FEMS Microbiology Reviews 46 (1987) 401-408
Published by Elsevier
401
FER 00079
The rumen anaerobic fungi
D o u g l a s O. M o u n t f o r t
Cawthron Institute, Nelson, New Zealand
Received 12 April 1987
Accepted 18 May 1987
Key words: Anaerobic fungi; Rumen; Plant structural material; Degradation; Fermentation
1. SUMMARY
The anaerobic fungi represent a new group of
organisms inhabiting the rumen ecosystem and
possess a life cycle alternating between a motile
flagellated form (zoospore) and a non-motile
vegetative reproductive form (thallus). In vivo
studies show extensive colonization of plant
material suspended in the rumen indicating the
fungi have a role in fiber digestion. Pure cultures
of anaerobic fungi ferment cellulose to give lactate,
acetate, CO 2 and H 2 as the major products.
Ethanol and formate may also be produced. Fermentation of cellulose by the fungi in coculture
with H2-utilizing methanogens results in a shift in
the fermentation pattern favouring the production
of H 2 (utilized in the formation of CH4) and
acetate at the expense of the electron-sink products, lactate and ethanol. It is postulated that the
methanogens in reducing the partial pressure of
H 2, facilitate an increased passage of reducing
equivalents towards the production of H 2 via a
pyridine-nucleotide (PN)-linked hydrogenase reaction. H 2 is believed to be produced in microbodies
of the fungi called hydrogenosomes which possess
all of the enzymes necessary for this function
Correspondence to: D.O. Mountfort, Cawthron Institute, Nelson, New Zealand.
including PN-linked hydrogenase. Absence of
mitochondria and key electron transport components in these organisms indicate a dependence
wholly on fermentative processes for growth.
Anaerobic fungi also participate in hemicellutose
and starch degradation but it is not yet clear
whether they have a role in the degradation of
lignin. Simple sugars (mono- and disaccharides)
are readily utilized and their uptake is subject to
similar regulatory constraints such as is found
with other micro-organisms.
Enzymological studies have revealed that
anaerobic fungi release substantial amounts of
endo-acting cellulase and protease, possibly giving
them a competitive advantage over rumen bacteria
in the degradation of plant structural material.
2. I N T R O D U C T I O N
Until recently it was believed that metabolic
transformations in the rumen were mediated by
bacteria and protozoa, and fungi were not considered as inhabitants of this ecosystem. However,
there have been developments from in vivo and in
vitro studies leaving little doubt that anaerobic
fungi make a significant contribution to rumen
metabolism, particularly in the digestion and subsequent fermentation of plant structural materials.
There have also been important advances in the
0168-6445/87/$03.50 © 1987 Federation of European Microbiological Societies
402
understanding of the life cycle mechanism of these
organisms and the controlling factors involved.
This review does not attempt to deal with all of
the recent developments in the field, but rather
focusses on those studies which have contributed
to a clearer understanding of the role of the fungi
in the rumen environment. Consequently those
physiological and morphological features pertinent to the anaerobic lifestyle of these organisms
will be discussed together with the evidence supportive of a role in digestion and fermentation of
plant structural components. It is anticipated that
this review should provide the basis for a reevaluation of previously held concepts concerning degradative processes in the rumen.
Liebetanz [1] and Braune [2]. They were essentially regarded as protozoa and received little attention until the studies of Orpin. Orpin [3] demonstrated that when an inducer obtained by extracting crushed oats, was added to the rumen
there was a decrease in the population density of
sporangial forms and an increase in the number of
flagellates. When a rumen fraction containing
sporangia was taken, then placed on a microscope
slide at 39°C, flagellates were liberated from
sporangia between 15 and 45 min after addition of
inducer. Later, Orpin [4] demonstrated that addition of inducer in vivo resulted in a two-stage
decrease in the population density of sporangia
coupled with a two-phase increase in the number
of flagellated forms. This was verified by further
in vitro studies with sporangial preparations which
showed an early release of flagellates 30 min after
addition of inducer, followed by a later release at
60 min. The flagellates from the first and second
phases were identified as the motile forms of
Neocallimastix frontalis and Sphaeromonas corn-
3. E V I D E N C E FOR T H E PRESENCE OF
F U N G I IN T H E R U M E N A N D T H E I R
L I K E L Y ROLE
Rumen flagellates possessing a spherical-toovoid cell with flagella were first described by
~
lmm
im
m
m
u
mm
mm
~
S
()
/I/flltilllll[ll/llff~
T/1//////H/1/////,
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Fig. 1. Diagrammatic representation of thallus development, release, and attachment of motile zoospores to plant material surface
envisaged for the life cycle of anaerobic fungi.
