FEMS Microbiology Ecology 85 (1991) 169-180 © 1991 Federation of European Microbiological Societies 0168-6496/91/$03.50 Published by Elsevier ADONIS 0168649691000708

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FEMSEC 00325

An anaerobic protozoon, with symbiotic methanogens, living in municipal landfill material

Bland J. F in lay i and T o m Fenche l 2

l Institute of Freshwater Ecology, The Ferry House, A rnbleside, Cumbria, U.K., and 2 Marine Biological Laboratory, University of Copenhagen, Helsingor, Denmark

Received 19 September 1990 Revision received and accepted 24 November 1990

Key words: Cyst; Ciliate; Metopuspalaeforrnis; Polymorphic life cycle; Methanogen; Landfill

1. SUMMARY

We have established that anaerobic protozoa do live in municipal landfill material although they probably spend much of the time encysted, especially in the drier ( < 40% water) sites. At least eight species were observed; they were readily isolated by adding anoxic water to dry landfill samples. The ciliate Metopus palaeformis was fre- quently isolated; it appears to be ubiquitous in anaerobic landfills. It has a polymorphic life cycle, it is positive for hydrogenase, each ciliate contains about 500 bromoethanesulfonate-sensitive meth- anogen symbionts (probably Methanobacterium formicicum), and maximum cell densities in cul- ture exceed 3000 per ml. The methanogens are not attached to the hydrogenosomes, neither do they undergo morphological transformation; the ciliate receives no measurable energetic advantage from its symbionts. The ciliate encysts in response to a

Correspondence to: B.J. Finlay, Institute of Freshwater Ecol- ogy, The Ferry House, Far Sawrey, Ambleside, Cumbria LA22 0LP, U.K.

shortage of food or water, and the methanogens remain viable within the cysts, When the proto- zoon excysts, the methanogens resume growth and cell division within the trophic form of the ciliate. Unlike free-living methanogens, the M. palae- formis -me thanogen consortium is not particularly sensitive to oxygen; the symbiotic methanogens remain viable following exposure of the con- sortium to atmospheric oxygen for several days. Dispersal of methanogen-bearing protozoan cysts through oxygenated environments is a potential mechanism of transfer between landfill sites and other anaerobic environments. Anaerobic proto- zoan consortia are theoretically capable of making a significant contribution to methane generation from wet landfill sites.

2. I N T R O D U C T I O N

Much of the solid domestic waste produced in western countries is disposed of by dumping and compaction in disused quarries and other depres- sions on land ('landfill') [1-3]. Over half of this material is organic in origin (paper products, plant

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biomass, etc.) and much of it subsequently under- goes anaerobic decomposition. The gaseous end- products are mainly carbon dioxide and methane, the latter typically accounting for 40-50% by volume [4]. This 'landfill gas' is currently ex- ploited as an economically viable energy source, particularly in the U.S.A., the U.K. and F.R.G. [2]. It is generally believed that free-living methanogens are the sole producers of the methane.

In the last few years a diversity of unrelated anaerobic protozoa have been shown to contain symbiotic methanogenic bacteria. There is much convincing evidence that the methanogens are usually Methanobacteriurn formicicum and that the symbiosis is based on the utilisation by the methanogens of hydrogen gas produced in the ciliates' hydrogenosomes [5-11]. These 'protozoan consortia' are found in a variety of anaerobic environments, including marine and freshwater sediments, the rumen of sheep and cattle and the hindguts of termites [12-19[. Since landfill material is a quantitatively important anaerobic habitat, from which anaerobic protozoa have not previ- ously been reported, we undertook the examina- tion of material from various sites in the search for similar consortia between protozoa and methanogens.

The work reported here describes the successful isolation of anaerobic protozoan consortia from landfill material. Special attention is paid to the polymorphic life cycle, ecology and symbiotic partners in one apparently ubiquitous ciliate species. The work was given some momentum by the continuing drive to optimise landfill gas pro- duction, by the recognition that our knowledge of landfill microbiology is far from complete, and by the assumption, so far untested, that anaerobic protozoa may, in time, be shown to have some economic significance.

