Influence of methylamine on anaerobic rumen bacterial growth and plant fiber digestion

Bioresource Technology 50 (1994) 253-257 Elsevier Science Limited Printed in Great Britain 0960-8524(94)00098-0

ELSEVIER

INFLUENCE OF METHYLAMINE ON A N A E R O B I C R U M E N BACTERIAL GROWTH A N D PLANT FIBER DIGESTION S. C. Ricke* Department of Poultry Science, Texas A &M University, College Station, Texas 77843-2472, USA

&

D. M. Schaefer & T. S. Chang Departments of Meat and Animal Science and Bacteriology, Universityof Wisconsin, Madison, W153706, USA (Received 15 April 1994; revised version received 25 August 1994; accepted 19 September 1994)

Pisulewski et al., 1981). One possible explanation for the discrepancy between the in vitro and in vivo results may be that specific dietary components give rise to antimetabolites of NH 3. Another base volatile nitrogen compound, methylamine, has often been detected in rumen fluid (Hill & Mangan, 1964; Itabashi & Kandatsu, 1978; Wallace, 1979). Methylamine has been found in the rumen contents of cattle and sheep fed various diets, with concentrations ranging between 0.45 and 1-2 mM (Hill & Mangan, 1964; Wallace, 1979). Rumen methylamine concentrations have been shown to peak approximately 5 h post feeding, when NH 3 concentrations are the lowest, and at various times after feeding can be equal to 2-36% of the rumen NH 3 concentration (Hill & Mangan, 1964; Patterson & Hespell, 1979). Methylamine is thought to be formed in the rumen via demethylation of the dietary constituents betaine and choline (Aft et al., 1982; Neill et al., 1978) and trimethylamine (Hippe et al., 1979). Both choline and betaine occur widely in plants (Aii et aL, 1982) and the main dietary source of choline in ruminant diets is phosphotidylcholine from plant membrane material (Neill et al., 1978). The consequences of the addition of methylamine and other N-alkyl amines on growth and metabolism of the rumen microbial population are unclear. They can be used by some rumen methanogens as methyl sources (Hippe et al., 1979; Neill et al., 1978; Patterson & Hespell, 1979) but apparently have no nutritional value for the saccharolytic rumen bacteria (Patterson & Hespell, 1979). However, this does not imply that methylamine is physiologically inert. Many investigators have used ~4C labelled methylammonium as an ammonium analog to measure ammonium transport (see Kleiner (1981) for reviews). Growth of nitrogen-limited Escherichia coli is completely inhibited by high concentrations of methylamine (Servin-Gonzalez et al., 1987). We also observed inhibition of growth rate in the rumen microorganism Selenomonas ruminantium when methylamine con-

Abstract Anaerobic rumen bacteria were screened for growth inhibition by methylamine. Under nitrogen-limited growth conditions only Ruminobacter amylophilus, Prevotella ruminicola and Megasphaera elsdenii were significantly inhibited. Ruminobacter amylophilus was inhibited more than any other species. Inhibition of R. amylophilus growth occurred at all increments in methylamine for all NH4C1 concentrations tested (0"2-1"8 mm). Dimethylamine was less inhibitory, while trimethyl-, ethyl- and diethylamine were not inhibitory. When methylamine was added to in vitro mixed culture, ruminal fermentations containing alfalfa, neutral detergent fiber disappearance and ammonia production were not affected. This suggests that although methylamine may inhibit specific rumen bacteria, it does not alter overall mixed ruminal microorganism activities with alfalfa as a substrate.

Key words: Methylamine, rumen, Ruminobacter amylophilus, ammonia, digestion.

INTRODUCTION

Ammonia-nitrogen (NH3-N) is a key intermediate in metabolic nitrogen transformations in the rumen. Rumen NH 3 concentration varies with diet and time after feeding and can range between 2 and 40 mM (Church, 1969; Hespell, 1984; Wohlt et al., 1976). Based on NH 3 saturation constants estimated for predominant species of rumen bacteria, Schaefer et al. (1980) found that 1 mM NH 3 was sufficient to support maximum specific growth rate. However, in vivo estimates for maximum microbial protein synthesis are much higher, ranging between 6"3 mM (Hume et al., 1970) and 19.7 m u (Leibholz & Kellaway, 1980; *To whom correspondence should be addressed. 253

254

S. C. Ricke, D. M. Schaefer, T. S. Chang

centration was increased above 10mM (Ricke & Schaefer, 1991 ). Our objective in this paper is to determine whether methylamine is a potential antimetabolite of NH 3 in the rumen, by screening additional rumen bacterial species for methylamine sensitivity and determining if methylamine influences NH 3 production and plant fiber degradation when it is added to in vitro mixed cultures. METHODS