403
munis, respectively. Based on the in vivo and in
vitro observations it was proposed that these
organisms were fungi rather than protozoa, and
that they possessed a life cycle alternating between
a motile flagellated form (zoospore) and a nonmotile vegetative, reproductive form (thallus consisting of a rhizoid bearing sporangia) [3,4], as
described in Fig. 1. Both N. frontalis and S.
communis were subsequently isolated and cultured
in undefined medium and were found to have
differing morphologies in their flagellate and
vegetative stages [3,4]. Later a third fungus was
described ( Piromonas communis) having a life cycle
similar to that of the previous two isolates but
with a larger flagellated form than S. communis
[5]. All three organisms were shown to contain
chitin in their cell walls [6].
Further in vivo studies revealed that plant
material suspended in the rumen in nylon bags
~
was extensively colonized by rumen fungi [5,7-9]
as evident from development of sporangia, and
the principal sites of invasion were on damaged
tissue. Flagellated forms identified as those of N.
frontalis and P. comrnunis were released from
sporangia when partially digested plant material
from nylon bags removed from the rumen, was
treated with inducer [5,7].
Because of the widespread colonization of plant
material by fungi it was suggested that they had a
role in fiber digestion perhaps as the initial colonizers in lignocellulose digestion [8-10]. Akin et
al. [11] observed that anaerobic fungi were prevalent in the rumen of sheep fed on the forage,
Digitaria pentzii which had been fertilized with
sulfur. Nylon bag trials showed extensive colonization of lignified cells of leaf blade sclerenchyma
tissue by fungi and it was likewise suggested that
these organisms had a role in lignocellulose di-
Hexoae
NAD
ox.,o.!2/"
~
~
Lactate
NAD
~ ~"~i~ NADH
Malate
Pyruvete
CoA
Malic
-enzyme ~
.
Pyruvate
MaIaIe ~ C O ~
NADPH
NADP
/
C02
' ,,.
----___
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..
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," ~ ' ~
....
Acetyl-CoA
I pyruvate'-FO [
[ oxidoreductaae]
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H~
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Fig. 2. Proposed scheme for the production of H 2 and other products from hexose fermentation by N. patriciarum (after Yarlett et al.
[19]). Enclosed area refers to reactions occurring within the hydrogenosome.
404
gestion. The importance of anaerobic fungi in
fiber digestion was illustrated by in vitro digestion
trials which showed that in the absence of actively
growing bacteria, they could remove about 60% of
forage material [11].
4. C E L L U L O S E F E R M E N T A T I O N
MEN ANAEROBIC F U N G I
BY RU-
Isolation of pure cultures of anaerobic fungi
enabled definitive studies to be carried out on the
digestion of cellulose by these organisms. Colonization of Whatman No. 1 filter paper strips was
demonstrated in several in vitro systems and this
was found to be associated with extensive pitting
of the paper [8,12]. Cellulosic digestion was
quantitated in time-course experiments which
showed that growth of N. frontalis on filter paper
strips was accompanied by a decrease in cellulose,
and at full growth, 50-70% of the initial cellulose
(100 m g / 1 2 ml of culture medium) had been
utilized [12,13].
The first comprehensive study on cellulose fermentation by a rumen fungus was carried out by
Bauchop and Mountfort [13]. They showed that
N. frontalis fermented cellulose to give 6 products.
Moles product as a percent of moles cellulosic
hexose were: acetate 72.7; carbon dioxide, 37.6;
formate, 83.1; ethanol 37.4; lactate, 67.0 and H 2,
35.3. This mixed acid type fermentation is similar
to that for coliform bacteria growing anaerobically
on glucose. Similar fermentations have been demonstrated with other isolates of rumen fungi, however in some cases ethanol a n d / o r formate were
not produced [14].