3. MATERIALS AND METHODS

Landfill material was collected from five sites in the U.K.: Lower Burgh, Lancashire (2, 4, 6, 8 m); Slackhead, Cumbria (0.5, 1.5, 2.5, 4.2, 7 m); Brogborough, Bedfordshire (one sample from each

of three test cells, No. 1, No. 2, No. 3); Stairfoot, Yorkshire (1, 3, 4.5, 5.5, 6.5 m); Backford Burrow, Cheshire (1, 3, 4.5, 5.5, 6.5 m). Extraction was by shell and auger (Lower Burgh, Slackhead), rotary drilling rig (Backford Burrow) or JCB excavator (Stairfoot). Samples were delivered directly into double, thick plastic bags, and sealed. The Brog- borough samples were posted to Windermere from Harwell. Duplicate bagged samples from Lower Burgh and Slackhead were placed in anaerobic jars filled with N 2 before being transported to the laboratory but this procedure made no difference to the success of subsequent isolation of anaerobic protozoa and the procedure was discontinued, A sample of fresh leachate was also collected from the edge of the site at Backford Burrow and returned to the laboratory in a stoppered bottle. Samples were stored at 10°C, with all laboratory procedures being carried out within 24 h of sam- ple collection. The Brogborough samples were re- ceived between 5 and 12 days from the date of collection. We were always careful to sample from the centre of the bulk landfill material im- mediately after it was brought to the surface and to pack the material tightly, with air excluded. before transporting it. If these precautions are observed, the material remains capable of gener- ating methane and the anaerobic protozoan con- sortia remain viable. Water content of landfill was calculated as evaporative weight losses (80°C, 24 h) from approximately 30 g of sample.

Direct observation of protozoa in landfill was difficult because the material was relatively dry and very heterogeneous, so we submerged 2 3 g from each sample in outgassed (95% N, :5% CO 2) mineral water (' Volvic'). This diluted material was then examined by light microscopy in Sedgewick- Rafter Cells. We used two methods to stimulate the growth of protozoa in landfill samples. The first method was aimed at the ciliates. Approxi- mately 1 g of landfill was inoculated, under a stream of nitrogen, into a 160 ml serum vial containing either anoxic 'Volvic' or anoxic SES medium [13], both at an initial pH of 7. The vials were incubated with a headspace of N~ at 20°C. One ciliate species (Metopus palaeforrnis) fre- quently found growing in these crude cultures was isolated with micropipettes into SES medium with

additional powdered cereal leaves (Sigma). Ciliate cell density was monitored by removing samples with a syringe, fixing the cells in 4% formaldehyde solution, and counting them in a Sedgewick-Rafter Cell.

The second method of cultivation was aimed at the small amoebae and the flagellates. Landfill was diluted ten-fold with anoxic 'Volvic'. 1 ml from each sample was pipetted onto a non-nutri- ent agar plate previously streaked with E. coli. The plates were incubated in an anaerobic jar filled with N 2, at 20°C.

Methanogenic symbionts in protozoa were de- tected by autofluorescence [11] after fixation in 4% formaldehyde solution. The cytochemical as- say for hydrogenase [20] was used with nitroblue tetrazolium as electron acceptor. Transmission electron microscopy was carried out after simulta- neous fixation in cold 3% glutaraldehyde and 1% OsO 4 for 20 min. (each fixative being prepared in a mixture of 100 mM phosphate buffer (pH 7), 1 mM sucrose and a trace of MgSO 4), followed by overnight fixation and staining in uranyl acetate (saturated solution in 70% ethanol, filtered through a 0.2-/~m membrane) in the dark, dehydration and embedding in Spurr resin. Sections were cut with diamond knives and then examined with a JEOL 100CX electron microscope. Other methods used t o observe stratified populations in Sedgewick- Rafter Cells are described in [21]. Additional methods used for the growth experiments are de- scribed within the RESULTS section.

Total numbers of free-living bacteria were enu- merated using the fluorochrome DAPI [15]. Bromoethanesulfonic acid (BES), the specific in- hibitor of methanogens [22] was used at a final concentration of 10 mM.

