Cellulose Fermentation by a Rumen Anaerobic Fungus in Both the Absence and the Presence of Rumen Methanogens

0099-2240/81/121103-08$02.00/0

Cellulose

Fermentation by

a

Rumen

Anaerobic

Fungus

in

Both the Absence and the

Presence

of Rumen Methanogens

THOMAS BAUCHOP'AND DOUGLAS 0. MOUNTFORT2t*

Applied Biochemistry Division,Department of Scientific and IndustrialResearch, PalmerstonNorth,'and

Cawthron

Institute,

Nelson,2

NewZealand

Received6May 1981/Accepted4August1981

The fermentation of cellulose by an ovine rumen anaerobic fungus in the

absence andpresence ofrumen methanogens is described. In the monoculture,

moles of productasapercentageof the moles of hexose fermentedwere:acetate, 72.7; carbon dioxide,37.6;formate,83.1;ethanol,37.4;lactate,67.0;andhydrogen, 35.3.Inthe coculture,acetatewasthe major product(134.7%), and carbon dioxide

increased (88.7%). Lactate and ethanol production decreased to 2.9 and 19%,

respectively, little formatewasdetected (1%),and hydrogen didnotaccumulate.

Substantial amounts ofmethanewereproducedinthe coculture (58.7%).Studies

with

[2-14C]acetate

indicated that acetatewas nota precursorof methane. The

demonstration ofcellulose fermentationbyafungusextends therangeof known

rumen organisms capable of participating in cellulose digestion and provides further supportforarole of anaerobic fungi inrumenfiber digestion. The effect

of themethanogensonthepatternoffermentation is interpretedas ashift in flow

of electrons away from electron sink products to methane via hydrogen. The

study providesa new example of intermicrobialhydrogen transfer and thefirst

demonstration ofhydrogenformationbyafungus.

Cellulose

digestion

is a common propertyof manyfungi, but until

recently

virtually all

fungi werefound to require oxygen for growth. As a

result

a

role

for

fungi

inrumenfermentation had

notbeen considered

previously. However,

obli-gately anaerobic fungi

were

recently

discovered

in the rumen

(18),

and the

finding

that

large

populations

were present attached to fibrous

plant fragments (3) led

to the

suggestion

that there

should be re-examination of

accepted

con-cepts of fiber

digestion.

The

subsequent

dem-onstration

of

cellulose

digestion by

rumen

an-aerobic

fungi (4,

5,

19)

was

additional

evidence forarole infiber

digestion. However,

cellulose

fermentation

hasnotbeendescribed and forms partof the present

investigation.

Our

discovery

thatrumen

methanogens

formedastable

cocul-ture with the rumen

fungus

led tothe

investi-gation

of their

effects

on the fermentation of

cellulose.

Recent studies

involving

intermicrobial

H2

transferto

methanogens (8,

16)

have indicated that in

complex

ecosystems,suchasthe rumen, microbial interactions may have

profound

ef-fects on the

pattern

of fermentation

products.

Coculture of

H2-utilizing

methanogens

with

ru-men

carbohydrate-fermenting

bacteria

pro-tPresent address:DepartmentofDairy Science, Univer-sity ofIllinois, Urbana,IL 61801.

duced a shift in the flow of electrons generated in

glycolysis,

awayfrom the formation of

elec-tron sink

products (lactate

and succinate) to methane via hydrogen. In each case this was accompanied byanincrease in acetate.The pat-tern

of fermentation products

in

the cocultures

could not account for the

regeneration

of oxi-dized

pyridine

nucleotide

required

in

glycolysis.

It has beenassumed,

therefore,

that the

carbo-hydrate fermenters

were

able

to

produce H2

directly from reduced pyridine nucleotide. How-ever,

calculations

have shown that the reaction reduced nicotinamide adenine dinucleotide

(NADH)

+H+-- oxidized nicotinamide adenine

dinucleotide

(NAD+)

+ H2 is

thermodynami-cally

unfavorable at

partial

pressures of

H2

above

10'

atm (0.1kPa) (26). Thus

only

under conditions of low

partial

pressures of H2 main-tained by the presence of

methanogens

could the

production

of

H2

occurfrom reduced

pyri-dine nucleotide. The coculture studies

(8, 16)

provided useful insight into the intermicrobial H2 transfer mechanisms that couldoccurinthe

rumenwhere the

steady-state

hydrogen

concen-tration is low

(12),

and theresultswere

consist-entwith

Hungate's hypothesis (11)

that

meth-anogenesis

may

provide

an alternative electron sinktothe

production

of reduced

products

from pyruvate.

