The predatory soil flagellate Heteromita globosa stimulates toluene biodegradation by a Pseudomonas sp

A model food chain was established to investigate the influence of grazing by flagellates on bacteria degrading toluene in batch culture. The rate of toluene consumed by a Pseudomonas sp. strain PS+ (max. 0.37 fmol cell(-1) h(-1)) was significantly higher in the presence of the bacterivorous flagellate Heteromita globosa (max. 1.38 fmol cell(-1) h(-1)). A maximum increase of up to 7.5 times was observed in the rate of toluene consumed by these bacteria during exponential growth of this flagellate. Carbon conversion efficiency (CCE) of bacteria to flagellate biomass was estimated to be 33.4% based on measured biovolumes and published values for carbon contents. However, the CCE for toluene-derived carbon was lower (max. 4.9%) when calculations were based on incorporation of [ring-U-(14)C]toluene into biomass of flagellates grazing on labelled bacteria. The findings suggest a potential role for flagellates in bioremediation processes.     

The importance of bacterivorous protists in the decomposition of stream leaf litter

1. Allochthonous organic matter, in the form of senesced leaves, is a major source of carbon supporting detrital food webs. While studies have documented the role of bacteria and fungi in the decomposition of leaf litter, little information is available regarding the role of protists in the decomposition process. 2. We tested the hypothesis that the presence of stream‐dwelling bacterivorous protists leads to an increased rate of leaf decomposition through grazing pressure on bacteria. We isolated live protists from decomposing leaves collected in a stream in Northern Virginia, U.S.A. (Goose Creek) and established laboratory cultures of common bacterivorous protists. 3. Recently senesced leaves from the field were used in laboratory microcosm experiments to determine if the rate of litter decomposition differed between four treatments: bacteria only, bacteria + flagellates, bacteria + flagellates + ciliates, autoclaved stream water (control). We determined the dry weight of leaf remaining, bacterial abundance, flagellate abundance and ciliate abundance for each replicate on days 0, 7, 14, 30, 60 and 120. 4. The rate of leaf decomposition was significantly higher in treatments with protists than without and bacterial abundance declined in protist treatments compared with bacteria only treatment. Weight loss in the presence of flagellates was three to four times higher when protists were present compared with treatments with bacteria alone. These results provide experimental evidence that protists could play a significant role in the detrital processes of streams.     

Successful predation of filamentous bacteria by a nanoflagellate challenges current models of flagellate bacterivory

Current models suggest that (i) filamentous bacteria are protected against predation by nanoflagellates, (ii) prey size is positively correlated with prey-predator contact probability, and (iii) contact probability is mainly responsible for size-selective predation by interception-feeding flagellates. We used five strains of filamentous bacteria and one bacterivorous nanoflagellate, Ochromonas sp. strain DS, to test these assumptions. The five strains, including one spirochete and four Betaproteobacteria strains, were isolated by the filtration-acclimatization method. All five strains possess flexible cells, but they differ in average cell length, which ranged from 4.5 to 13.7 micro m. High-resolution video microscopy was used to measure contact, capture, and ingestion rates, as well as selectivity of the flagellate feeding. Growth and feeding experiments with satiating and nonsatiating food conditions, as well as experiments including alternative well-edible prey, were performed. In contrast to predictions by current models, the flagellate successfully consumed all the tested filamentous strains. The ingestion rate was negatively correlated with bacterial length. On the other hand, the lengths of the filamentous bacteria were not positively correlated to the contact rate and capture rate but were negatively correlated to ingestion efficiency. In experiments including alternative nonfilamentous prey, the flagellates showed negative selection for filamentous bacteria, which was independent of food concentration and is interpreted as a passive selection. Our observations indicate that (i) size alone is not sufficient to define a refuge for filamentous bacteria from nanoflagellate predation and (ii) for the investigated filamentous bacteria, prey-predator contact probability could be more influenced by factors other than the prey size.     