A characteristic of all rumen fungal fermentations reported so far is the production of H 2. The
hydrogen-producing system of the fungi may be
similar to that of other rumen H 2 producers because coculture with H2-utilizing methanogens
produced a shift in the fermentation pattern towards the formation of more oxidized products
such as acetate [13], as had been previously observed with rumen bacterial H 2 producers in
methanogenic coculture [15,16]. It has been postulated that reducing equivalents otherwise used in
the production of electron-sink products such as
lactate and ethanol in monoculture, are in cocultures, diverted to CH 4 via H 2 in a pyridinenucleotide (PN)-linked hydrogenase reaction
which becomes favourable through a reduction in
the tt 2 partial pressure by the methanogens [17].
The site of H 2 production in the rumen fungi may
be on intracellular organelles called hydrogenosomes [18,19]. These structures are distinct from
mitochondria and bear a similarity in morphology
to H2-evolving organelles possessed by certain
anaerobic protozoa [20,21]. Hydrogenase, pyruvate : ferredoxin oxidoreductase, N A D P H : ferredoxin oxidoreductase, and 'malic' enzyme were
found to be enriched in a hydrogenosomal fraction obtained from Neocallimastix patriciarum
which is closely related to N. frontalis, and it was
proposed that these enzymes were involved in H 2
production according to the scheme in Fig. 2 [19].
Absence of glucose-6-phosphate dehydrogenase
activity together with the demonstration of the
presence of key glycolytic enzymes in cell-free
extracts led to the suggestion that glycolysis was
the sole mechanism for hexose fermentation in
this organism [19]. Surprisingly the enzymes of
hexose fermentation in N. frontalis have not yet
been studied, but it is probable that the pathways
are similar to those outlined for N. patriciarum.
Electron transport components such as cytochromes and menaquinone have not been detected
in anaerobic fungi (J. Macy, personal communication) and microscopic studies suggest that
mitochondria are absent [18,22]. It therefore appears that these organisms are wholly dependent
upon fermentative processes for growth.
5. D E G R A D A T I O N OF O T H E R PLANT FIBER
C O M P O N E N T S BY R U M E N ANAEROBIC
FUNGI
Anaerobic fungi appear to have a role in the
degradation of other fiber components apart from
cellulose. Mountfort et al. [23] demonstrated that
more
CH 4 was produced than could be accounted
for by cellulose during the fermentation of barley
straw leaf strips by N. frontalis in coculture with
methanogens, indicating that another component,
perhaps hemicellulose, was also fermented. The
405
h.emicellulose, xylan, has been shown to support
growth of N. frontalis [12], but the fermentation of
this substrate has not been investigated. It is probable however that the range of fermentation products is similar to that obtained from cellulose
fermentation by this organism. N. frontalis has
been tested for its ability to grow on pectin and
polygalacturonic acid and neither substrate was
found to be utilized [12,24].
Because of the repeated suggestions made by
various workers concerning the role of anaerobic
fungi in ligno-cellulose digestion [8-10,11,23], the
possibility that these organisms degrade lignin has
attracted interest. Studies with 10-ml cultures of
N. frontalis grown on 0-100 mg Whatman No. 1
filter paper strips in the presence of 1 - 2 mg
[fl-y-14C]lignin (sp, act. 3.4 × 10 4 dpm •mg -1) or
[U-14C]lignin (sp. act. 5 × 104 d p m . m g - 1) showed
that although active growth had occurred on the
cellulose (full growth, 6 - 7 days), < 0.05% of the
label from lignin was incorporated into volatile
fatty acids and CO 2 after 6 weeks incubation and
no more than 5% was detected in the soluble
(30000 × g) fraction of the culture fluid [25]. The
result suggested that N. frontalis did not have an
active role in lignin degradation.
Akin [26] likewise suggested that anaerobic
fungi may not degrade lignin on the basis of
observations from in vitro fiber degradation experiments carried out (i) in the absence, and (ii)
in the presence of antibiotics used to inhibit
bacterial growth. No appreciable change was found
in the amount of lignin degraded between the two
types of system. Lignin degradation by anaerobic
fungi was also considered unlikely because fiber
degradation by these organisms was inhibited by
the presence of 0.1% p-coumaric, sinapic and ferulic acids [27].