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BAUCHOP MOUNTFORT

The present results show that therumen

an-aerobic fungi can be grouped physiologically with the fewspecies of bacteria capableof

par-ticipating in the primary fermentation ofplant fiber in therumen.Cocultureof thefungus with methanogens resultedin an enhancedcellulose fermentationrateandan elevated acetate

pro-duction, an indication of the importance of in-termicrobial H2 transfer in the rumen

fermen-tation.

MATERIALS AND METHODS

Isolationprocedures.Sheeprumencontents were

strainedthroughcheesecloth, anda5% inoculumwas

transferred into7ml of standard medium(3)

contain-ing an antibiotic mixture(benzylpenicillin, 103 IU/ml;

streptomycin sulfate, 102 IU/ml). The culture was

incubated at 38°C, and growth of the fungus was

monitored by direct examination with a dissecting

microscope(Olympus). After three subculturesasmall

inoculum fromanactively growingfungal culture was

transferred to anaerobic broth (standard medium

without agar).Asingle thalluswasthen removedfrom

the culturewith a1-ml syringe witha 1.5-in (ca. 3.8

cm) 22-gauge needle and washed by transferring it

fourtimes through successive tubesto remove

con-taminating zoospores. The thallus was then

trans-ferredtostandard mediumandincubated.Microscopic

examination of theresultant culture indicatedasingle

fungus, based onthemorphology of the thallus and

zoospores.However, further examination by light and

ultraviolet epifluorescence microscopy revealed that

thefunguswasin coculture withmethanogenic

bac-teria. The coculturewasmaintained and kept to

in-oculateexperimental media.Pureculture of the

fun-gus, free from methanogens, was obtained by the

treatmentof thecoculture withchloramphenicol (30

,ug/ml) andsubsequent passage through three

subcul-tures.Thefungus was depositedinthe culture

collec-tion at the Department of Scientific and Industrial

Research, Palmerston North, New Zealand and

des-ignatedasstrainPN1.Taxonomy of the organism has

yettobecompleted.

Culture conditions.Culturesweregrown

anaero-bicallyat 38°C in tubes ofmediumprepared bythe

method ofHungate (13). The gas phase was 70% N2

and30%CO2.

Salt solutions (11) wereused in medium

prepara-tion. Solution A contained (wt/vol): KH2PO4, 0.3%;

NaCl,0.6%; (NH4)2SO4, 0.3%; CaCl, 0.03%;andMgSO4,

0.03%.SolutionB contained0.3%(wt/vol)K2HPO4. Themediumused was modified from Bauchop (3)

and hadthefollowingcomposition:solution A, 165 ml;

solutionB,165ml;cell-freeovinerumen fluid, 170ml;

distilled water, 500 ml; NaHCO3, 5 g; yeast extract

(BBLMicrobiology Systems,Cockeysville,Md.), 1g;

peptone(DifcoLaboratories,Detroit,Mich.), 1g;

cys-teine-HCl,0.2g;Na2S *9H2O,0.1g;and resazurin, 0.001

g.Medium (12 ml) wasdispensed anaerobicallyinto

anaerobic culture tubes(140 by 16mm;A.H.Thomas)

eachcontaining100mgofWhatmanno.1filterpaper

strips (about 15 by2 mm). Tubes were sealed with

butylrubberstoppers,and themediumwassterilized

byautoclaving. Sodium sulfidewasautoclaved

sepa-rately and added to culture media when cooled to

room temperature. The final pH of the culture

me-diumwas6.9.

Culturetechniques. The techniques described by

Hungate (13) were adapted for the maintenance and

subculturing of the fungus alone and

fungus-plus-methanogen cultures.Experimental mediawere

inoc-ulatedby transferring 1/20 the volume ofa6-day-old

cultureoffungus aloneorfungus plus methanogens.

For the former inoculation, this was equivalent to

approximately2.5 x 103colony-forming units (CFU)

offungus per ml ofinoculating culture, and for the

latter, itwasequivalentto 3 x103 CFU of thefungus

and1.4 x107CFU ofmethanogens per ml of inoculum.