Bacterial feeders, the nematode Caenorhabditis elegans and the flagellate Cercomonas longicauda, have different effects on outcome of competition among the Pseudomonas biocontrol strains CHA0 and DSS73

How bacterial feeding fauna affects colonization and survival of bacteria in soil is not well understood, which constrains the applicability of bacterial inoculants in agriculture. This study aimed to unravel how food quality of bacteria and bacterial feeders with different feeding habits (the selective feeding flagellate Cercomonas longicauda versus the non-selective feeding nematode Caenorhabditis elegans) influence the abundance of two bacteria that compete for resources in simple model communities. Microcosms consisted of either one gfp-tagged bacterial strain (Pseudomonas fluorescens DSM50090 or one of two biocontrol strains P. fluorescens CHA0 or Pseudomonas sp. DSS73) or combinations of two bacterial strains. DSM50090 is a suitable food bacterium, DSS73 is of intermediate food quality, and CHA0 is inedible to the bacterial feeders. Bacterial and protozoan cell numbers were measured by flow cytometry. In the presence of flagellates, CHA0 increased its abundance as compared to the other biocontrol strain DSS73 or to DSM50090, which were both eaten by the flagellates. In contrast, the number of CHA0 declined as compared to DSS73 when the model community was subjected to nematode predation pressure. Hence, the results suggested that the outcome of competition among bacteria depended on their ability to cope with the prevailing bacterial predator.     

A common soil flagellate (Cercomonas sp.) grows slowly when feeding on the bacterium Rhodococcus fascians in isolation, but does not discriminate against it in a mixed culture with Sphingopyxis witflariensis

Flagellates are very important predators on bacteria in soil. Because of their high growth rates, flagellate populations respond rapidly to changes in bacterial numbers. Previous results indicate that actinobacteria are generally less suitable than proteobacteria as food for flagellates. In this study, we investigated the growth of the flagellate Cercomonas sp. (ATCC 50334) on each of the two bacteria Sphingopyxis witflariensis (Alphaproteobacteria) and Rhodococcus fascians (actinobacteria) separately and in combination. The growth rate of the flagellate was lower and the lag phase was longer when fed with R. fascians than when fed with S. witflariensis. This supports our initial hypothesis that the actinobacterium is less suitable as food than the alphaproteobacterium. However, after longer periods of growth the peak abundance of flagellates was higher on R. fascians, indicating that the food quality of bacterial prey depends on the time perspective of the flagellate-bacterial interaction. There was no evidence that the flagellates selected against the actinobacterium when feeding in mixed cultures of the two bacteria. Experiments where flagellates were fed with washed bacterial cells or with bacteria growing with different substrate concentrations suggested that the low food quality of R. fascians is related both to the intrinsic cell properties and to the extracellular metabolites.     

Successful Predation of Filamentous Bacteria by a Nanoflagellate Challenges Current Models of Flagellate Bacterivory

Current models suggest that (i) filamentous bacteria are protected against predation by nanoflagellates, (ii) prey size is positively correlated with prey-predator contact probability, and (iii) contact probability is mainly responsible for size-selective predation by interception-feeding flagellates. We used five strains of filamentous bacteria and one bacterivorous nanoflagellate, Ochromonas sp. strain DS, to test these assumptions. The five strains, including one spirochete and four Betaproteobacteria strains, were isolated by the filtration-acclimatization method. All five strains possess flexible cells, but they differ in average cell length, which ranged from 4.5 to 13.7 μm. High-resolution video microscopy was used to measure contact, capture, and ingestion rates, as well as selectivity of the flagellate feeding. Growth and feeding experiments with satiating and nonsatiating food conditions, as well as experiments including alternative well-edible prey, were performed. In contrast to predictions by current models, the flagellate successfully consumed all the tested filamentous strains. The ingestion rate was negatively correlated with bacterial length. On the other hand, the lengths of the filamentous bacteria were not positively correlated to the contact rate and capture rate but were negatively correlated to ingestion efficiency. In experiments including alternative nonfilamentous prey, the flagellates showed negative selection for filamentous bacteria, which was independent of food concentration and is interpreted as a passive selection. Our observations indicate that (i) size alone is not sufficient to define a refuge for filamentous bacteria from nanoflagellate predation and (ii) for the investigated filamentous bacteria, prey-predator contact probability could be more influenced by factors other than the prey size.     

