APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1994, p. 3718-37230099-2240/94/$04.00+0Copyright © 1994, American Society for Microbiology

Effects of Ruminal Protozoa on Cellulose Degradation andthe Growth of an Anaerobic Ruminal Fungus,

Piromyces sp. Strain OTS1, In VitroDIEGO P. MORGAVI,* MASARU SAKURADA, MUNETAKA MIZOKAMI,

YOSHIFUMI TOMITA, AND RYOJI ONODERA

Laboratory ofAnimal Nutrition and Biochemistry, Faculty ofAgriculture, Miyazaki University,1-1 Gakuen-Kibanadai-Nishi, Miyazaki 889-21, Japan

Received 1 March 1994/Accepted 8 August 1994

An anaerobic rumen fungus, Piromyces sp. strain OTS1, was incubated in the presence or absence of a mixed,A-type, protozoal population obtained from a goat, in a medium containing filter paper cellulose as energy

source and antibiotics to suppress bacterial growth. Fermentation end products, cellulose degradation, andchitin as an indicator of fungal biomass were examined. In the presence of protozoa, total volatile fatty acids,notably propionate and butyrate, increased, and lactate decreased. In fungus-protozoan coincubations, formatewas not detected at the end of the experiment and the amount of reducing sugars remained low throughout theincubation period. The fungal growth in the coincubations was negatively affected. While protozoal predationon zoospores was one mechanism of inhibition, mature fungal cells were also affected. Total cellulosedegradation was greater in fungal monocultures, but the amount of cellulose degraded per unit of fungalbiomass was 25% larger in the coincubations. The negative effects that the protozoal predatory activity had on

the fungal growth and subsequently on the amount of cellulose degraded by Piromyces sp. strain OTS1 were

partially attenuated by the protozoal fibrolytic activity or by an enhanced fungal activity due to a more

favorable environment.

The anaerobic fungi that inhabit the gastrointestinal tract ofruminants and other herbivores have the ability to degradecellulose and other structural polysaccharides (14). Theircontribution to fiber degradation in the host animal is poten-tially perhaps the most important role of these microorgan-isms. They degrade plant fiber in axenic cultures as efficientlyas bacteria (3), but the effects of other rumen microorganismson the fungal growth and their ability to degrade structuralpolysaccharides in the complex rumen ecosystem are notcompletely understood.

Interrelationships between ruminal bacteria and fungi havebeen extensively studied. The degradation of structural po-lysaccharides and fungal growth are stimulated when fungi are

cocultured with methanogenic bacteria (5, 7). Some otherfungus-bacterium associations have been reported (6, 7, 18, 33,42). The interaction effects range from synergism to antago-nism depending on the kind of fungal and bacterial species andthe type of substrate used.The interrelationships between ruminal protozoa and fungi

have not been as well studied as that of ruminal bacterium-fungus interactions. Protozoal predatory activity on rumen

fungi has been described previously (31), and this is possiblythe reason why in defaunated animals fungal counts are usuallyhigher than those in faunated ones. However, increased fungalcounts have been found in fauna-free animals after refaunation(41). These apparently contradictory data suggest that theprotozoan-fungus interrelationship could be more complexand not limited only to a predator-prey one. For instance, thestabilizing role that protozoa have on the physico-chemical

* Corresponding author. Mailing address: Laboratory of AnimalNutrition and Biochemistry, Faculty of Agriculture, Miyazaki Univer-sity, 1-1 Gakuen-Kibanadai-Nishi, Miyazaki 889-21, Japan. Phone:0985 58 2811. Fax: 0985 58 2884.

characteristics of the ruminal environment (39) may have a

beneficial action for fungi.The objective of the present study was to investigate how

cellulose degradation and growth of an anaerobic ruminalfungus, Piromyces sp. strain OTS1, cultured in vitro are af-fected by ruminal protozoa.

MATERIALS AND METHODS

Microorganisms. A fungus, Piromyces sp. strain OTS1, was

isolated from the rumen of a ruminally fistulated goat by themethod of Joblin (19). Its morphology was examined by lightmicroscopy. Transmission electron microscopy was used toinvestigate some ultrastructural characteristics of the zoo-spore.Mixed rumen protozoa were obtained from a ruminally

fistulated goat (Japanese native breed, 35 kg) fed twice a day,at 0900 and 1700 h, a ration consisting of 300 g of alfalfa(Medicago sativa) hay cubes and 100 g of concentrated feed(17% protein, 72% total digestible nutrients, a-Dairy Mix;Chubu-Shiryo, Chita, Japan). Ruminal contents were collectedbefore the morning feeding and strained through four layers ofsurgical gauze. These strained rumen fluids were poured into a

separating funnel that had been gassed with 02-free 95%N2-5% CO2 and incubated at 39°C for up to 60 min to allowsmall feed particles to buoy up and protozoa to sediment at thebottom. Most of the lower portion was then anaerobicallycollected and centrifuged at 500 x g for 5 min. The superna-tant was carefully decanted and discarded. The protozoalpellet was washed by centrifugation (500 x g, 3 min) 10 timeswith MB9 buffer solution (28), in order to remove bacteria andfungal zoospores as completely as possible, and resuspended in50 ml of MB9 buffer solution plus a mixture of antibiotics(chloramphenicol sodium succinate, ampicillin sodium, andstreptomycin sulfate, each at a concentration of 0.1 mg/ml of