Colony-forming units of thefungus inmono- or

cocul-ture weredetermined after4days of incubationinroll

tubes inoculated with 5 x 10-2 to 5 x 10-4 ml of

culture. Colony-forming units of the methanogens

were determined after 21 days of incubation in roll

tubes inoculated with 5 x 10-5 to 5 x 10-7 ml of

coculture. Glucose (0.1%; wt/vol) was used as the

growth substrate for the fungus and H2-CO2 (80:20

[vol/vol]inthe gasphase) for themethanogensinthe

roll tubes.

For studiesonthepurity of thecultures,theywere

roll-tubed in media described above, but with 0.2%

glucosereplacing celluloseasthesubstrate, orin AC

medium. The agarconcentrationwas2%(wt/vol).

Quantitative analysis of substrate and

prod-ucts in culture media. Cellulosewasdetermined by

the methodof Updegraff(21). Whatmanno. 1 filter

paper contained 99.7% cellulose. Soluble

carbohy-drates weredeterminedbyamodification of the

an-throne method (1), in whichglucosewasusedasthe

standard.

Forthe determination ofmethane, hydrogen, and

CO2 (headspace), 1-ml gas samples were removed from

culturetubes and analyzedon aFisher-Hamilton gas

partitioner equipped withathermalconductivity

de-tector.For methane andCO2 analyses, gaswas

frac-tionated at 25°C on dual columns; column 1 was

packed with2-ethylhexylsebacateonColumnpak, and

column 2waspacked with molecular sieve 13x.

He-lium was the carrier gas. For hydrogen analysis, gas

was fractionated on column 2, and argon was the

carrier gas.Gaseswerequantified by comparison with

thepeakheights of known standards.

Total CO2 in culture tubes wasdetermined

gravi-metrically asBaCO3. Culture medium inthe sealed

culture tubes wasacidified topH2byinjection of 0.4

ml of50% (vol/vol) H2SO4 to ensure that all of the

CO2wasliberated from the medium. The stopper was

piercedwith twosyringe needles (19 gauge), and CO2

was flushedfromthe culture tubes byslowly bubbling

nitrogen through the medium for 1 h; CO2 in the

effluent gaswastrappedin 3 MNaOH(10ml). Alkali

was then quantitatively removed from the trap and

madeupto 20ml withC02-freewater. Trapped CO2

wasprecipitatedasBaCO3 by the addition of 1 ml of

12% (wt/vol) BaCl2. The precipitate wascollectedby

centrifugation at 5,000 xg for 10min at 0 to 2°C,

suspended in 10 ml ofC02-free ethanol-H20 (50:50

[vol/vol]),andcentrifugedas before.TheBaCO3pellet

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was suspended in 2 ml ofethanol-H20 mixture and

collected on tared glass fiber filter disks (Millipore

Corp., Bedford, Mass.). The disks plus BaCO3 were

dried toconstant weight in a desiccator at 60°C. In

culture tubes where gas samples were removed for

analysis by gas chromatography, CO2 determined in

these samples was added to the CO2 determined as

BaCO3 to give totalCO2.

Formeasurement of volatile fatty acids and ethanol,

cells were removed by centrifugation at 5,000 x g for

20 min at 0 to20C. A 2-ml portion of thesupernatant

was acidified with 0.1 ml of 6 NHCI, and the volatile

fatty acids (except formic acid) and ethanol were

de-terminedby gas chromatography. A glass column (2

mby4mm)packed with 3% (wt) Carbowax20M-0.5%

(wt)H3PO4on60/80 meshCarbopak B (Supelco, Inc.,

Bellefonte, Pa.) wasused in aTracor 560 gas

chro-matographequipped with a flame ionization detector.

The column temperature was 160°C, and nitrogen was

the carrier gas.Fatty acids and ethanol were identified

andquantitated by comparison of the retention times

andpeak height with those of known standards.

For-mate was determined as described previously (10).