4. RESULTS

4.1. Water content and actioe protozoa The water content of 21 landfill samples from

five sites varied between 11 and 46% (mean, 26% water). The range with depth at any one site was almost as great (e.g. 18% at 1 m, 46% at 5.5 m and 26% at 6.5 m in samples from Stairfoot). This variation owes much to the complex hydrological

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processes within landfill, and to the extreme het- erogeneity and variable rate of decomposition of landfill material ('landfill ' often does not resemble any natural soil; it often contains large quantities of identifiable waste (newspapers, plastic, glass, etc.) interspersed with decaying organic matter). The two wettest samples were from 5.5 m at Stairfoot, and from 6 m at Lower Burgh (46 and 41% water respectively). In these samples alone we found a few protozoa, upon direct observation of the fresh material. In each case the protozoa were anaerobic flagellates, one of which was probably Chilomastix sp. Several cells of the ciliate Discom- orphella sp. were observed in surface run-off from Stairfoot.

4.2. Cultiuation and isolation of protozoa A variety of flagellates, amoebae and ciliates

developed in landfill material from four of the five sites if water (' Volvic') or aqueous culture medium (SES plus powdered cerophyll) was added to the untreated material, followed by anaerobic incuba- tion. We obtained very poor growth of protozoa in material from the fifth site (Brogborough). This was the only landfill material that was not dealt with by us immediately after collection. The posi- tive results from the other sites indicate that bacterial growth in untreated landfill is sufficient to support protozoa ('Volvic' is virtually free of dissolved organic matter), that landfill material is not toxic to protozoa, that the protozoa were probably encysted at the time of sampling, and that they require nothing more than some ad- ditional water to effect their excystment. The species identity of many of these protozoa is still being examined. They include two Mastigamoeba species, Heteromita sp., Chilomastix sp., Phreat- amoeba sp. and at least one other unidentified heterotrophic flagellate. We also observed cysts, but not trophs, of at least one Acanthamoeba sp. The identity of the most frequently observed species is certain; Metopus palaeformis Kahl 1927 is a ciliate, it grew up in material from all four positive sites, and its description and ecology are the principal subjects of this paper. It was isolated from all depths in Slackhead and Stairfoot, from 4 and 6 m in Lower Burgh and from a sample of leachate from Backford Burrow.

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4.3. Metopus palaeformis The ciliate is remarkable in the variety of forms

it takes during the course of its polymorphic life cycle. Trophic cells look like the individuals in Figs. 1 and 2 when they are well-fed. Towards the end of population growth, they begin to starve, becoming long and thin. This change in shape is accompanied by a significant reduction in cell volume, from about 40-60000 /~m 3 in well-fed cells to 10-20000 /~m 3 when starved (Fig. 3). Several features of the trophic cells do remain relatively constant - - the mouth is restricted to the anterior quarter of the body length, somatic ciliation is sparse, and there is always a typical heterotrich series of adoral membranelles and the small undulating membrane characteristic of Metopus (Fig. 2) [23]. Starvation does not always produce long and thin cells; some encyst (Figs. 4 and 5) without first becoming long and thin. The polymorphic life cycle also includes the occasional production of 'giants ' from normal trophic cells (Figs. 10 and 11). The giants contain large num- bers of methanogens, they eat bacteria and they do not appear to eat other ciliates. We do not yet know which factors induce their production.

The endosymbiotic bacteria in M. palaeformis are easily visualised by their autofluorescence in response to UV or violet excitation (Figs. 6 and 7). T h e r o d - s h a p e d bac te r i a are d i s t r ibu ted throughout the cytoplasm of all trophic cells; they are also retained when the ciliate encysts (Fig. 8). The number of fluorescing bacteria is positively correlated with the size of the host cell, ranging from 116 to 845 (mean 492) in an analysis of 37 trophic cells. There are no ectosymbiotic bacteria.

The cytochemical test for hydrogenase in M. palaeformis was positive. The activity was loca-

lised in the hydrogenosomes (Fig. 9), which in this ciliate are roughly spherical (Fig. 19).

The anaerobic behaviour of the ciliate was con- firmed by introducing a culture, with bacteria, into a Sedgewick-Rafter chamber. An oxygen gradient quickly developed, largely through bacterial activity (Fig. 12). The microaerobic bacteria showed a typical response to low 0 2 tension [21] by aggregating in a tight band at the oxic-anoxic boundary (between arrows). The ciliates avoided the microaerobic layer and the layer of water on either side of it. We can safely say that the ciliate seeks out anaerobic conditions.