3718

Vol. 60, No. 10

on Septem

ber 18, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

RUMINAL FUNGUS-PROTOZOAN INTERACTIONS IN VITRO 3719

suspension). These protozoal suspensions were incubated at390C for 12 h to further reduce the associated bacterialpopulation (25, 29). The gas phase was 02-free 95% N2-5%CO2. After incubation, the suspensions were centrifuged (500x g, 5 min), and the pellet was washed three times with an

autoclaved MB9 buffer solution and resuspended at 15 to 20%(vol/vol) concentrations in the same buffer. These protozoalsuspensions were used as inocula. Samples were fixed in methylgreen formalin salt (MFS) solution (29) for protozoal countingand population type determination.Growth conditions. Piromyces sp. strain OTS1 was main-

tained on medium 10 of Caldwell and Bryant (8) at 39°C withsubculturing every 5 to 6 days. The medium used in theexperimental cultures was based on medium 10 containing allnormal constituents (e.g., Trypticase, yeast extract, hemin), butsoluble carbohydrates were replaced with 100 mg of filterpaper strips (ca. 8 by 3 mm; no. 5A; Toyo Roshi Co., Ltd.,Tokyo, Japan) per tube. The medium also contained a mixtureof antibiotics (the same as above, each at a concentration of 0.1mg/ml of medium) that was added before the fungal inocula-tion. Experimental cultures (8 ml per tube) were started bytransferring 1 ml of a 2-day-old fungal culture. The approxi-mate amount of zoospores after a 2-day incubation under theseconditions is about 4 x 103 to 5 x 103 zoospores per ml.The protozoal suspensions (0.5 ml) were added into the

experimental cultures at days 1, 4, and 7 of the incubationunder aseptic and anaerobic conditions. In tubes in which fungiwere grown alone, 0.5 ml of an anaerobic and autoclaved MB9buffer solution was added instead of the protozoal suspension.

Uninoculated controls were carried out throughout the exper-

imental period. Incubations were carried out at 39°C for 10days. The gas used was 02-free CO2.The aseptic anaerobic techniques, methods of medium

preparation, and sterilization used have been described else-where (8, 16, 17). The antibiotics were dissolved in anaerobicdistilled water prepared by boiling and bubbling with 02-free

N2 gas. The solution was filter sterilized with a 0.2-,um-pore-diameter pyrogen-free filter membrane (Dismic-25 cs; ToyoRoshi Co., Ltd.) before addition to the culture tube.

Sampling and dry matter determination. Samples were

taken at 1, 4, 7, and 10 days of incubation after the addition ofthe protozoal suspension or MB9 buffer solution. At eachsampling time, six tubes of experimental cultures and threetubes for control were used.The pH of the medium was measured immediately after the

tubes were opened; supernatants, 0.5 ml for volatile fatty acid(VFA) determination and 3 ml for other analysis, were frozenand kept at -20°C until processing. The remaining contents ofthe tubes were filtered through a nylon filter, used for thedetermination of crude fiber. The filter did not retain most ofthe protozoa, except for some large ciliates. The retentate waswashed with approximately 50 ml of distilled water and trans-

ferred to weighed aluminum dishes by being washed down withdistilled water. From optimization experiments, it was foundthat dried weights were more reproducible when aluminumdishes were used instead of the sample being dried in the samenylon filter. The dishes were kept overnight in an oven set at

80°C as a predrying treatment and dried at 135°C for 2 h. Theywere then weighed, and the amount of filter-paper loss was

calculated.Analytical methods. Fungal growth was estimated by the

amount of cell wall chitin. After the dry matter determination,

chitin in the dried material was determined by a colorimetricmethod (9). VFAs in the medium were measured by gas

chromatography using a Yanaco gas chromatograph (G2800-F;Yanagimoto Seisakusho, Kyoto, Japan) fitted with a hydrogen

flame detector. The column was packed with 10% polyethyleneglycol 6000 (60 to 80-jim-size mesh), the injection port anddetector temperature were set at 200°C, and the columntemperature was set at 135°C. The carrier gas was N2. Sampleswere treated with metaphosphoric acid in H2SO4, with a finalconcentration of 5% (wt/vol) in 1N H2SO4, kept overnight at4°C, and centrifuged (18,500 x g, 60 min). The supernatantswere used for VFA determination. Formate was detected bythe method of Sleat and Mah (34), lactate was measured witha commercial kit (Boehringer, Mannheim, Germany), andreducing sugars were measured by the neocuproine method(12), with glucose as the standard. A method for cellulosedetermination (37) was used to monitor small fragments offilter paper suspended in the medium.

