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Keywords:

  • Rumen ciliate;
  • Methanogen;
  • Symbiosis

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Methanogenesis per ciliate protozoan and most probable numbers (MPN) of methanogens per ciliate were measured and observed to be affected by feeding. The close similarity observed between the changes in apparent methane production and the changes in the number of methanogens per ciliate indicated that apparent methane production by ciliates depended primarily on the number of methanogens associated with them. Large changes in the MPN of methanogens associated with ciliates occurred rapidly. It is likely that these changes reflect the number of methanogens that associated extracellularly with the ciliates. Methanogens associated with ciliates may have differed from the free living methanogens in halophilism and their resistance to ammonium ions.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Methanogenesis is an important metabolic activity of the rumen microflora. Ruminal methanogens utilize reducing equivalents produced by fermentative microorganisms such as Ruminococcus spp., Neocallimastix spp. and ciliate protozoa. Normally, the association of methanogens with hydrogen-producing organisms is by attachment or by floc formation [1, 2]. Among the hydrogen-producing organisms of rumen origin, ciliate protozoa are the only organisms for which such an association has been demonstrated [3]. Recently, the presence of endosymbiotic methanogens in certain rumen ciliates has also been demonstrated [4] and there is little doubt that they contribute to ruminal methanogenesis. The contribution of ciliate protozoa and their adherent methanogens to ruminal methanogenesis has been estimated to be a few nmol per ciliate per day [5, 6]. Since the number of methanogens colonizing the protozoan cell surface may vary with the surrounding physico-chemical conditions such as hydrogen partial pressure [7], the contribution of this consortium to ruminal methanogenesis may change during a day.

Here we report that the contribution of the ciliates to ruminal methanogenesis may be modulated by the number of ciliate-associated methanogens. We also report on the responses of these methanogens to chloride and ammonium ions.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Animals and diets

Three ruminally fistulated Suffolk sheep were used as experimental animals. They were fed timothy hay supplemented with 33% of a commercial concentrate feed (Coop dairy 14, Kumiai Shiryo, Tokyo). The concentrate feed contained maize (47%), wheat bran (39%), and soybean cake (8%); the remainder consisted of molasses, calcium carbonate, calcium phosphate and sodium chloride. This feed also contained other microelements. Water and mineral blocks (Green Kouen, Nihon Zenyaku Kogyo, Fukushima, Japan) were also present.

2.2Culture of ciliates

Rumen ciliate protozoa were cultured according to Ushida and Jouany [6]. Briefly, samples of rumen contents were removed through a fistula prior to (0 h), and 1, 2 and 3 h after the morning feed. The contents were strained through double layers of gauze and then subjected to gravity sedimentation in a separating funnel. The resultant ciliate fractions were recovered on nylon mesh (10 μm pore size). The protozoa were washed in Coleman Simplex buffer [8] and collected in 100 ml of the same buffer. Precautions were taken to avoid contact with air and to keep the protozoa warm. Portions (10 ml) of these treated samples were inoculated into serum bottles containing 25 ml Coleman Simplex buffer, 15 ml cell-free rumen fluid, 0.5 g dried grass powder (timothy hay), 0.5 g soluble starch and 2 ml of an antibiotic mixture. Two different antibiotic mixtures were used; one contained penicillin G and streptomycin, and the other contained penicillin G, streptomycin and chloramphenicol. The antibiotics were purchased from Sigma (St. Louis, MO, USA). The concentration of each antibiotic in the stock solution was 1 mg ml−1. Ciliates were incubated at 39°C for 48 h in the presence of CO2.

2.3Enumeration of methanogens present in the ciliates fraction

The most probable numbers (MPN) of methanogens were estimated from ciliate fractions. Fractionation of rumen fluid was performed according to the method of Coleman [9]. Ciliate fractions were recovered by centrifugation at 150×g for 2 min from 300 ml rumen contents that were collected prior to (0 h), and 1, 2 and 3 h after the morning feed. The resultant pellets were homogenized in 200 ml of physiological saline using a Biomixer homogenizer (Nissei, Nara, Japan) and subjected to serial 10-fold dilutions. All manipulations were conducted in an anaerobic chamber (Coy Laboratory, MI, USA) with H2/CO2/N2 (10/10/80) with the exception that, during centrifugation, the tubes were pre-filled with CO2. Paynter-Hungate medium [10] under a H2/CO2 (80/20) gas mixture was used for the enumeration of methanogens. The initial gas pressure was increased to ca. 200 kPa after inoculation. Three tubes were allotted for each dilution. Tubes that produced methane (>10 ppm) after 14 days incubation were considered to be methanogen-positive. MPN were estimated as described by Clarke and Owens [11].