6. E N Z Y M E S R E L E A S E D BY N. frontalis INVOLVED IN THE DEGRADATION
OF
PLANT STRUCTURAL COMPONENTS
6.1. Cellulase
Subsequent to the studies showing that
anaerobic fungi could ferment cellulosic materials,
investigation of their cellulases represented a new
area of interest. It was considered that the cellulases might possess unique properties in facilitating deep mycelial penetration and rapid colonization of plant material by the fungi giving them a
competitive advantage over bacteria. N. frontalis
growing on Whatman No. 1 filter paper strips was
shown to release relatively large amounts of
carboxymethylcellulase (CMCase) into the culture
fluid and activities of up to 170 U . m1-1 (IU
represents 1/~g glucose equivalents r e l e a s e d / m i n )
were obtained for cultures grown on 2.5 g cellulose-m1-1 [28]. As much as 10.4 × 104 U of
C M C a s e - g - 1 of cellulosic substrate fermented
could be obtained at full growth in cultures with
an initial cellulose concentration of 0.6 mg - m l - ~.
Elevation of cellulose in the culture medium led to
a decline in CMCase yield which was accompanied by an accumulation of glucose. Subsequently
it was demonstrated that elevated levels of glucose
repressed CMCase production [28]. In contrast to
the production of CMCase, that of exoglucanase,
determined as the activity of culture fluid towards
alkali-treated Avicel or Sigmacell micro-crystalline
cellulose, was low (maximal activity about 100-fold
less than CMCase) and treatment of fungal rhizoid
with various solubilizing agents failed to give an
increase in activity [28]. A possible explanation for
these results could have been that neither Sigmacell or Avicel was suitable for measurement of
exoglucanase activity. Therefore to closer approximate 'true' exoglucanase activity, cellooligosaccharide (an oligomer mixture, chain length,
> 6) prepared by partial hydrolysis of cellulose,
was used as a substrate. Activity of the culture
fluid towards this substrate was found to be 6-fold
higher than towards Avicel or Sigmacell, indicating that it could have been the more appropriate
in the measurement of exoglucanase [28]. However, it was also conceivable that N. frontalis
produced several types of exoglucanase. Possession of a ' b a t t e r y ' of these enzymes by the fungi
might give them an ecological advantage in the
digestion and subsequent fermentation of cellulosic fiber in the rumen. An additional competitive advantage might be gained from the release of
substantial amounts of endo-acting cellulase such
as demonstrated with N. frontalis [28]. One consequence would be the production of a range of
406
short-chained oligosaccharides which would be
freely available for utilization by other rumen
microbiota, including bacteria. Thus it might be
envisaged that after primary attack on plant
material by the fungi there would be a secondary
attack on partially digested carbohydrate involving other organisms in a similar way to that described for rumen interbacterial systems [29].
6.2. Proteases
Proteolytic activity has been determined in the
culture fluid of a strain of N. frontalis, and it
appears to be due to a metalloprotease since activity was inhibited by metal chelators such as
phenanthroline and E D T A [30]. Inhibition by
E D T A could be reversed by addition of Zn 3+,
Cu 2+ or Co 2+. It was suggested that the protease(s)
functioned either (i), in the provision of amino
acids for growth, (ii), in the modification of the
activities of other enzymes (i.e. cellulases), or (iii),
in the degradation of plant structural protein
thereby facilitating invasion of plant tissue by
fungal rhizoid. In vitro studies in which N. frontalis was added to a mixed rumen bacterial culture
(including the important proteolytic strains) fermenting solid substrate, indicated that the fungi
may contribute up to 50% the total proteolytic
activity in the rumen [31]. It is of interest that the
more important rumen cellulolytic bacteria are not
actively proteolytic, while the rumen fungi possess
both of these functions. Thus besides the characteristic properties of their cellulases (Section
6.1), the combined cellulolytic-proteolytic functions of the rumen fungi may represent an additional property which provides them with an
advantage over competing bacteria, particularly if
the fungal proteases function in degradation of
plant structural protein.
6.3. Other fibre degrading enzymes
Although it is known that anaerobic fungi can
attack structural plant polysaccharide apart from
cellulose very little study has been directed towards the enzymes involved. An enzyme preparation from N. frontalis has been shown to attack
xylan initially producing oligomers of chain length,
3-10, and subsequently xylose, indicative of a
hydrolysis proceeding first via xylanase then fl-D-
xylosidase [24]. The characteristics of these enzymes have not been investigated. Similarly, there
has been no investigation of the enzymes involved
in starch degradation by anaerobic fungi.