Protozoa fixed in MFS were appropriately diluted in thesame solution and counted with a Fuchs-Rosenthal hematocy-tometer in triplicate. The presence of fungal zoospores in theprotozoal suspensions was also checked with the aid of thehematocytometer chamber. To help zoospore visualization, 0.2ml of lactophenol blue solution (Merck, Darmstadt, Germany)was added to 1 ml of the protozoal suspension.

Statistical analysis of the data was performed by t test (35).

RESULTS AND DISCUSSION

Microorganisms. Under normal feeding conditions, fungiand protozoa are usually present together with bacteria in therumen. Although not essential to the life of the host animal,they appear to play an important role in the degradation ofnutrients, particularly fibrous materials (21, 31). The interre-lationships between them, in the absence of bacteria, can bestudied only in vitro. To overcome the problem that protozoacannot be cultivated for extended periods of time in axenicconditions, a protozoal inoculation every 3 days was necessaryto maintain a relatively constant population. Survival under theconditions studied was about 3 days, coincident with thatreported by Coleman for the protozoon Entodinium caudatum(10).The inocula were prepared so that the final protozoal

concentration resembled in vivo counts. Populations were oftype A (13), composed predominantly of small entodiniomor-phids (95%). Large entodiniomorphids and holotrichs ac-counted for 2 to 4% and 1.5 to 2.5% of the total, respectively.Total numbers of protozoa inoculated at 1, 4, and 7 days ofincubation were 22.3 x 105, 7.0 x 105, and 33.6 x 105. Thedensity of the 4-day inoculum was low compared with that ofdays 1 and 7. However, the total number of protozoa inocu-lated was within the normal ranges found in ruminants andmay well reflect in vivo variations within the same animal(reference 7a, cited in reference 39). In the fraction of rumenfluid used to collect protozoa, fungal zoospores were alsoprobably present, but zoospores were eliminated, or theirnumber was reduced significantly, after the centrifugationwashing procedures because their smaller size compared withprotozoa, and also because of protozoal predation during the12-h incubation with antibiotics (unpublished observation).Zoospores were not seen under microscopic examinations. Amore sensitive cultivation technique was not considered nec-essary to assess the fungal contamination. Contaminatingruminal bacteria, on the other hand, were eliminated by thecombination of the three antibiotics, which are very effective(29). Bacteria which might have survived were also inhibited bythe presence of antibiotics in the experimental medium, and,although it is acknowledged that preformed bacterial enzymesare still active, they could not play a significant metabolic rolein this experiment.

VOL. 60, 1994

on Septem

ber 18, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

3720 MORGAVI ET AL.

TABLE 1. Fermentation of filter paper cellulose by Piromyces sp.strain OTS1 in the absence or presence of rumen protozoaa

Treatment Piromyces sp. Piromyces sp. strain OTS1strain OTS1 plus rumen protozoa

Acetate 18.76 ± 1.95 26.54 ± 8.78Propionate 1.76 ± 0.32 13.11 ± 7.68Isobutyrate b 2.01 ± 1.00Butyrate 0.40 ± 0.07 6.92 ± 0.61Isovalerate 0.30 ± 0.04 3.88 ± 0.97Valerate 2.29 ± 0.99Total VFA 21.22 + 2.30 54.77 ± 18.83Acetate/propionate ratio 10.6 2.0Formate 16.90 ± 1.41Lactate 13.86 ± 1.17c 0.08 ± 0.02cGlucose 8.67 ± 1.81 0.79 ± 0.13c

a All determinations are expressed as micromoles per milliliter and are themean ± SD of six samples analyzed after 10 days of incubation. Values betweentreatments are different at the P < 0.01 level, except acetate at P < 0.1.b_, not detectable.Cn = 4.

Piromyces sp. strain OTS1 produces a highly branchedmonocentric rhizoid. The sporangial development is mainlyexogenous, but zoosporangia presenting an endogenous spo-rangial development are also seen. Zoospores are globose and5.5 to 7.5 ,um in diameter and are uniflagellates. Thesestructural, and some ultrastructural, characteristics of thezoospore (e.g., lack of ribosomal aggregates and presence ofovoid hydrogenosomes) suggest that the isolate is likely to bePiromyces communis (4, 15, 22, 30), but this remains to beconfirmed.

