectophotometrically. With this method, the amount of

gl ucose released f r om the seed could be c ompared to the

percent germ i nation. The amount of reducing sugar produced

from the seeds in the presence of rumen bacteria increased

as exposure time increased. Seeds treated with k.

cellobioparum, grown in thioglycollate broth, liberated less

reduc i ng sugars t h an se ed s treated wi th k . cellobioparum

grown in H 10 broth. The results indicated that H 10 broth

stimulates the bacterium to synthesize more cellulase than

when grown in thioglycollate broth. This increase in

cellulase synthesis results in more degradation of the testa

41

leading to higher germination. Also, seeds treated with B.

flayefaciens libe~~ted more reducing sugar s than those seeds

treated with ~. cellobiogarum which is co related with the

fact that B. flayefaciens produces higher concentrations of

cellul se than does ~. cellobiogarum. One of the most

obvious observations from this experiment is the fact that

an increase of exposure time between the bacteria and the

seeds inc reased the amo unt of reducing sugar liberated.

This increase in exposure time allows the bacteria tv

produce more cellulase which can degrade the cellulose

polymers and libera t e more glucose .

Seed Germination with Enzyme Extracts

The cellulase system of B. flavefaciens has been

studied extensively (Halliwell and Bryant, 1963; Bryant,

1973; and Gardner, 1987 ) . These investigators have shown

that B. flayefaciens produces both an endocellulase and an

exocellulase. Other studies have shown that the

cellulolytic activity of B. flave f aciens is constitutively

expressed, and the degradation of both cel l ulose and

cellobiose occurs simultaneously (Hiltner and Dehority, 1983

and Pettiphen and Latham, 1979). As for ~. cellobiogarum,

characterization of cellulase it synthesi zes has not been

accomplished. ~. cellobiogarum is an isolate from the rumen

(Hungate, 1966) and has cellulolytic activity (Hungate,

1950). The result of the present study showed that ~.

cellobiogarum can degrade CHC, filter paper and to some

extent Avicel. These results imply that ~. cellobiogarum

definitely has an endocellulase activ i ty and to a lesser

degree an exocellulase activity.

42

Endocel lulase is produced by all cellulolytic micro­

organisms and is also referred to a s endo- B-1,4-g1ucanase,

B -1,4-g1ucan glucanohydrolase, CMCase or Cx cellulase.

Acid-swollen cellulo s e and carboxymethyl cellulose (CMC) are

the preferred substrate of endocellulase and does not

degrade crystalline cellulose with any efficiency.

Endoc ellulases degrade cellulose polymers by random internal

cuts and produce cellod extrins, cellobiose and glucose.

Exocellulase has been described in mycelial fungi;

however, the presence of the enzyme in bacterial species is

somewhat controversial. The enzyme is also referred to as

exo- B-1,4-g1ucanase, B-1,4-g1ucan cellobiohydrolase,

Avicelase, C1 cellulase or cellobiohydrolase. The prefe r red

substrate for exocellulase is crystalline cellulase and

degrades CMC ineffectively. Exoc ellulase degrades cellul ose

through end-wise cleavage generating cellobiose (written

communication, from Dr. B. A. White, University of Illinois,

Urbana, Illinois).

The cell-free enzyme extract prepared from ~.

cellobioparum grown in thioglycollate broth had a positive

effect upon seed germination. The germ i nation rate of the

seeds increased as exposure time with the enzyme extract

increa s ed. In addition, more seeds germin~ted when treated

with the cell-free extract prepared from ~. cellobioparum

grown in M 10 broth than cells grown in thioglycollate broth

43

indicating that a greater concentration of c llulase was

present in the extract prepared from cells grow in H 10

than in the thioglycollate broth. In suppo r t of this

conclusion, the amount of reducing sugar l I berated from the

seeds by the cellulase extract was sim i lar to that from the

rumen bacteria. With the cell-free enzyme extract of R.

flavefaciens, an increase in contact time between seeds and

extract liberated more reducing sugar; however, there was a

decrease i n germination s exposure time was i ncreased.

Thi ~ suggested excessive seed degradation occurred due to a

greater concentration of the cellulase enzyme and greater

length of exposure t i me. The inhibition assays, using HgC1 2

and increased incubation temperature, reduced the amount of

reducing sugars liberated or completely stopped the

cel l ul ase act i vity. These results support the conclusion

that a cel l ulase enzyme was present in the extract.

Seed Germination with Use of Commercial Cellulases

The results obtained from the concurrent use of the

purified commercial endocellulase pr oved that the cellulase

from the rumen microorganisms is involved in stimulating

germination of E. americanus seeds. Also, it was shown that

the endocellulase, not the exocellulase, is the key enzyme

that induced germina tion. As the data indicated from the

endocellu ase assay, a more dil ute enzyme preparation yields

less reducing sugar and takes longer contact time with the

seeds for the induction of germination. A more concentrated

enzyme preparation yields more reducing s ugar and results in

44

less contact time needed with the seeds fo germination to

be induced. The optimum exposure time of th e endocellulase

with the seeds was between 4 hand 10 h . After 10 h there

was a plateauing of the r educing sugar liberated and a

reduction of seed germination . Apparently the endocellulase

had exhaus ted its supply of subst rate resul ti ng in extreme

degradation of the seed leading to the death of the embryo.

The cellulolytic microflora in the r umen possibly are

synthesizing cellulases that are degrading the cellulose of

the seed coat causing sufficient scarifica ti on of the seed

resulting in imbibition. This process leads to normal

germination of t he seed. By germinating the seeds,

different genotypes of t he plant can be grown and possibly

lead to a better var iety for landscaping. Also, the

cellulolytic bacter i a tested in this research could possibly

be used to stimulate germination in other plants. Other

cellulolytic micro flora of the rumen could possibly induce

germinat ion of E. americanus seeds.

45

SUMMARY

Cellulolyt i c bacter i a of rumen fluid were shown to be

the key fa c tor i n breaking down dormancy and stimulating

germina tion of £. americanus seeds. Onl y seeds treated with

v i able cultures of ~. cellobioparum and l. flayefaciens

germinated. Unconcentrated c ell-free e xt r act and autoclaved

cel l s d i d not stimul ate germination. Concentrated enzyme

extracts prepared from bacteria grown i n M 10 broth and

thiog l ycoll a te broth were tes t ed for stimulation of seed

germination. These enzyme extracts were successful in

induc i ng germinat i on of the seeds and liberating reducing

sugars from the seed coat. Apparently the endocellulase

synthesized by both cellulolyt i c bacteria is induc i ng

germ i na ti on. The purified endocellulase from £. funiculo s um

was a l so successful i n s timulating germination of the seeds.

The concentration of reducing sugars l iberated from the seed

coat and the percent of seed germinating were directly

re la ted to exposure t i me of seeds to the endocellulase.

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