Some further features support the identification of the endosymbionts as methanogens: their growth is inhibited by the specific methanogen inhibitor BES (Figs. 13 and 14), and their mor- phology (rods with conically-pointed ends (Fig. 15)) and intracytoplasmic membranes (Fig. 16) are characteristic of Methanobacterium [24]. There is no evidence that the methanogens are attached to the hydrogenosomes (Fig. 17) or that the methanogens and hydrogenosomes form clusters. The ciliate appears to digest some of its sym- bionts; bacteria resembling the symbionts are sometimes seen in various stages of digestion within the food vacuoles (Figs. 17 and 18). The methanogens which are retained in the cyst are apparently identical to those in the trophs (Fig. 20).

4.4. Polymorphic life cycle The main elements of the polymorphic life cycle

of Metopus palaeformis in anaerobic culture are shown in Fig. 21. The culture volume was 80 ml in a 160-ml serum vial with an headspace of N 2 / C O 2 (95 : 5). The carbon source for the bacteria was 50

Figs. 1-12. Photographs of Metopus palaeformis from landfill. Fig. 1: well-fed trophic cell (scale bar 20/~m); Fig. 2: mouth, showing typical short metopid undulating membrane (arrow) (scale bar 20 # m); Fig. 3: starved cell, the nucleus (arrow) also becomes long and thin (scale bar 20/Lm); Fig. 4: cysts. The nucleus (arrow) is visible (scale bar 20/tm); Fig. 5: cyst, showing outer protective coat (scale bar 20 /zm); Fig. 6: autofluorescing methanogens in well-fed cell (scale bar as in Fig. 1); Fig. 7: autofluorescing methanogens in starving cell (scale bar as in Fig. 3); Fig. 8: autofluorescing methanogens in a squashed cyst (scale bar as in Fig. 5); Fig. 9: positive response to cytochemical assay for hydrogenase in ciliate's hydrogenosomes (arrow) (scale bar 20 t~m); Figs. 10 and 11: normal tropkic cell and two 'giants'; Fig. 12: avoidance of oxygen in a Sedgewick-Rafter Cell. A band of microaerobic bacteria (between arrows) marks the boundary between oxygenated water (above) and anoxic water (below). The ciliate distribution is sharply delimited at a point where the cells can still detect some oxygen but the population is randomly distributed within the underlying anaerobic

zone (the squares have sides of 1 mm).

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Figs. 13-20. Photographs of Metopus palaeforrnis from the Slackhead landfill site. Figs. 13 and 14: autofluorescence of symbiotic methanogens, before (Fig. 13) and after (Fig. 14) treatment with BES (scale bar 20 p~m); Fig. 15: endosymbiotic methanogen (scale bar 0.5 ~m); Fig. 16: internal membranes of endosymbiotic methanogen (permanganate fix) (scale bar as in Fig. 15); Fig. 17: probable digestion of endosymbiotic methanogens in food vacuole (arrow) (scale bar 2 ~m); Fig. 18: digestion of endosymbiotic methanogen in food vacuole (scale bar 0.5 /~m); Fig. 19: lack of attachment between endosymbiotic methanogens and hydrogeno-

somes (arrows to latter) (scale bar 1 ~m); Fig. 20: symbiotic methanogens within cyst of ciliate (scale bar 1 /~m).

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TIME (h) Fig. 21. Population growth and polymorphic life cycle of an anaerobic culture of Metopus palaeformis. Following the establishment of the culture, the trophic cells fed and increased in size. As they continued to divide they became smaller, and eventually long and thin, or they encysted. Introduction of a new bacterial food supply at points A and B (see text) st imulated a repetition of the cycle.

Hatched lines indicate estimated trends.

mg ground cereal leaves (Sigma). The growth of these bacteria, and their consumption by the ciliates, fuelled growth and reproduction of the latter. The ciliates reached their maximum cell size at the end of stationary phase; thereafter, their average size decreased. Most cells ended up as long thin forms while about 5% transformed into cysts.

The cause of population decline was probably starvation. A supplement of 57 mg cellobiose (arrow A) led to new ciliate growth and reproduc- tion. Thereafter, the pattern of population devel- opment was repeated. At point B, the gas-tight seal was removed and replaced with cotton wool. After several days there was another increase in

ciliate number, probably in response to the stimu- lation of microaerobic food bacteria (see below).