Fermentation of cellulose by Piromyces sp. strain OTS1 inmonocultures or in the presence of protozoa. Table 1 showsthe fermentation products at the end of the incubation period.Acetate accounted for approximately 90% of the total VFAsproduced by Piromyces sp. strain OTS1 under the conditionsstudied. This VFA is always abundantly produced by Piromycesspp. grown on various sources of carbohydrates (6, 7, 36).Detection of propionate, although low, is rather unusual foranaerobic fungi and can be a consequence of the culturemedium composition. When protozoa were present in theincubation, the total production of VFAs increased signifi-cantly (P > 0.01). In addition, a shift in the proportion ofindividual fatty acids produced was detected with a reducedacetate-to-propionate ratio. Butyrate, isobutyrate, valerate,and isovalerate, which were absent or almost absent in fungalmonocultures, increased when the fungus was cultivated in thepresence of protozoa (Table 1). Protozoa are able to digestfungal material (24), and apart from the fungus itself, nutrientsfrom the culture medium (including cellulose), fungal fermen-tation products, and protozoa themselves may also have beenutilized. The main VFAs, produced by protozoa in vitro withconventional substrates, like starch, as a carbon source, areacetate and butyrate (39). These VFAs also predominatedwhen mixed protozoal suspensions were incubated alone;propionate was the third most abundant VFA closely followingbutyrate, and isobutyrate was also detected, but not valerateand isovalerate. Increased formation of isobutyrate, valerate,and isovalerate in the coincubations seems, therefore, to bemainly a consequence of the protozoal utilization of aminoacids of fungal origin. The finding of propionate as the secondmost abundant VFA in Piromyces sp.-protozoan cultures isinteresting. Propionate and butyrate are the main VFAsproduced from lactate metabolism by entodiniomorphid pro-tozoa (27). It is worth noting that the increase in propionate in

16 ** **

14-

1 2

E 4 ,, ,

10

0 2 4 6 8 1 0Incubation Time (day)

FIG. 1. Lactate in the culture medium of the anaerobic ruminalfungus Piromyces sp. strain OTS1 incubated alone (M) or in thepresence of a mixed protozoal population obtained from a goat (0).Values are the mean t SD of five samples, except at day 7 forPiromyces sp. strain OTS1-protozoan coincubation and day 10 for bothtreatments where n = 4. **, significant (P < 0.01).

the medium is coincident with the depletion of lactate (seebelow).Formate is an end product that can be used as an indicator

of active fungal growth (23). Formate concentrations increasedthroughout the incubation period without statistical differencesbetween cultures (data not shown), but formate was notdetected at the end of the incubation period in culturescontaining protozoa (Table 1). Although in defaunated ani-mals decreased formate concentrations were detected (1),there is not much information about the metabolism of for-mate by rumen protozoa.

Lactate is another end product produced by anaerobic fungi(6, 7, 36). Piromyces sp. strain OTS1 produced predominantlyD-lactate (75 to 85%). In fungus-alone incubations, totallactate accumulated throughout the incubation period (Fig. 1and Table 1). Lactate metabolism by protozoa differs betweengroups. While holotrichs produce lactic acid, mainly L-lacticacid, as a fermentation product (32, 40), the entodiniomor-phids metabolize it (27). Lactate concentration in the coincu-bations showed no differences with fungus-alone incubationsuntil day 4 but decreased to basal level at day 7 of theexperimental period (Fig. 1). Although L-lactate is attackedmore rapidly than the D-isomer by entodiniomorphid protozoa(27), the proportion of L-lactate increased from 30% at day 1to 61% at day 10, probably reflecting holotrich protozoalactivity.

T'he amount of glucose in the medium increased in thefungal monocultures throughout the incubation. Glucose con-centration in the second half of the incubation period couldpartially repress fungal cellulase production (26). In the pres-ence of protozoa, glucose levels remained low; this may havefavored the activity of polysaccharidases (Fig. 2). Although theholotrichs represented a small percentage of the total proto-zoal population used, they rapidly assimilate and metabolizesoluble sugars (39) and probably played a major role in thisparticular parameter.The pH of the incubation medium decreased when the

fungus was incubated alone. From an initial 6.64 ± 0.01 at day1, it reached pH of 5.88 ± 0.04 after 10 days of incubation. Theaddition of the MB9 buffer solution probably prevented a

APPL. ENvIRON. MICROBIOL.

on Septem

ber 18, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

RUMINAL FUNGUS-PROTOZOAN INTERACTIONS IN VITRO 3721

10

8

6

4

E0o0)

EEa.