Lactatewasconvertedtomethyl-lactate and

deter-mined bygas-liquid chromatography. A 2-ml sample

ofculture mediumsupernatantorlactic acidstandard,

1mlof 0.2%(wt/vol) malonic acid as internal standard,

1mlof 50%(vol/vol) sulfuric acid, and2ml ofdistilled

water were added to the chamber ofa liquid-liquid

extraction apparatus.Carboxylic acidswereextracted

with diethylether for6h, and the extract was then

reducedto avolume of-1mlby rotaryevaporation.

Anhydrous sodium sulfatewasadded(approximately

20mg), andmethylationwasachievedbythe addition

of diazomethane in ether. The methyl esters were

separatedat 1200Con aglass column (2mby4mm)

packed with 10%diethylene glycol succinate on

100-to 120-meshGasChromQ, connectedto aflame

ioni-zation detector.Nitrogenwasthe carrier gas.

Methyl-lactate insampleswasdetermined from the ratios of

the peak heights of dimethylmalonate standards to

those ofmethyl-lactate samplesand standards.

Radioactive incubations with

[2-'4C]acetate

andanalyses.To determineacetate as aprecursorof

methane during cellulose fermentation by the

cocul-tures, 2,ICi of

[2-14C]acetate

(56 mCi/mmol) was

addedtoculture tubes atzeroincubation time.

Fer-mentation wasterminated at variousstages of

incu-bationbetween0and 168 hby theinjection of1mlof

3NNaOH into culturemedia,and theCO2inthe gas

phasewasabsorbed.Aftercompleteabsorption ofC02,

asascertainedby gaschromatographicprocedures, the

resulting vacuum in the tubes was relieved by the

injection of nitrogen with a glass piston syringe to

allow quantitative gas sampling. Tubes were

main-tained at2°C,and the total volume ofgaswas

deter-mined by recording the excess gas forced into the

syringe. For analysis of radioactive methane, 1-ml

volumesof gas were injectedinto scintillation vials

sealedwithbutyl septum stoppers and countedin 20

ml oftoluene-based scintillantasdescribedbyZehnder

etal.(27).To ensurethatamaximal amountof

meth-anewasdissolved,vials wereagitated vigorously.

As-say ofthegasatmosphereabove thescintillationfluid

revealed that 80 ± 3% of the methane added was

absorbed and therefore counted. Corrections were

madefor methane not absorbed and for quenching.

Counting efficiencywas 86±2%.

Foranalysis of radioactive acetate, media were

cen-trifuged at 5,000 x g for 15 min at 0 to 2°C. The

proceduresfor the isolation ofradioactiveacetatefrom

thesupernatant and the determination of radioactivity

were based onpreviously described methods (17).

Quantitativemeasurementsof methane and acetate

forspecific radioactivity determinations were asinthe

preceding section.

Microscopy. Examination of the monoculture of

the fungus and coculture with methanogens was by

light microscopy and scanning electron microscopy.

Details ofsample preparation for scanning electron

microscopy were as previously described (3). Because

of their characteristic fluorescence at 420 nm, the

methanogenswere alsoexamined byultraviolet,

epi-fluorescence microscopy.

Chemicals. All chemicalswereobtained from

com-mercial sources andwereof reagentgrade. The

radi-oisotope,

[2-14C]acetate

(specific activity, 56 mCi/

mmol), wasobtained from the RadiochemicalCenter,

Amersham,England.

RESULTS

Description of anaerobic fungus.

The an-aerobic

fungus strain

PN1 appears

closely

re-lated

tothe

rumen phycomycete Neocallimastix

frontalis

(18). The

phylogenetic

position

of these

fungi

remains to be

determined, although

the modeof

zoosporogenesis

from the monocentric, rhizoid-bearing

thallus

is

comparable

withthat of many

chytrids. Figure

1A shows a

thallus

of the

fungus consisting

of a

sporangium

with a

highly

branched rhizoid. The size

of the

spo-rangia

varied depending

on the stage

of

devel-opment

of the

thalli,

but in

glucose medium (3)

it was up to 200 ,um in

length

and 140 t,m in width. With

cellulose

as

substrate,

sporangia

up to180

t,m

long

wereobtained.

Figure

1B shows a

single

zoospore. The zoospores were oval to bean

shaped

(length,

14 to 18

,im;

width,

12 to

14,um)

and contained between9and12

flagella.

The body of the zoospore in

Fig.

1B is

slightly

out

of

focus,

which makes it appear

larger.

In older

cultures

zoospores

characteristically

round

off.