4.5. Oxygen inhibition In a crude test of the oxygen sensitivity of the

ciliate-methanogen consortium, we observed the response of cultured cells to an elevation of the p O 2. The results are shown in Fig. 22. Two anaerobic cultures were established in serum vials. At 118 h, one culture was poured into a Petri dish, to a depth of 2 -3 mm. More than 50% of the trophic cells died in the first day but most of the remainder survived for 6 days, albeit with reduced motility. The number of fluorescing endosym- bionts also decreased, to about 10% of the original

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Fig. 22. Oxygen inhibition of population growth in Metopus palaeformis. Two anaerobic cultures were established at time zero. One was transferred to a Petri dish, with exposure to atmospheric oxygen, at 118 h, then returned to anaerobic culture at 260 h. Average numbers of symbiotic methanogens (per 100 /*m 2 of ciliate flattened on a membrane) are shown.

Hatched line indicates the estimated trend.

number. Some weakly fluorescing methanogens were probably retained but their autofluorescence was difficult to discriminate from the background. Ultrastructurally, however, these weakly fluoresc- ing methanogens appeared to be normal (e.g. the cyst shown in Fig. 20). At 260 h the contents of the Petri dish were returned to an anaerobic serum vial. The resulting yield of ciliates was almost identical to that in the permanently anaerobic control, so exposure to oxygen produced no last- ing damage, neither to the ciliates nor to their symbionts.

4. 6. Nature of the symbiosis M. palaeformis appears to receive little if any

energetic advantage from its methanogen sym- bionts. The relevant results were obtained from the growth experiment which is illustrated in Fig.

23. Three anaerobic serum vial cultures were established. Each contained 55 ml SES medium and 50 mg ground cereal leaves. At 112 h, one of the cultures was opened and half the contents transferred to another vial containing BES (final concentration 10 mM). Each of these vials was plugged with cotton wool, which encouraged a vigorous ' microaerobic' bacterial growth in a liquid depth of about 12 mm. At the same time (112 h), one of the other anaerobic cultures also received BES, so producing four treatments in all: micro- aerobic, microaerobic + BES, anaerobic, and anaerobic + BES. All four cultures were incubated without stirring or shaking at a constant 20 °

Several results are clear: (a) BES inhibited the growth of symbiotic methanogens, reducing their

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Fig. 23. Population growth of Metopus palaeformis, with and without access to oxygen, and in the presence and absence of the methanogen inhibitor BES. Numbers of autofluorescing methanogens per 100 /zm 2 of ciliate flattened on a membrane, at 235 h, were: anaerobic, 25; microaerobic, 20; anaerobic+ BES, 2.8; microaerobic+BES, 2.1. The total number of free- living bacteria (DAPI counts) in the culture medium at 180 h and 235 h, were, respectively, 1.7 x 108 and 1.3 X 108 per ml (anaerobic): 3.4x108 and 3.2x108 per ml (microaerobic). Microaerobic and anaerobic ciliate generation times were 28 h

and 35 h respectively.

number by about 90% (see Fig. 23). The remaining 10% were weakly fluorescent and may not have been functional; (b) ciliate growth rate in the presence of BES was indistinguishable from the controls; and (c) ciliate growth rate was higher and the final yield of ciliates was about twice as great in the microaerobic cultures. This was prob- ably due to the increased abundance of free-living bacteria ( 'food' for the ciliates) in the micro- aerobic cultures (see legend of Fig. 23). Allowing for the probable anaerobic niches (pieces of de- composing grass etc.) in the microaerobic cultures it is likely that the ciliates would have remained effectively anaerobic, with occasional access to low levels of (energetically irrelevant) oxygen.

5. DISCUSSION

There is little doubt that the landfill ciliate we have isolated and identified as Metopus palaefor- mis is the species described by Kahl [25], but whereas he believed the species to contain several varieties with distinct forms, we have shown, by observing the organism in culture, that the varie- ties are morphological variants of the same species. This polymorphic life cycle is similar in many respects to the recently-described life cycle in the ciliate Pseudocohnilembus pusillus [26], particularly with regard to the observation that starvation in the latter also induces encystment in only a frac- tion of the population.

The advantages of encystment in an environ- ment as variable as landfill are obvious, and the benefits are the same as those enjoyed by soil protozoa in general, the vast majority of which also encyst when the soil dries out. Other Metopus species are relatively common in freshwater [15,27] and marine sediments [29], where water is not limiting but encystment in these species is prob- ably rare. The water content of the landfill we examined was much lower than in lake sediment which usually contains more than 50% water. [e.g. 28]. It is likely that M. palaeformis remains en- cysted for much of the time it spends in such dry landfills.