2

0

**

**

350

U1)

t-

Cc._

0.

300

250

200

1 50

100

50

00 2 4 6 8

Incubation Time (day)10

FIG. 2. Reducing sugars, expressed as micromoles of glucose per

milliliter, in the culture medium of the anaerobic ruminal fungusPiromyces sp. strain OTS1 incubated alone (-) or in the presence of a

mixed protozoal population obtained from a goat (0). Values are themean ± SD of six samples, except in Piromyces sp. strain OTS1-protozoan coincubation at day 10 where n = 4. **, significant (P <0.01).

further decrease of the pH. In Piromyces sp.-protozoan cul-tures, the pH decreased during the first half of the incubationand increased thereafter; at the end of the incubation, it was6.31 ± 0.06. This initial decrease was similar to that of fungusalone (Fig. 3). The MB9 buffer solution used to prepare theprotozoal suspensions also had a stabilizing effect on the pH;however, the increase detected in the second half of theincubation period has to be a consequence of the protozoa.

Effect of ruminal protozoa on the growth and degradation ofcellulose of Piromyces sp. strain OTS1. Fungal growth in themonoculture showed an exponential phase until day 4. Fromday 4 until the end of the incubation, a decelerated growthphase was recorded. When the fungus was incubated in the

7

6.5

Q 6

5.5

5

**

**

0 2 4 6 8 10

IncubationTime (day)FIG. 3. Changes in the pH of the culture medium throughout the

incubation period in the anaerobic ruminal fungus Piromyces sp. strainOTS1 incubated alone (-) or in the presence of a mixed protozoalpopulation obtained from a goat (0). Values are the mean ± SD of sixtubes. **, significant (P < 0.01).

**

0 2 4 6 8 10IncubationTime (day)

FIG. 4. Amount of chitin detected in cultures of the anaerobicruminal fungus Piromyces sp. strain OTS1 incubated alone (-) or inthe presence of a mixed protozoal population obtained from a goat(0). Culture medium contained filter paper cellulose as carbon source.Values are the mean ± SD of six samples, except at day 1 where n =5. **, significant (P < 0.01).

presence of protozoa, the exponential growth phase was notaffected, but after that, a steady decline in the amount of chitinwas observed, accounting for a 42% reduction at the end of theincubation period (Fig. 4). The amount of chitin detected atday 10 of the incubation in the fungal monocultures was morethan twice that of the Piromyces sp. protozoan coincubations.The fungal zoospores that were inoculated into the medium

at the onset of the incubation period developed into zoospor-angia before the first protozoal inoculation and were probablyresponsible for the similar exponential growth phase observedin both incubations. This indicates that fungal growth wasaffected by protozoal predation on zoospores. Indeed, thenumber of zoospores was smaller in the coincubations than inthe fungus-alone incubations, although the counts were toolow, and varied too much among the same group of samples, tobe evaluated statistically. The sharp decline in the amount ofchitin detected in Piromyces sp.-protozoan cultures from day 4until the end of the incubation, however, cannot be attributedsolely to autolysis (unpublished data), and predation of fungalcells had to take place. Protozoal ingestion of Piromycescommunis rhizoids and sporangia (20) and the fact that thesemicroorganisms possess chitinases along with other enzymescapable of degrading fungal cell walls (25) support this obser-vation. Utilization of nutrients in the incubation medium byprotozoa was another factor likely to contribute to the reducedfungal growth in the coincubations.

Figure 5 shows the percentage of dry matter (cellulose)disappearance. In monocultures, it progressed linearly until itreached a 50% reduction at day 10 of the incubation. Piromycessp. strain OTS1 showed less degradation ability than otherPiromyces strains (6, 7, 36). The results can be attributed to thedifferences of the culture media and to the fact that the isolate,which was not primarily selected by its cellulolytic activity, wasnot previously adapted to the filter paper medium. In fungus-protozoan cultures, until day 4 of the incubation the disappear-ance was similar to that of the Piromyces sp. strain OTS1monocultures, but from day 4 until the end of the incubation,the rate of disappearance was lower. Dry matter disappearancewas about 35% at day 10 of incubation (Fig. 5). In optimization

VOL. 60, 1994

on Septem

ber 18, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

3722 MORGAVI ET AL.

0)c 50

coCD 40cL.*0^3

a)0U 2

CL5c30

0-2

1 0

0

**

c

'3O0._

CLu

iL

= acn._-

0.2

0.16

0.12

0.08

0.04

0

0 2 4 6 8Incubation Time (day)

10

FIG. 5. Degradation of filter paper cellulose by the anaerobicruminal fungus Piromyces sp. strain OTS1 incubated alone (U) or inthe presence of a mixed protozoal population obtained from a goat(0). Values are the mean ± SD of six samples, except at day 1 wheren = 5. *, significant (P < 0.05); **, significant (P < 0.01).

experiments, protozoa alone did not degrade cellulose underthe conditions studied. Similar results were reported withmedium-sized (30 to 70 jim) protozoa (reference 20a, cited inreference 14).