Colonies in roll tubes inoculated with

actively

fermenting

fungal

culture

probably

developed

from zoospores, since these were numerous in

the culture fluid

(>103

per ml of

6-day-old

cul-ture), whereas free

sporangia

(detached

from solid

cellulose substrate)

were either

absent,

or

in low

numbers,

and

cellulose

was not

usually

transferred in the inoculum.

Stability

of

fungus-methanogen

cocul-ture. The

fungus-methanogen

coculture

iso-lated was stable and was maintained in

liquid

42,

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mX

',-

A

p

___

B

FIG. 1. (A) Light micrograph of rumen anaerobic fungus. Thallus

showing

sporangium

and highly

branched rhizoid. (B)Multiflagellatedzoosporeoftherumenanaerobicfungus.

medium

containing cellulose

forover 1yearby routine transfer every6

days.

During this time therewas noloss of

cellulolytic

or

methanogenic

activity.

Methanogenic population

in

the

cocul-ture. Colonies inroll tubes with

H2-CO2

(80:20

in the gas

phase),

inoculated with 5 x

10-5

to 5

x

i0'

mlof stable

fungus-methanogen coculture

(6

days

old)

were

of

onetypeand

became visible

after1week of

incubation.

Surface colonies

were

translucent, circular

with

entire

margins,

off-whitetoyellow in

color,

and reacheda

diameter

of

approximately

2 mmafter21

days

of incuba-tion. Deep

colonies

were

lenticular

anddidnot

increase in size

beyond

1mmin

diameter.

When

colonies

were

repeatedly picked

and

examined

by ultraviolet

epifluorescence microscopy,

the bacteria exhibited characteristic

blue-green

flu-orescence of

methanogens

at420nm.

The cells

weregram

positive,

nonmotile oval rodsorcocci,

usually

0.7ymin

width,

withsomecellsreaching

1 ,um, and 0.8 to 1.6

,um

in

length,

occurring

predominantly in pairs but occasionally in chains. These bacteriacan

clearly

beidentified in the

scanning

electron

micrograph

of the co-culture (Fig. 2). Isolated colonies grew

well

on H2-CO2 (80:20 in the gas phase), slowly on for-mate, butnotonacetate,although acetate stim-ulated

growth

on

H2-CO2.

The

colony-type,

mor-phology,

and substrates utilized indicated that the methanogens were most likely Methano-brevibacter

ruminantium.

Also present in the mixed culture but not shown in Fig. 2 were large

FIG. 2. Scanning electron micrographof coculture

of anaerobicfunguswithrumenmethanogens

show-ingmethanogens andfungal hyphaegrownonpaper.

cocci (about 1.5

Am

in

size),

occurring

singly,

in pairs, and in

clusters,

and

exhibiting

blue-green fluorescence at 420 nm. We wereunsuccessful in

isolating

these organisms, but their morpholog-ical features and

fluorescence

at 420 nm sug-gested they were

methanogens

resembling Methanococcus. Direct counts of these orga-nisms by

epifluorescence microscopy

indicated that

they

accountedfor<1% of the total

meth-anogenic population.

The organism resembling M. ruminantium designated as Methanobrevi-bactersp. strain

RAl,

accountedfor >99% of the

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H2 FUNCTION IN FUNGUS-METHANOGEN INTERACTION 1107

total

methanogens in

the

coculture,

consistent

with the

presence

of only

one

colony

type inthe

roll

tubes.

Fermentation of cellulose. The fermenta-tion ofcellulose by the rumen anaerobic fungus alone

resulted

in theformation of six products, acetate,

lactate, formate,

ethanol, C02, and H2 (Fig. 3). The lag time for lactate production was longer than for the other products.Fermentation terminated about 300 h after inoculation. In the coculture, the major products were acetate, car-bon dioxide, and methane (Fig. 4). Ethanol and lactate production decreased. Hydrogen was not detected duringfermentation (detection limit, 5 x

10-4

atm), and formate occurred in only trace amounts. The coculture fermentation termi-nated

after

about 200 hof incubation. For both the mono- andcocultures, the pH

of

the medium decreased from 6.9 at the

beginning

of incuba-tion to 6.0 atthe end of fermentation. The rate and extent

of cellulose degradation

weregreater in the

coculture

than in the monoculture(Fig. 3 and 4). In thecoculture, after 100 h, more than 70% of the initial cellulose had been

degraded,

and in the monoculture, less than 10% had been

degraded.