Small but Manifold – Hidden Diversity in “Spumella‐like Flagellates”

Colourless, nonscaled chrysophytes comprise morphologically similar or even indistinguishable flagellates which are important bacterivors in water and soil crucial for ecosystem functioning. However, phylogenetic analyses indicate a multiple origin of such colourless, nonscaled flagellate lineages. These flagellates are often referred to as “Spumella‐like flagellates” in ecological and biogeographic studies. Although this denomination reflects an assumed polyphyly, it obscures the phylogenetic and taxonomic diversity of this important flagellate group and, thus, hinders progress in lineage‐ and taxon‐specific ecological surveys. The smallest representatives of colourless chrysophytes have been addressed in very few taxonomic studies although they are among the dominant flagellates in field communities. To overcome the blurred picture and set the field for further investigation in biogeography and ecology of the organisms in question, we studied a set of strains of specifically small, colourless, nonscaled chrysomonad flagellates by means of electron microscopy and molecular analyses. They were isolated by a filtration‐acclimatisation approach focusing on flagellates of around 5 μm. We present the phylogenetic position of eight different lineages on both the ordinal and the generic level. Accordingly, we describe the new genera Apoikiospumella, Chromulinospumella, Segregatospumella, Cornospumella and Acrispumella Boenigk et Grossmann n. g. and different species within them.     

自由生活的异养鞭毛虫多样性及生态功能

随着对水生态系统结构与功能的深入研究,自由生活的异养鞭毛虫在水生态系统中的作用成为当前生态学领域的热点之一.已有的研究结果表明,异养鞭毛虫物种丰富,摄食方式多样,周转迅速,是微食物环的重要成分,在水生态系统中起着十分重要的作用.异养鞭毛虫物种多样性及生态学方面的系统研究将有助于对水生态系统结构、功能和过程深入了解.综述了异养鞭毛虫多样性、群落结构、摄食生态学以及在氮、磷循环中的作用,分析了其在生态系统中的功能.     

Impact of violacein-producing bacteria on survival and feeding of bacterivorous nanoflagellates

We studied the role of bacterial secondary metabolites in the context of grazing protection against protozoans. A model system was used to examine the impact of violacein-producing bacteria on feeding rates, growth, and survival of three common bacterivorous nanoflagellates. Freshwater isolates of Janthinobacterium lividum and Chromobacterium violaceum produced the purple pigment violacein and exhibited acute toxicity to the nanoflagellates tested. High-resolution video microscopy revealed that these bacteria were ingested by the flagellates at high rates. The uptake of less than three bacteria resulted in rapid flagellate cell death after about 20 min and cell lysis within 1 to 2 h. In selectivity experiments with nontoxic Pseudomonas putida MM1, flagellates did not discriminate against pigmented strains. Purified violacein from cell extracts of C. violaceum showed high toxicity to nanoflagellates. In addition, antiprotozoal activity was found to positively correlate with the violacein content of the bacterial strains. Pigment synthesis in C. violaceum is regulated by an N-acylhomoserine lactone (AHL)-dependent quorum-sensing system. An AHL-deficient, nonpigmented mutant provided high flagellate growth rates, while the addition of the natural C. violaceum AHL could restore toxicity. Moreover, it was shown that the presence of violacein-producing bacteria in an otherwise nontoxic bacterial diet considerably inhibited flagellate population growth. Our results suggest that violacein-producing bacteria possess a highly effective survival mechanism which may exemplify the potential of some bacterial secondary metabolites to undermine protozoan grazing pressure and population dynamics.     

Comparison of growth efficiencies of protozoa growing on bacteria deposited on surfaces and in suspension

Bacteria were deposited in tubes as compact pellets by centrifuging suspensions of cultured Vibrio at stationary phase. Numbers and protein biomass of flagellates added to these tubes and of the Vibrio, were followed and compared with the growth of the same and other protists on identical, uncentrifuged Vibrio. The flagellates Bodo saliens and Caecitellus parvulus, which could not be seen to multiply in tubes of suspended bacteria, grazed deposited bacteria actively as did the more versatile flagellate Cafeteria roenbergensis. The growth of these flagellates and their consumption of deposited bacteria were very similar to those of the flagellate Pteridomonas danica or the ciliate Uronema marinum fed with suspended bacteria, although deposit-feeders grew more slowly. Gross growth efficiencies (30-60%) of deposit-feeding flagellates were similar to those of the suspension-feeding protists. Caecitellus consumed 55 Vibrio to produce one flagellate, while 4,500 Vibrio were consumed to produce one Uronema. Surface-feeding flagellates are shown to be efficient bacterivores, capable of restricting the numbers of bacteria deposited on surfaces just as other protozoa control numbers of suspended bacteria.     