7. U T I L I Z A T I O N OF S O L U B L E CARBOH Y D R A T E S BY A N A E R O B I C F U N G I A N D
THE REGULATORY
MECHANISMS INVOLVED
Most studies attempting to provide a better
understanding of the role of fungi in the rumen
ecosystem have been directed towards utilization
of plant fiber components by these organisms.
However, the observation by Orpin and Letcher
[12] that rumen fungi do not digest cellulose in the
presence of glucose, coupled with studies showing
repression of cellulase by glucose [28] suggest that
in vivo, soluble carbohydrate may be preferentially utilized. N. frontalis has been shown to
grow actively on cellobiose, D-fructose, D-xylose,
maltose, sucrose, and glucose giving growth yields
of 40-50 mg dry w t . . m m o l of hexose ~ and 20
m g . mmol pentose 1 [32]. Studies on the utilization of paired substrates indicated that the uptake
of fructose and xylose was inhibited by glucose by
a catabolic regulatory mechanism [32]. It is not
clear what the role for catabolite regulation is in
anaerobic fungi but one possibility is that it provides a means by which direct competition with
other organisms for the same substrate can be
removed in situations where there is an overlapping range of substrates in critical stages of the
host animal's feeding cycle. However, in the rumen the levels of soluble carbohydrate are usually
very low [29] reflecting rapid utilization due to
competition by various microflora. Thus it may be
important to determine whether the regulatory
mechanisms observed in the in vitro studies also
play a role under substrate-limiting conditions
such as occurs in the rumen. Regulation of the
utilization of one simple sugar by another might
also effect the degradation of various plant fiber
components by anaerobic fungi. Thus the inhibition of xylose utilization by glucose may lead to
accumulation of xylose which would in turn repress xylan breakdown in a similar way to repres-
407
sion of cellulase by glucose [28]. This possible
'secondary regulatory effect' should merit further
investigation.
8. C O N C L U D I N G R E M A R K S
The rumen anaerobic fungi represent a new
group within the fungal kingdom and it is perhaps
surprising that so much time has elapsed before
they attracted the more widespread attention of
researchers. Physiologically they can be grouped
with the few species of bacteria which participate
in the digestion and fermentation of plant fiber in
the rumen. Whether the fungi are truely primary
invaders of plant tissue facilitating secondary attack by fermentative bacteria is open to question.
However, since they release substantial amounts
of endo-acting cellulase and protease [28,30] this
most likely provides the means by which they can
readily attack plant structural material. It should
be recognised however that at least in the case of
N. frontalis the pattern of cellulase production is
similar to that observed for several rumen cellulolytic bacteria [33].
The role of anaerobic fungi in the degradation
of hemicelluloses has received meagre attention
and much more information is required in the
elucidation of the fermentation patterns and the
enzymes involved. Based on the findings from a
limited number of in vitro studies [25,26] it appears that anaerobic fungi are unlikely to play an
important role in lignin degradation. This would
remove one possibility which could otherwise explain any advantages these organisms have over
competing bacteria in fiber digestion.
The estimation of the contribution of the
anaerobic fungi to overall rumen metabolism still
needs to be assessed. In vivo determination of
fungal biomass and associated enzymatic activities
would be required and this would not be without
difficulties particularly in situations where there is
extensive penetration of plant tissues by fungal
rhizoid. The nature of the life cycle of these
organisms would pose additional problems.
Perhaps the most interesting features of the
rumen fungi reside in (i), their simple life cycle;
(ii), the absence of certain structural organelles
such as mitochondria and (iii), the presence of
hydrogenosomes. All three reflect the unique
adaptation of these organisms to an anaerobic
lifestyle. Possession of hydrogenosomes appears
central to the production of H 2 and further studies should be conducted to determine the significance of these organelles to other aspects of
metabolism including energy conservation.
Finally, the characteristics of anaerobic fungi
raise new questions concerning their evolution and
perhaps more importantly, the evolution of fungi
in general. In future years there should be some
interesting debate concerning the position that
this group occupies in the fungal evolutionary
time-scale.
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