It is known that the enzymatic activities of fungi, combinedwith the particular penetrating growth of the rhizoidal system,lead to weakening and particle size reduction of plant fiber (2,31). We tried to check whether Piromyces sp. strain OTS1 alsoproduced small filter paper particles that could be ingested byprotozoa. Suspended cellulose particles in the medium in-creased throughout the incubation in Piromyces cultures: 110

15, 86 ± 18, 210 ± 66, and 290 ± 28 jig per tube ± standarddeviation (SD) at 1, 4, 7, and 10 days of incubation, respec-tively. This increment was also noted under direct microscopicexamination. In Piromyces sp.-protozoan cultures, values wereerratic, probably because ciliates were included in the samples.However, at the end of the incubation period, when protozoawere not likely to influence the sample, amounts ot celluloseparticles suspended in the coincubations were lower comparedwith cultures without protozoa (150 ± 10 versus 290 ± 28 jigper tube). Though the amount of suspended cellulose was lessthan 1% of the total, small cellulose particles are readilyingested by protozoa (11). In the rumen, this kind of positiveinteraction can be enhanced by the mechanical effects ofrumination on fungus-weakened plant fragments.The amount of cellulose degraded per unit of chitin in-

creased throughout the incubation time in both cultures (Fig.6). Cultures of Piromyces sp. strain OTS1 alone degraded 160jig of cellulose per jig of chitin by the end of the incubationperiod. In the presence of protozoa, 202 jig of cellulose per jigof chitin was degraded, showing a 25% increase over fungus-alone incubations.Rumen protozoa adversely affected the growth of the Piro-

myces sp. strain OTS1 in vitro, and the total amount ofcellulose degraded was smaller in fungus-protozoan coincuba-tions. This negative effect seems to be mainly a consequence ofthe predatory activity of the protozoa. Predation on zoosporeswas indirectly confirmed, and there was evidence that theprotozoa also affected mature zoosporangia in some ways; one

of these can be the degradation of fungal cell walls by

0 2 4 6 8Incubation Time (day)

10

FIG. 6. Degradation of cellulose as a percentage of filter paperdisappearance per unit of fungal biomass in Piromyces sp. strain OTS1incubated alone (U) or in the presence of a mixed protozoal popula-tion obtained from a goat (0).

protozoal enzymes (25). On the other hand, the amount ofcellulose degraded per unit of chitin was greater in thecoincubations. This could be attributable to protozoal fibrolyticactivity or to the fact that the utilization of fungal degradationproducts by the ciliates may have resulted in an enhancedfungal activity. This positive interrelationship can becomemore prominent with the more complex plant substrates,because both protozoa and fungi ferment hemicellulose moreeffectively than cellulose (14, 38) and because of the effect ofholotrichs on soluble sugars. The number of large cellulolyticentodiniomorphids, only 2 to 4% of the total in this study, isalso likely to influence the amount of cellulose degraded.Although the total amount of cellulose degraded by Piromy-

ces sp. strain OTS1 was adversely affected by the mixedprotozoal population studied as a result of predation, a syner-getic interaction was also detected. It remains to be investi-gated whether the negative effects of protozoa on fungalgrowth can be attenuated by reducing the number and manip-ulating the type of protozoa in the incubation, resulting in anoverall improvement of the amount of cellulose degraded.

ACKNOWLEDGMENT

Diego P. Morgavi thanks the Ministry of Education, Science, andCulture of Japan (Monbusho) for the award of a research studentship.

REFERENCES1. Abou-Akkada, A.R., and K. El-Shazly. 1964. Effect of absence of

ciliate protozoa from the rumen on microbial activity and growthof lambs. Appl. Microbiol. 12:384-390.

2. Akin, D. E., G. L. R. Gordon, and J. P. Hogan. 1983. Rumenbacterial and fungal degradation of Digitaria pentzii grown with orwithout sulfur. Appl. Environ. Microbiol. 46:738-748.

3. Akin, D. E., and L. L. Rigsby. 1987. Mixed fungal populations andlignocellulosic tissue degradation in the bovine rumen. Appl.Environ. Microbiol. 53:1987-1995.

4. Barr, D. J. S., H. Kudo, K. D. Jakober, and K.-J. Cheng. 1989.Morphology and development of rumen fungi: Neocallimastix sp.,Piromyces communis and Orpinomyces bovis gen. nov., sp. nov.Can. J. Bot. 67:2815-2824.