Impact of Violacein-Producing Bacteria on Survival and Feeding of Bacterivorous Nanoflagellates

We studied the role of bacterial secondary metabolites in the context of grazing protection against protozoans. A model system was used to examine the impact of violacein-producing bacteria on feeding rates, growth, and survival of three common bacterivorous nanoflagellates. Freshwater isolates of Janthinobacterium lividum and Chromobacterium violaceum produced the purple pigment violacein and exhibited acute toxicity to the nanoflagellates tested. High-resolution video microscopy revealed that these bacteria were ingested by the flagellates at high rates. The uptake of less than three bacteria resulted in rapid flagellate cell death after about 20 min and cell lysis within 1 to 2 h. In selectivity experiments with nontoxic Pseudomonas putida MM1, flagellates did not discriminate against pigmented strains. Purified violacein from cell extracts of C. violaceum showed high toxicity to nanoflagellates. In addition, antiprotozoal activity was found to positively correlate with the violacein content of the bacterial strains. Pigment synthesis in C. violaceum is regulated by an N-acylhomoserine lactone (AHL)-dependent quorum-sensing system. An AHL-deficient, nonpigmented mutant provided high flagellate growth rates, while the addition of the natural C. violaceum AHL could restore toxicity. Moreover, it was shown that the presence of violacein-producing bacteria in an otherwise nontoxic bacterial diet considerably inhibited flagellate population growth. Our results suggest that violacein-producing bacteria possess a highly effective survival mechanism which may exemplify the potential of some bacterial secondary metabolites to undermine protozoan grazing pressure and population dynamics.     

Effects of hydrophobic and electrostatic cell surface properties of bacteria on feeding rates of heterotrophic nanoflagellates

The influence of cell surface hydrophobicity and electrostatic charge of bacteria on grazing rates of three common species of interception-feeding nanoflagellates was examined. The hydrophobicity of bacteria isolated from freshwater plankton was assessed by using two different methods (bacterial adhesion to hydrocarbon and hydrophobic interaction chromatography). The electrostatic charge of the cell surface (measured as zeta potential) was analyzed by microelectrophoresis. Bacterial ingestion rates were determined by enumerating bacteria in food vacuoles by immunofluorescence labelling via strain-specific antibodies. Feeding rates varied about twofold for each flagellate species but showed no significant dependence on prey hydrophobicity or surface charge. Further evidence was provided by an experiment involving flagellate grazing on complex bacterial communities in a two-stage continuous culture system. The hydrophobicity values of bacteria that survived protozoan grazing were variable, but the bacteria did not tend to become more hydrophilic. We concluded that variability in bacterial cell hydrophobicity and variability in surface charge do not severely affect uptake rates of suspended bacteria or food selection by interception-feeding flagellates.     

Effects of Hydrophobic and Electrostatic Cell Surface Properties of Bacteria on Feeding Rates of Heterotrophic Nanoflagellates

The influence of cell surface hydrophobicity and electrostatic charge of bacteria on grazing rates of three common species of interception-feeding nanoflagellates was examined. The hydrophobicity of bacteria isolated from freshwater plankton was assessed by using two different methods (bacterial adhesion to hydrocarbon and hydrophobic interaction chromatography). The electrostatic charge of the cell surface (measured as zeta potential) was analyzed by microelectrophoresis. Bacterial ingestion rates were determined by enumerating bacteria in food vacuoles by immunofluorescence labelling via strain-specific antibodies. Feeding rates varied about twofold for each flagellate species but showed no significant dependence on prey hydrophobicity or surface charge. Further evidence was provided by an experiment involving flagellate grazing on complex bacterial communities in a two-stage continuous culture system. The hydrophobicity values of bacteria that survived protozoan grazing were variable, but the bacteria did not tend to become more hydrophilic. We concluded that variability in bacterial cell hydrophobicity and variability in surface charge do not severely affect uptake rates of suspended bacteria or food selection by interception-feeding flagellates.     