At theend

of

fermentation, 82%

of the initial

cellulose

hadbeen

degraded

inthe cocul-ture, and 53% had been

degraded

inthe

mono-culture.

Table

1 comparesthe fermentation

products

of cellulose

in the mono-

and cocultures.

In

the

O 100 200 300

TIME(h)

FIG. 3. Fermentationofcellulosebyrumen

anaer-obic fungus. Cellulose is expressed as total milli-grams, andproducts areexpressed astotal

micro-moles in thegrowth medium (12 ml). Media were

inoculated with 0.6 mlof fungalculture (2.5 x 103

CFUper ml). Values for acetate, formate, ethanol,

and lactate werecorrectedforamounts transferred

in the inoculum, andwith theexception ofethanol and lactate, the amountspresent in uninoculated media.

TIME (h)

FIG. 4. Fermentation of cellulose by cocultureof

anaerobicfungus withrumenmethanogens. Cellulose

is expressed as totalmilligrams, andproducts are

expressedastotal micromoles in thegrowth medium

(12ml).Mediawereinoculatedwith 0.6 mlof

fungus-methanogencoculture (3x103CFUoffungusperml;

1.4 x 107 CFU of methanogensper ml). Valuesfor

acetatewerecorrectedfor theamountspresent in the

inoculumand inuninoculatedmedia.

coculture, carbon dioxide and acetate increased

by

51 and 62

mol/100

of

hexose,

respectively. Lactate and ethanol decreased by 64 and 18.4 mol/100 mol of hexose, respectively. The de-creaseinhydrogen and formate in the coculture reflected their utilization in methane production. To determine whether acetate, the only other

likely

methaneprecursor, contributed to meth-ane in the coculture, fermentation was carried out in the presence of

[2-14C]acetate.

Data on

specific radioactivity

ratios

(Table

2) indicated that acetate contributed less than 1% of the methane formed during

cellulose

fermentation and was therefore not a significant precursor. Thus theamountofH2

required

for the forma-tion of methane

(58.7

mol of

CH4/100

mol of

hexose),

including

H2

produced

via formate cleavage, would be 235

mol

(4H2+

CO2

-- CH4

+

2H20).

Other

possible products

of

cellulose

fermen-tation

by

the

fungus,

propionate, butyrate, or succinatewere notdetected

(<0.1

mol/100

mol of hexose

fermented),

and there was no

accu-mulation

of

soluble carbohydrates

inthe

mono-or

cocultures

during

the fermentations.

Recombined culture

experiments.

Iso-lated colonies of

Methanobrevibacter

sp. strain

RA1,

were transferred to culture tubes inocu-lated with thefungus alone. After

completion

of growth

(approximately

170

h),

asascertained

by

methane production, the recombined cultures

wereinoculated into fresh media. The inoculum contained

approximately

3 x 103CFU of

fungus

per ml and 1.2 x

107

CFU of

methanogen

per

VOL. 42,1981

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1108

ml. At the completion ofmethanogenesis (168 h), products in the culturemediawereanalyzed, and unutilized cellulose was determined. The formationofproductswassimilar to the stable coculture (Table 1), except that ethanol

de-creased to9.6mol/100 mol of hexose. CO2was not determined. Formate and hydrogen were

absent, and lactate was low (3.5 mol/mol of hexose). Mean values foracetate and methane

were 138.5 and 59 mol/100 mol ofhexose,

re-spectively. At the end offermentation, 87% of

the initialcellulosehadbeendegraded. DISCUSSION

The gut anaerobicfungipossessseveral

prop-erties not found in otherfungithatare

undoubt-edly related to their obligately anaerobic

life-style, perhaps itself the most unusual of their

properties. Much of the interest in thesefungi

TABLE 1. Fermentationof cellulose by anaerobic

fungus in the absence andpresenceofrumen

H2-formate-utilizingmethanogensa

mol/100mol of hexoseunits'