The ciliate's capacity for dispersal must also be enhanced by its relative insensitivity to oxygen;

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both the trophic cell and the cyst can apparently withstand limited exposure to atmospheric oxygen, and dispersal through oxygenated water or even in the air (in the case of cysts) must be considered probable. This ease of dispersal between anaerobic habitats would help to explain the apparent ubiqu- ity of the ciliate in landfill sites and various other aquatic environments (work in preparation). The mechanism of oxygen detoxification in the ciliate is unknown. It may rely on oxygen consumption by the hydrogenosomes although this is probably trivially small in cysts. It is also likely that the oxygen sensitivity of key enzymes is significantly reduced within the anabolic state of the cyst, which may contain only bound water. The thick- ness of the cyst wall must also be a barrier to the inwards diffusion of oxygen.

Since the hydrogenosomes contain an hydro- genase we can assume that, as in other anaerobic protozoa [9], M. palaeformis disposes of reducing equivalents as hydrogen gas, which is captured and oxidised by the methanogens. But this ciliate is unusual in that the hydrogenosomes and methanogens do not appear to be attached to each other, as they are in other Metopus species [11], and the methanogens do not undergo a morpho- logical transformation when associated with hy- drogenosomes [14]. The absence of attachment and transformation is, however, consistent with our observation that this ciliate obtains no mea- surable energetic advantage from its symbionts (Fig. 23). In other ciliate species, in which the two partners are attached and the bacteria do trans- form, the ciliate benefits through enhanced growth, probably from the secretion of low molecular weight organic compounds by the methanogens. It is gradually becoming apparent that the level of functional integration between hydrogenosomes and methanogens varies between species. In Plagiopyla [30], the two partners operate almost as a single functional organelle, and for their mutual benefit. At the opposite extreme is the apparently passive co-existence of partners in M. palaeformis in which the benefits of the association are heavily balanced in favour of the methanogens, although the ciliate does digest at least some of its methanogens. It would be interesting to know if the methanogens within the encysted ciliate were

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an additional insurance against prolonged starva- tion.

It is likely that the availability of water often restricts the growth of protozoa in landfill. We know that by simply adding water we can stimu- late excystment and growth. The question then arises - - could protozoan consortia be a signifi- cant source of methane production in landfill sites? Our best available model is M. palaeformis grow- ing in crude culture. Culture densities in which the number of symbiotic methanogens is at least 1.5 × 10 6 per ml are readily obtained. If similar densi- ties could be obtained in wet landfill material, the likely methane production would be about 0.9 m 3 C H 4 / t o n n e / y e a r (based on values of methane production by Methanobacterium formicicum; see [31]). Alternatively, if we assume that the methanogens go through two generations for each ciliate generation and that M. formicicum grows with a yield of 4.8 g dry wt per tool C H 4 pro- duced [32] the rate would be about 0.3 m 3 C H 4 / tonne /year . These figures, for ideal conditions, are at the low end of our range of measurements of total net methane production from three land- fill sites (0.3 to 18 m 3 C H a / t o n n e / y e a r ; in pre- paration). However, an equally significant role of these anaerobic protozoa may derive from the variety of ways in which they interact with the rest of the microbial community. We have tentative evidence (in preparation) that the grazing and flocculation activities of anaerobic ciliates in par- ticular lead to an overall stimulation of anaerobic microbial activity and to an increased rate of turnover of organic matter. The role of landfill ciliates would then be similar to the 's t imulation' role already established for protozoa in aerobic communities [33].

A C K N O W L E D G E M E N T S

The research reported here received financial support from the Department of Energy ( E / 5 A / C O N / 1 2 5 6 / 2 0 9 9 ) , The Natural Environment Re- search Council (U.K.) and the Danish Natural Science Research Council (11-8391), and assis- tance from the Energy Technology Support Unit (Harwell) (Dr. P. Lawson and Dr. S. Evans). We

are grateful to Mrs. R. Hindle and B. Simon for assistance, to K.J. Clarke for help and advice with electron microscopy, and to J.A. Winlow. E. Neelon, P. Procter, D.E. Cowen and B. Croft for making it possible for us to collect samples of landfill.

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