Protistan bacterivory in an oligomesotrophic lake: Importance of attached ciliates and flagellates

Seasonal and depth variations of the abundance, biomass, and bacterivory of protozoa (heterotrophic and mixotrophic flagellates and ciliates) were determined during thermal stratification in an oligomesotrophic lake (Lake Pavin, France). Maximal densities of heterotrophic flagellates (1.9 × 10³ cells ml^–1) and ciliates (6.1 cells ml^–1) were found in the metalimnion. Pigmented flagellates dominated the flagellate biomass in the euphotic zone. Community composition of ciliated protists varied greatly with depth, and both the abundance and biomass of ciliates was dominated by oligotrichs. Heterotrophic flagellates dominated grazing, accounting for 84% of total protistan bacterivory. Maximal grazing impact of heterotrophic flagellates was 18.9 × 10⁶ bacteria 1^–1h^–1. On average, 62% of nonpigmented flagellates were found to ingest particles. Ciliates and mixotrophic flagellates averaged 13% and 3% of protistan bacterivory, respectively. Attached protozoa (ciliates and flagellates) were found to colonize the diatom Asterionella formosa. Attached bacterivores had higher ingestion rates than free bacterivorous protozoa and may account for 66% of total protozoa bacterivory. Our results indicated that even in low numbers, epibiotic protozoa may have a major grazing impact on free bacteria. Correspondence: C. Amblard.     

Channeling of bacterioplanktonic production toward phagotrophic flagellates and ciliates under different seasonal conditions in a river

The objective of this study was to analyze the flux of biomass through the communities of bacteria and phagotrophic protists in the cold and warm conditions occurring seasonally in Butrón River. Bacterial and heterotrophic protistan (flagellate and ciliate) abundance was determined by epifluorescence direct counts; protistan grazing on planktonic bacteria was measured from fluorescently labeled bacteria uptake rates; and the estimate of bacterial secondary production was obtained from [³H]thymidine incorporation rates. The abundance of bacterial, flagellate, and ciliate communities was similar during cold and warm situations. However, we observed that estimates of dynamic parameters, i.e., secondary bacterial production and protistan grazing, in both situations were noticeably different. In the warm situation, grazing rates of flagellates and ciliates (bacteria per protist per hour) were, respectively, 7 times and 18 times higher than those determined in the cold situation, and the grazing rates of the protistan communities (bacteria per protists present in 1 ml of water per hour) increased up to 5 times in the case of flagellates and 42 times in the case of ciliates. Estimates of bacterial secondary production were also higher during the warm situation, showing a ninefold increase. The percentage of bacterial production preyed upon by flagellates or ciliates was not significantly different between the two conditions. These results showed that in the different conditions of a system, the flux of biomass between the trophic levels may be quite different although this process may not be reflected in the abundance of each community of bacteria, flagellates, and ciliates. Offprint requests to: J. Iriberri.     

Viruses and flagellates sustain apparent richness and reduce biomass accumulation of bacterioplankton in coastal marine waters

To gain a better understanding of the interactions among bacteria, viruses and flagellates in coastal marine ecosystems, we investigated the effect of viral lysis and protistan bacterivory on bacterial abundance, production and diversity [determined by 16S rRNA gene polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE)] in three coastal marine sites with different nutrient supplies in Hong Kong. Six experiments were set up using filtration and dilution methods to develop virus, flagellate and virus+flagellate treatments for natural bacterial populations. All three predation treatments had significant repressing effects on bacterial abundance. Bacterial production was significantly repressed by flagellates and both predators (flagellates and viruses). Bacterial apparent species richness (indicated as the number of DGGE bands) was always significantly higher in the presence of viruses, flagellates and both predators than in the predator-free control. Cluster analysis of the DGGE patterns showed that the effects of viruses and flagellates on bacterial community structure were relatively stochastic while the co-effects of predators caused consistent trends (DGGE always showed the most similar patterns when compared with those of in situ environments) and substantially increased the apparent richness. Overall, we found strong evidence that viral lysis and protist bacterivory act additively to reduce bacterial production and to sustain diversity. This first systematic attempt to study the interactive effects of viruses and flagellates on the diversity and production of bacterial communities in coastal waters suggests that a tight control of bacterioplankton dominants results in relatively stable bacterioplankton communities.     