2.4Estimation of response to chloride and ammonium ions

The effects of ammonium chloride and sodium chloride on methanogenic activity in rumen fluid were examined using strained rumen fluid (SRF) and ciliate-free SRF. Rumen contents were sampled with a fistula from a sheep 1 h after the morning feed. The contents were strained through four layers of gauze. Half the SRF was centrifuged at 150×g for 2 min to remove ciliates. Portions (2 ml) of SRF and ciliate-free SRF were incubated with 8 ml McDougall's buffer [12] containing 2 g l−1 each of soluble starch, glucose and cellobiose at 39°C for 12 h. Five concentrations (0, 2.5, 5.0, 7.5, 10.0 g l−1) of NaCl and NH4Cl were prepared. The initial gas (H2/CO2=80/20) pressure was increased to ca. 200 kPa.

2.5Analyses

Methane and hydrogen were analyzed by gas chromatography [13]. Organic acids (succinic, lactic, formic, acetic, propionic, n-butyric, isobutyric, n-valeric, isovaleric and capronic acids) in the culture supernatants were analyzed by ion exclusion HPLC [14]. All chemicals were purchased from Wako Junyaku Co. (Osaka, Japan) unless otherwise stated. Statistical analyses were achieved by ANOVA and Tukey's test where appropriate [15].

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1Apparent methanogenesis by ciliates

The predominant ciliate populations in the cultures were identified as Polyplastron multivesiculatum, Ophryoscolex caudatus, Isotricha spp. and Entodinium spp. A similar profile was observed in cultures prepared from the three sheep at four different times (0, 1, 2 and 3 h after feeding) (Table 1). Most genera persisted throughout incubation except for Isotricha spp. Methane, and not hydrogen, was detected in these ciliate cultures when treated with an antibiotic mixture containing penicillin G and streptomycin. However, hydrogen was detected in the cultures treated with an antibiotic mixture containing penicillin G, streptomycin and chloramphenicol. The ciliate inocula prepared 1 h after feeding appeared to produce copious amounts of methane (approximately 6 nmol ciliate−1 day−1), almost twice as high as the amount produced by the other ciliate preparations (Fig. 1a). However, hydrogen production was observed to be relatively constant (Fig. 1b). Varying amounts of formate were produced during methane production and these became constant when methane evolution was inhibited (Fig. 1c). Relatively high concentrations of formate were produced (approximately 120 nmol ciliate−1 day−1) in the cultures of ciliates prepared from samples collected prior to feeding, but low concentrations (20 nmol ciliate−1 day−1) were produced in the other cultures. Total acid production was higher when methane production was allowed and the highest production was recorded from the ciliate culture that was prepared from the sample collected 1 h after feeding (Fig. 1d).

Table 1.  Numbera of predominant ciliates in cultures for gas determination and in samples for enumeration of methanogens
GenusSampling time (time after morning feed)
0 h1 h2 h3 h
  1. aNumbers are expressed in logarithm (ml−1). Means±S.D. for the three sheep.

  2. bNumber of ciliates in cultures at the beginning of incubation.

  3. cNumber of ciliates in cultures at the end of incubation.