5. Bauchop, T., and D. 0. Mountfort. 1981. Cellulose fermentationby a rumen anaerobic fungus in both the absence and the presenceof rumen methanogens. Appl. Environ. Microbiol. 42:1103-1110.

6. Bernalier, A., G. Fonty, F. Bonnemoy, and P. Gouet. 1992.Degradation and fermentation of cellulose by the rumen anaero-

APPL. ENvIRON. MICROBIOL.

on Septem

ber 18, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

RUMINAL FUNGUS-PROTOZOAN INTERACTIONS IN VITRO 3723

bic fungi in axenic cultures or in association with cellulolyticbacteria. Curr. Microbiol. 25:143-148.

7. Bernalier, A., G. Fonty, and P. Gouet. 1991. Cellulose degradationby two rumen anaerobic fungi in monoculture or in coculture withrumen bacteria. Anim. Feed Sci. Technol. 32:131-136.

7a.Boyne, A. W., J. M. Eadie, and K. Raitt. 1957. The developmentand testing of a method of counting rumen ciliate protozoa. J.Gen. Microbiol. 17:414-423.

8. Caldwell, D. R., and M. P. Bryant. 1966. Medium without rumenfluid for nonselective enumeration and isolation of rumen bacte-ria. Appl. Microbiol. 14:794-801.

9. Chen, G. C., and B. R. Johnson. 1983. Improved colorimetricdetermination of cell wall chitin in wood decay fungi. Appl.Environ. Microbiol. 46:13-16.

10. Coleman, G. S. 1962. The preparation and survival of almostbacteria-free suspensions of Entodinium caudatum. J. Gen. Micro-biol. 28:271-281.

11. Coleman, G. S. 1992. The rate of uptake and metabolism of starchgrains and cellulose particles by Entodinium species, Eudiplo-dinium maggii, some other entodiniomorphid protozoa and natu-ral protozoal populations taken from the ovine rumen. J. Appl.Bacteriol. 73:507-513.

12. Dygert, S., L. H. Li, D. Florida, and J. A. Thoma. 1965. Determi-nation of reducing sugar with improved precision. Anal. Biochem.13:367-374.

13. Eadie, J. M. 1962. The development of rumen microbial popula-tions in lambs and calves under various conditions of management.J. Gen. Microbiol. 29:563-578.

14. Fonty, G., and K. N. Joblin. 1991. Rumen anaerobic fungi: theirrole and interactions with other rumen microorganisms in relationto fiber digestion, p. 655-680. In T. Tsuda, Y. Sasaki, and R.Kawashima (ed.), Physiological aspects of digestion and metabo-lism in ruminants. Academic Press, Inc., San Diego, Calif.

15. Ho, Y. W., D. J. S. Barr, N. Abdullah, S. Jalaludin, and H. Kudo.1993. A new species of Piromyces from the rumen of deer inMalaysia. Mycotaxon 47:285-293.

16. Holdeman, L. V., E. P. Cato, and W. E. C. Moore. 1977. Anaerobelaboratory manual, 4th ed. Virginia Polytechnic Institute and StateUniversity, Blacksburg.

17. Hungate, R. E. 1969. A roll tube method for cultivation of strictanaerobes, Methods Microbiol. 3B:117-132.

18. Irvine, H. L., and C. S. Stewart. 1991. Interactions betweenanaerobic cellulolytic bacteria and fungi in the presence of Meth-anobrevibacter smithii. Lett. Appl. Bacteriol. 12:62-64.

19. Joblin, K. N. 1981. Isolation, enumeration, and maintenance ofrumen anaerobic fungi in roll tubes. Appl. Environ. Microbiol.42:1119-1122.

20. Joblin, K. N. 1990. Bacterial and protozoal interactions withruminal fungi, p. 301-309. In D. E. Akin, L. G. Ljungdahl, J. R.Wilson, and P. H. Harris (ed.), Microbial and plant opportunitiesto improve lignocellulose utilization by ruminants. Elsevier Sci-ence Publishing Co., Inc., New York.

20a.joblin, K. N., and G. E. Naylor. Unpublished results.21. Jouany, J. P., and K. Ushida. 1990. Protozoa and fibre digestion in

the rumen, p. 139-150. In S. Hoshino, R. Onodera, H. Minato, andH. Itabashi (ed.), The rumen ecosystem. The microbial metabo-lism and its regulation. Japan Scientific Societies Press, Tokyo.

22. Li, J., B. Heath, and T. Bauchop. 1990. Piromyces mae andPiromyces dumbonica, two new species of uniflagellate anaerobicchytridiomycete fungi from the hindgut of the horse and elephant.Can. J. Bot. 68:1021-1033.