Size-selective grazing on bacteria by natural assemblages of estuarine flagellates and ciliates

The small average cell size of in situ bacterioplankton, relative to cultured cells, has been suggested to be at least partly a result of selection of larger-sized cells by bacterivorous protozoa. In this study, we determined the relative rates of uptake of fluorescence-labeled bacteria (FLB), of various cell sizes and cell types, by natural assemblages of flagellates and ciliates in estuarine water. Calculated clearance rates of bacterivorous flagellates had a highly significant, positive relationship with size of FLB, over a range of average biovolume of FLB of 0.03 to 0.08 microns3. Bacterial cell type or cell shape per se did not appear to affect flagellate clearance rates. The dominant size classes of flagellates which ingested all types of FLB were 3- to 4-microns cells. Ciliates also showed a general preference for larger-sized bacteria. However, ciliates ingested a gram-positive enteric bacterium and a marine bacterial isolate at higher rates than they did a similarly sized, gram-negative enteric bacterium or natural bacterioplankton, respectively. From the results of an experiment designed to test whether the addition of a preferentially grazed bacterial strain stimulated clearance rates of natural bacterioplankton FLB by the ciliates, we hypothesized that measured differences in rates of FLB uptake were due instead to differences in effective retention of bacteria by the ciliates. In general, clearance rates for different FLB varied by a factor of 2 to 4. Selective grazing by protozoa of larger bacterioplankton cells, which are generally the cells actively growing or dividing, may in part explain the small average cell size, low frequency of dividing cells, and low growth rates generally observed for assemblages of suspended bacteria.     

Effects of Eutrophication on Flagellates Associated with Eichhornia crassipes: An Experimental Approach

The effects of nutrient enrichment on density, biomass and on the relative contribution of pigmented and heterotrophic flagellates to the total abundance of all flagellates associated with Eichhornia crassipes were evaluated through mesocosm experiments over 33 days. Two triplicate treatments (nitrogen and phosphorus addition and a control treatment) were colonized, producing six mesocosm systems. The only variable that showed significance was flagellate density. Density increased significantly with increased nutrient concentrations in both flagellate groups, suggesting the importance of bottom‐up control mechanisms. However, the time lag and the low magnitude response to density, as well as the lack of significant variation in biomass with respect to fertilization, suggested that a complex relationship is responsible for influencing the structure of flagellate communities associated with vegetation during nutrient enrichment in these food webs. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Marine biofilm bacteria evade eukaryotic predation by targeted chemical defense

Many plants and animals are defended from predation or herbivory by inhibitory secondary metabolites, which in the marine environment are very common among sessile organisms. Among bacteria, where there is the greatest metabolic potential, little is known about chemical defenses against bacterivorous consumers. An emerging hypothesis is that sessile bacterial communities organized as biofilms serve as bacterial refuge from predation. By testing growth and survival of two common bacterivorous nanoflagellates, we find evidence that chemically mediated resistance against protozoan predators is common among biofilm populations in a diverse set of marine bacteria. Using bioassay-guided chemical and genetic analysis, we identified one of the most effective antiprotozoal compounds as violacein, an alkaloid that we demonstrate is produced predominately within biofilm cells. Nanomolar concentrations of violacein inhibit protozoan feeding by inducing a conserved eukaryotic cell death program. Such biofilm-specific chemical defenses could contribute to the successful persistence of biofilm bacteria in various environments and provide the ecological and evolutionary context for a number of eukaryote-targeting bacterial metabolites.     

Studies on the rumen flagellate Sphaeromonas communis.

The rumen flagellate Sphaeromonas communis showed a significant increase in population density 1 to 2 h after the host sheep commenced feeding, followed by a reduction in numbers to the pre-feeding level after a further 2 to 3 h. The life-history of the organism was shown to consist of a motile flagellate which germinated to produce a vegetative stage comprising a limited rhizoidal system on which up to three reproductive bodies were borne together with (in vitro) other spherical bodies of unknown function; in vivo, the reproductive bodies were stimulated to liberate flagellates by a component of the diet of the host. The vegetative stage strongly resembled that of certain species of aquatic phycomycete fungi, and the flagellates may therefore by zoospores. Flagellates liberated in vivo lost their motility within 2 to 3 h and developed into the reproductive vegetative phase, producing a rapid decrease in numbers of flagellates. Conditions of maximum flagellate production (pH 6.5, 39 degrees C, presence of CO2, absnece of oxygen) approximated to those found in the rumen. The organism was cultured in vitro in an undefined medium in the absnece of bacteria and other flagellates.