Product Plusrumen

Fungusalone methanogens

Acetate 72.7±4.9 134.7±8.5 Lactate 67.0±2.8 2.9±0.1 Ethanol 37.4 ± 1.5 19.0±1.0 Methane 0.0 58.7±0.9 Carbondioxide 37.6±7.0 88.7±5.0 Hydrogen 35.3±2.5 <0.05 Formate 83.1±2.8 1.0±0.07 Carbonrecovery(%) 90.3 77.5 Hydrogenrecovery 87.0 75.6 (%) Oxidation-reduction 1.44 1.14 index

a Determinedatthecompletionoffermentation,as

ascertained bynofurtherincrease inhydrogen

(mon-oculture) ormethane (coculture). Thepercentagesof

cellulose degradedinthemono-andcoculture were 53

and82%, respectively.

bValuesare meansof duplicatedeterminations, and

where theerror ispresented, +1standarddeviation.

cDetermined according to Barker (2).

sofarhas concerned the extent of their role in

ruminantdigestion.But since thesearetheonly fungiknowntobeobligate anaerobes,the com-plete spectrum of theirproperties is of

consid-erableintrinsic interesttomicrobiologists. Thedegradationofcellulosebythe monocul-ture providesthe firstexampleofa mixed-acid

typeoffermentation infungi,and theproducts

weresimilartothose thatareproduced by

coli-form bacteria during anaerobic growthon

glu-cose.Hydrogenformation has not beenreported previously infungi, andtheunderlying

mecha-nism of hydrogenformationneedstobestudied

in detail. Inaddition, thepathwaysforlactate,

acetate, and ethanol alsorequire studyto

deter-mine whetherornottheyaresimilar to known pathways for the formation of these products.

All of the fungal fermentation products are

known to be formedbyrumenbacteriaalso,and

in therumentheymaybeutilizedbyeither the

hostanimal or further fermented by other

ru-menmicrobes.

The present work confirms reports (4, 5, 19)

of cellulose digestion by the rumen anaerobic

fungiand thus extends the knownrange of

cel-lulolyticmicrobescapableofparticipatinginthis

primary fermentative process central to rumi-nant digestion. Glucose did not accumulate in

the medium during growth on cellulose,which

may indicate that the cellulase is associated

closely with the fungal hyphae and is not

re-leased into the medium. Thisagreeswith results

of microscopic examination of strips of paper undergoing fermentation, which showed that

digestion occurred only in the areas in close proximity to fungal rhizoid (4). Until recently the major cellulolytic microbes found in the

rumen havebeen anaerobic bacteria, and they

havelongbeenaccepted asthe main agents of

cellulosedigestion.The close association offungi

with fibrous plant fragments suggested that

fungihadarole in fiberdigestionintherumen (3). Theextentofthis role required

demonstra-tion of the appropriate enzymatic activities, as

wellasdetermination ofthemassoffungal

veg-etative tissues within the tissues of digested

plant fragments.The firstof theserequirements,

TABLE 2. Comparison of specificradioactivities ofmethane with [2-

4C]acetate

during the fermentation of cellulosebycocultureof anaerobic fungus with rumen H2-formate-utilizingmethanogensa

Incubation Acetate CH4

14CH4

(dpm)

[2-'4C]acetate Sp act of CH4 Sp act of acetate Sp act ratio time(h) (pmol) (smol() (dpm) (dpm/,umol)

(dpm//imol)

CH4/acetate

0 10.8 -b 4.30 x 10 - 3.9 x 105

-72 395 178 1.37 x 104 3.72 x106 76.9 9.1 x103 8.4 x 10-3

168 537 335 1.83x 104 3.37 x 106 54.6 6.3 x 103 8.7 x10-3

aTubes contained12mlofmedium with 100 mg of Whatman no. 1filter paper strips as substrate (cellulose).

A

2-,Ci

amountof[2-'4C]acetate(56mCi/mmol) was added at zero time. Results are mean values of duplicate

tubes.

b-,Not done.

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H2 FUNCTION FUNGUS-METHANOGEN INTERACTION the ability to ferment cellulose, has now been

demonstrated. It may be important also that

fungi,

by nature of their mode of growth involv-inghyphal extension, possess the ability to pen-etratedeeply into tissuesnormally inaccessible to bacteria, and this suggests a special role for anaerobicfungi in rumen fiber digestion.