23. Lowe, S. E., M. K. Theodorou, and A. P. J. Trinci. 1987. Cellulasesand xylanase of an anaerobic rumen fungus grown on wheat straw,wheat straw holocellulose, cellulose, and xylan. Appl. Environ.Microbiol. 53:1216-1223.

24. Morgavi, D. P., R. Onodera, and T. Nagasawa. 1993. In vitrometabolism of chitin and protein from ruminal fungi by ruminalprotozoa. Anim. Sci. Technol. 64:584-592.

25. Morgavi, D. P., M. Sakurada, Y. Tomita, and R. Onodera. 1994.Presence in rumen bacterial and protozoal populations of enzymescapable of degrading fungal cell walls. Microbiology 140:631-636.

26. Mountfort, D. O., and R. A. Asher. 1989. Production of polysac-charides by the rumen anaerobic fungi from the rumen, p.139-144. In J. V. Nolan, R. A. Leng, and D. I. Demeyer (ed.), Theroles of protozoa and fungi in ruminant digestion. PenambulBooks, Armidale, Australia.

27. Newbold, C. J., A. G. Williams, and D. G. Chamberlain. 1987. Thein-vitro metabolism of D,L-lactic acid by rumen microorganisms. J.Sci. Food Agric. 38:9-18.

28. Onodera, R., and C. Henderson. 1980. Growth factors of bacterialorigin for the culture of the rumen oligotrich protozoon, Ento-dinium caudatum. J. Appl. Bacteriol. 48:125-134.

29. Onodera, R., H. Yamaguchi, C. Eguchi, and M. Kandatsu. 1977.Limits of survival of the mingled rumen bacteria in the washed cellsuspension of rumen ciliate protozoa. Agric. Biol. Chem. 41:2465-2466.

30. Orpin, C. G. 1977. The rumen flagellate Piromonas communis: itslife-history and invasion of plant material in the rumen. J. Gen.Microbiol. 99:107-117.

31. Orpin, C. G. 1984. The role of ciliate protozoa and fungi in therumen digestion of plant cell walls. Anim. Feed Sci. Technol.10: 121-143.

32. Prins, R. A., and W. VanHoven. 1977. Carbohydrate fermentationby the rumen ciliate Isotnicha prostoma. Protistologica 13:549-556.

33. Roger, V., E. Grenet, J. Jamot, A. Bernalier, G. Fonty, and P.Gouet. 1992. Degradation of maize stem by two rumen fungalspecies, Piromyces communis and Caecomyces communis, in purecultures or in association with cellulolytic bacteria. Reprod. Nutr.Dev. 32:321-329.

34. Sleat, R., and R. A. Mah. 1984. Quantitative method for colori-metric determination of formate in fermentation media. Appl.Environ. Microbiol. 47:884-885.

35. Snedecor, G. W., and W. G. Cochran. 1980. Statistical methods,7th ed. The Iowa State University Press, Ames.

36. Teunissen, M. J., A. A. M. Smits, H. J. M. Op den Camp, J. H. J.Huis in't Veld, and G. D. Vogels. 1991. Fermentation of celluloseand production of cellulolytic and xylanolytic enzymes by anaero-bic fungi from ruminant and non-ruminant herbivores. Arch.Microbiol. 156:290-296.

37. Updegraff, D. M. 1969. Semimicro determination of cellulose inbiological material. Anal. Biochem. 32:420-424.

38. Ushida, K., J. P. Jouany, and D. I. Demeyer. 1991. Effects ofpresence or absence of rumen protozoa on the efficiency ofutilization of concentrate and fibrous feeds, p. 625-654. In T.Tsuda, Y. Sasaki, and R. Kawashima (ed.), Physiological aspects ofdigestion and metabolism in ruminants. Academic Press, Inc., SanDiego, Calif.

39. Williams, A. G., and G. S. Coleman. 1992. The rumen protozoa.Springer-Verlag New York Inc., New York.

40. Williams, A. G., and C. G. Harfoot. 1976. Factors affecting theuptake and metabolism of soluble carbohydrates by the rumenciliate Dasitricha ruminantium isolated from ovine rumen contentsby filtration. J. Gen. Microbiol. 96:125-136.

41. Williams, A. G., and S. E. Withers. 1993. Changes in the rumenmicrobial population and its activities during the refaunationperiod after the reintroduction of ciliate protozoa into the rumenof defaunated sheep. Can. J. Microbiol. 39:61-69.

42. Williams, A. G., S. E. Withers, and K. N. Joblin. 1991. Xylanolysisby cocultures of the rumen fungus Neocallimastix frontalis andruminal bacteria. Lett. Appl. Bacteriol. 12:232-235.

VOL. 60, 1994

on Septem

ber 18, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from