Numbers in cultures for gas determination
Polyplastron3.1±0.42b3.1±0.443.0±0.432.8±0.35
 (2.7±0.11)c(2.9±0.13)(2.8±0.09)(2.9±0.04)
Ophryoscolex2.9±0.462.9±0.462.6±0.442.4±0.38
 (2.9±0.04)(2.3±0.06)(2.9±0.03)(2.8±0.16)
Isotricha2.8±0.532.6±0.542.4±0.422.3±0.50
 (0.2±0.40)(0.2±0.40)(0.5±0.40)(0.3±0.35)
Entodinium2.9±0.542.9±0.562.8±0.552.9±0.57
 (2.1±0.26)(2.2±0.06)(2.3±0.03)(2.4±0.16)
Numbers in samples for enumeration of methanogens
Polyplastron3.3±0.153.4±0.143.4±0.213.2±0.10
Ophryoscolex3.2±0.113.1±0.093.1±0.313.0±0.21
Isotricha3.4±0.073.1±0.133.1±0.272.8±0.06
Entodinium5.7±0.145.6±0.075.6±0.275.5±0.22
image

Figure 1. Changes in (a) apparent methane emission, (b) hydrogen production, (c) formate production and (d) production of total organic acids per ciliate in incubations in vitro. Samples of rumen contents were removed prior to (0 h), 1, 2 and 3 h after the morning feed. The values are expressed as metabolite production (mmol or nmol) per ciliate per 48 h. •: methanogenesis not inhibited, ■: methanogenesis inhibited. Total organic acids include formate, acetate, propionate, isobutyrate, butyrate, isovalerate, valerate, succinate and lactate. Means±S.D., n=3. Values with different superscripts differ significantly (P<0.05).

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3.2Number of methanogens associated with ciliates

The values shown in Fig. 2 are the MPN of methanogens divided by the number of ciliates present in the same sample. The number of methanogens associated with ciliates similarly varied with time after feeding (Fig. 2) in all three sheep. The numbers of methanogens associated with ciliates increased after feeding up to 100–1000 MPN per ciliate and then declined to the initial levels of 0.5–3 MPN per ciliate. Values less than 1.0 indicate that all the ciliates were not colonized by methanogens. Although there was a considerable difference in the number of methanogens associated with ciliates in sheep 1 when compared to the other two sheep, the composition of ciliate genera was similar in all three sheep.

image

Figure 2. Changes in the number of ciliates (ml−1) and methanogens per ciliate (MPN ciliate−1). Samples of rumen contents were removed prior to (0 h), 1, 2 and 3 h after the morning feed. Number of methanogens, ♢: sheep 1, ▵: sheep 2, □: sheep 3. •: number of ciliates, means±S.D. for the three sheep. Most probable number (MPN) of methanogens was divided by the number of ciliates.

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3.3Response of methanogens to chloride and ammonium ions

When the ciliates were removed from the SRF, no response to NaCl was observed. In contrast, methane production was stimulated in the presence of 5.0–7.5 g l−1 NaCl in the SRF (Fig. 3a). Such concentrations correspond to about 0.1 M in the culture fluid. Ammonium salts reduced methane production in the cultures from both SRF and ciliate-free SRF, but the responses of these two cultures were different; methanogenesis declined gradually in the latter cultures, while it decreased suddenly at the highest ammonium level (10 g l−1) in the former cultures (Fig. 3b).

image

Figure 3. Effect of sodium chloride (a) and ammonium chloride (b) on methanogenesis. ■: endosymbiotic and free-living methanogens (ciliates present), •: free-living methanogens (ciliates absent). Means±S.D., n=3. Values with different superscripts differ significantly (P<0.05).

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4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

The apparent methanogenesis of ciliate protozoa was measured using inocula prepared from samples collected prior to and 1, 2 and 3 h after feeding. Due to the repeated washing steps during inoculum preparation, the free-living methanogens were likely to be removed. Therefore, the resulting methane produced can be attributed to methanogens closely associated with ciliates, including those living inside [4] and those attached to the outside [3] of the ciliate cells. Two factors should be considered in order to understand the fluctuations observed: the metabolic activity of ciliate protozoa and the number of methanogens in and on the protozoal cells. Earlier studies suggested that the number of methanogens attached to the outside of protozoal cells was modulated by the hydrogen partial pressure of the surrounding microenvironment [7].