The

coculture

consisting of an anaerobic fun-gus

and

methanogenic bacteria presumably re-sulted from the insensitivity of the methanogens

to the antibiotics used in the isolation of the fungus. However, in the rumen it seems likely that the methanogens would be metabolically associated with the anaerobic fungi as well as other H2-producing organisms. The coculture interaction provides the first example of H2

transfer

between a fungus and a bacterium. Pre-viousstudies on H2 transfer have involved

inter-bacterial

systems (7, 8, 15, 16, 20, 23), although the association of methanogenic bacteria with rumen ciliates suggests the possibility of H2

transfer

between protozoa and bacteria (22). Production of hydrogen is the key to the

co-metabolism

of the fungus with methanogens, resulting inadecrease inelectron sink products,

lactate

and ethanol. To explain this decrease, it is necessary to

postulate

the presence of a hy-drogenase whichcatalyzes the production of

hy-drogen

from reduced pyridine nucleotide at low

partial

pressure

of

the gas (26). Thus the meth-anogens,

by

removing H2 and maintaining low

partial

pressures,would facilitate the production of H2 from reducedpyridine nucleotide by the

fungus. Reducing

equivalents

whichwould oth-erwise be

used

in the formation

of lactate

and

ethanol would

be

diverted

to methane via hy-drogen in the coculture. The proportion of

meth-ane

expected

from theelectron shift via reduced pyridine nucleotide may be calculated by assum-ingthat the

only

other source of hydrogen was from reactions

leading

to the formation of

ethanol

andacetateand that thiswas

equivalent

tothesumof these

products

(153.7

mol/100

mol

of hexose).

The

assumption

wasbasedon

mon-oculture data

(Table

1), from which thesumof formate and

hydrogen

was

almost

equal

tothe

sumofacetateand

ethanol), suggesting

a

com-mon

intermediate

for the formation of the

2-carbon

products, perhaps

acetyl

coenzyme A. Thus, out of the

total

hydrogen required

for methane

production

(235 mol for 58.7 mol of

CH,),

about 35% would be

expected

tobe

pro-vided from reduced

pyridine

nucleotide from electron shift.

The carbon and

hydrogen

recoveries in the coculture were less than in the monoculture. One

possible explanation

is that one or more

products

from the

fungus

were assimilated into cell biomass of the

methanogens.

Previous

stud-ies have shown that both

CO2

and acetate may contribute to

cell

carbonof methanogens (6, 9,

24,

25) and that as much as 60% of the

cell

carbon in M.

ruminantium

maybederived from acetate (6). The

oxidation-reduction

index for bothculture fermentations was higher than the

theoretical

value of 1.0. The deficiency in

re-duced products

mayhave

been due

tothe

for-mation

of

cells

withan

oxidation-reduction

state

morereduced than that of cellulose (14). In the

coculture,

cellulose degradation

com-menced

earlier,

the rate was

faster,

and the

quantity digested

was greaterthan in the

mon-oculture

(82versus

53%).

These effects

might

be

explained

by increased

growth resulting

from higher energy

yields

via the "acetate"

path-way.Inaddition,

coculture

withthe

methanogen

resulted in the

removal

ofanumber of the

fungal

fermentation products,

some of which

might

have

been

inhibitory

to

growth

of the

fungus.

Mostof the

cellulose degradation

inthe

cocul-ture

occurred

in the

period

between 50

and

100 h;

perhaps application of

this systemto

biocon-version processes,

including cellulase

produc-tion, maywarrantfuture

investigation.

Inthe rumen many aspectsof

cellulose

diges-tion are

still inadequately understood,

and the presentwork nowraises

questions

on

accepted

concepts. The

demonstration

of cellulose

fer-mentation is further support forarole of

anaer-obic

fungi

in

fiber

digestion

in the rumen and extends the known range of

cellulose-fermenting

microbes there. The

knowledge

that similar

fungi

arepresentin the

foregut

and

hindgut

in

a

wide

range

of

herbivorous

animals

(4) likewise

raises the

question

of the extentof the

role

of these

fungi

infiber

degradation.

The

discovery

of

a

fungus-methanogen

combination in

stable

coculture and the resultant

altered fermentation

pattern

obtained

bring additional information

to

understanding

some of the

complexities

of the

rumen

fermentation.

ACKNOWLEDGMENTS

WethankRodAsher andRayWillsfortheir expert tech-nical assistance.

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