The MPN of methanogens per ciliate were also affected by the time after feeding (Fig. 2), showing similar fluctuation patterns in all three experimental sheep. Although the number of ciliates did not significantly change during this period (Table 1, Fig. 2), the changes in the number of methanogens per ciliate reflected the number of methanogens associated with the ciliate. The similarity between the changes in apparent methane production and those observed in the number of associated methanogens suggests that the intensity of apparent methane production by ciliates depended primarily on the number of methanogens closely associated with them. It was difficult to distinguish one ciliate genus from the others on the basis of the intensity of association of methanogens because mixed ciliates were used in the experiment. Since the contribution of ciliates to ruminal methanogenesis varied among the different genera [6], the intensity of the association may depend on the ciliate genera. A cell of the large ciliates like Polyplastron and Isotricha may be more heavily colonized by methanogens due to their larger cell volumes, surfaces and metabolic activities.

The current results suggest that the number of methanogens associated with ciliates was modulated by feeding. In this experiment, the maximal number of methanogens per ciliate reached 103–104. The values are similar to those observed in several aquatic protozoa, such as Metopus striatus, Plagiopyla frontata and Mastigella sp., but much lower than for Pelomyxa palustris, reported to have 9.8×108 endosymbiotic methanogens per cell [16]. However, both endosymbiotic and adherent methanogens were included in the present results.

The large amount of change in methanogen numbers (100–1000-fold) occurring in the 3 h after feeding suggests a rapid change in the number of methanogens extracellularly associated with the ciliates. It can also indicate the rapid and intensive engulfment of methanogens by the ciliates. Methanogens should maintain their activity for at least 1 h after engulfment in the latter case. The resistance of methanogens against predation and digestion by ciliates has not been determined so far.

Characterization of the methanogens that associate with rumen ciliate protozoa has not been reported. Preliminary observations showed that Methanobrevibacter spp. of rumen origin failed to establish interspecies hydrogen transfer with P. multivesiculatum even though these methanogens are dominant species in the rumen [17]. This suggests that the methanogens associated with the ciliates may have special characteristics. Indeed, a specific relation between endosymbiotic methanogens and their host ciliates has been pointed out in the case of some anaerobic ciliates of ruminal and other environmental origin [4, 17].

To test the hypothesis that free-living and ciliate-associated methanogens are different in physiological characteristics, we examined the halophilism and resistance to ammonium salt of methanogens, because halophilism and NH4Cl sensitivities have been shown to be different among the species [18]. When ciliates were removed from the SRF, the effect of NaCl and NH4Cl on the methane production rate was different from that observed with SRF. Methanogens in the SRF were present in at least two fractions, free-living and ciliate-associated. When the ciliates were removed from the SRF, only the free-living methanogens remained. In these cultures, an excess of hydrogen was supplied, so that the activities of the ciliates and the hydrogen-producing bacteria could be ignored. Although the sizes of the two methanogenic populations were not determined, the difference in halophilism and tolerance to ammonia appear to have resulted from the physiological difference between the free-living and ciliate-associated methanogens. The former methanogens may be less halophilic and less tolerant to ammonia than the latter. It is noteworthy that Methanobrevibacter spp., the predominant ruminal methanogens, are not halophilic [19]. These methanogens may be present primarily in the free-living pool. Among the ciliate-associated methanogens, the endosymbiotic strains may have different characteristics to those that adhere externally.

Formate accumulated only in the protozoal cultures prepared prior to feeding (0 h) when an antibiotic mixture without chloramphenicol was added. This suggests that the mixed ciliates produced formate during fermentation in this experiment. The absence of formate in the methanogenic culture of ciliates taken after feeding suggests that the methanogens present used this acid. However, as formate accumulated in the culture prepared before feeding, methanogens present in this culture were probably not using it. In this particular inoculum (0 h), the number of extracellularly associated methanogens in the ciliate preparations was relatively small (Fig. 2). Thus, it may be that the methanogens that colonize ciliates before and after feeding have different preferences for electron donors. Although the characterization of endosymbiotic methanogens in rumen ciliates has not yet been reported [4], endosymbiotic strains may be very special as indicated above for the endosymbiotic methanogens of aquatic protozoa [20].

Endosymbiotic methanogens in rumen ciliate protozoa have been discovered only recently. Further research is required to understand the relationship between rumen ciliates and methanogenic bacteria.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

The authors are grateful to Dr. G.J. Faichney for his critical reading of the manuscript. This study was partly supported by a Grant-in-Aid for Scientific Research (Grant No. 07660364) from the Ministry of Education, Science, Sport and Culture, Japan.

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  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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