All strains of rumen bacteria were obtained from M. P. Bryant and B. A. White (Department of Animal Sciences, University of Illinois, Urbana, IL) except Streptococcus bovis strains C277 and JB1, which were obtained from G. A. Broderick and R. J. Wallace (Microbiology Department, Rowett Research Institute, Bucksburn, Aberdeen, Scotland) and J. B. Russell (Department of Microbiology, Cornell University, Ithaca, NY), respectively. Maintenance, confirmation of culture purity and anaerobic technique have been described previously (Ricke et al., 1988). Streptococcus bovis strains were grown in the defined medium described by Ricke et al. (1988). All other cultures were grown in defined media with glucose or cellobiose (glucose for all organisms except R. albus and R. flavefaciens) as the carbon source (final pH 6"5), as described by Schaefer et al. (1980), except volatile fatty acid stock solutions were not adjusted to pH 7 with NaOH. Selenomonad strains were grown on the defined medium described by Ricke et al. (1988), except FeSO4.7H20 was deleted. Stock solutions of NH4C1 and amine-HCl compounds were made with deionized, distilled water and stored at 4°C. Culture inocula were pre-adapted to test media via at least two serial transfers of 0"1 ml inoculum to 4.0 ml of test medium followed by 12 h (overnight) growth periods. Prevotella ruminicola and Megasphaera elsdenii were shifted to growth on NHnC1 as the sole nitrogen source by culturing first on medium containing 0"025% cysteine and transferring until consistent growth was obtained in a nitrogen-free basal medium (Schaefer et al., 1980) supplemented with NH4C1. In studies examining the effect of methylamine and other amines on NH4Cl-limited growth of R. amylophilus strain H18, all nutrients were kept at the same concentration, as reported by Schaefer et al. (1980), except maltose, which was increased to 15 mM. Failure to grow in a cysteine medium containing no NH4C1 established that cysteine was not used as a nitrogen source by R. amylophilus strain H18, and that it could be used as the reducing agent. Culture growth was monitored on a Spectronic 70 spectrophotometer (Bausch and Lomb, Rochester, NY) with absorbance at 600 nm (A600), with a light length of 10 mm and maximum growth rate calculated from duplicate runs of triplicate tubes, as described previously (Ricke & Schaefer, 1991). Maximum optical density (MOD) was defined as the highest reading during the stationary phase after logarithmic growth.

For in vitro mixed culture fermentations, an inoculum was obtained from a rumen-cannulated Jersey cow fed a hay diet. A 50% (v/v) collection of sofid and liquid rumen contents was taken from the cow, 4 h after feeding. The contents were transported to the nearby laboratory in stoppered jars in a styrofoam insulated container, blended and filtered through eight layers of cheesecloth into a prewarmed (39°C) container under a continuous stream of CO2. An anaerobic buffer (McDougall, 1948) was boiled under a stream of CO2, stoppered and autoclaved before cysteine.HC1.H20 was added. Strained rumen fluid was then mixed with the buffer at a 2: 3 ratio. The fiber substrate consisted of immature alfalfa, ground to obtain a homogenous mixture of leaves and stems and contained 26% recoverable neutral detergent fiber (NDF) residue (Goering & Van Soest, 1975; Robertson & Van Soest, 1977). The in vitro rumen fermentation technique used was modified from Barnes (1967). Triplicate sterile 50 ml tubes containing 1 g of alfalfa, 12.5 ml of rumen fluid-buffer mixture and 2.5 ml of methylamine solution were incubated at 39°C in a convection oven. Final methylamine concentrations were 0, 0"05, 0"5 and 5 mM. Tubes were vented and mixed every 12 h for each experiment and fermentation was stopped by adding 15 ml NDF solution (Goering & Van Soest, 1975)followed by storage at 5°C until fiber analysis. Fiber disappearance was measured using NDF anlaysis (Goering & Van Soest, 1975) with the enzyme modification of Robertson and Van Soest (1977). Before deactivation of the fermentations with NDF solution, a 1 ml sample of liquid was removed from each incubation tube using a syringe and a 20 gauge needle. Bacterial growth in these 1 ml samples was stopped with 0"1 ml of concentrated sulfuric acid. A phenol-hypochlorite assay (Broderick & Kang, 1980) was used to estimate the concentration of N H 3 in incubation tubes after determining that methylamine and cysteine did not interfere with N H 3 measurements. The total sum of squares for the effect of methylamine on rumen bacterial species was partitioned among species, M O D or growth rate, species × M O D or species x growth rate, and error. The total sum of squares for the effect of other amines on R. amylophilus H18 was partitioned among amine, M O D or growth rate, amine x M O D or amine x growth rate, and error. The total sum of squares for the effect of methylamine concentration on mixed culture fermentation was partitioned among percent fiber disappearance or NH 3 concentration and methylamine concentration and time of incubation. If significant (P<0.05) sources of variation were found, multiple comparisons between treatments were made by the least significant difference method (Canner & Swanson, 1973). RESULTS Data are shown in Table 1 for the effects of NH4CI and methylamine additions on M O D of NH4Cl-grown

Methylamine and rumen bacteria

255

Table 1. Effects of ammonium chloride and methylamine (MA) on maximum optical density (MOD) of ammonia-limited cultures of rumen bacteria

Bacteria

Ruminobacteramylophilus lYevotella ruminicola Butyrivibrio fibrisolvens Megasphaera elsdenii Ruminococcus albus Ruminococcusflavefaciens Selenomonas ruminantium subsp, lactilytica subsp, ruminantium Streptococcus bovis

Strain

1 mM NH4C1

1 mM NH4CI + 10 mM MA

11 rnM NH4C1

11 mM NH4CI + 10 mM MA

H18 70 B14 D1 T 81 7 FD1

0"96 b 0"88 ~ 0-82 b 0"53 b 0-88 b 0.48 b 0.50 b,'

MOD (A~oo)a 0'37' 0"37' 0-65' 0.45 h 0"58 c 0-49 b 0.42'

1"32a 1"40a 1"40 d 0.69 " 1.40 d 1.45 ' 0.59'

1"32 d 1"40a 1"40d 0-69' 1"40d 1"45 ' 0"50 b,'

PC18 GA192 C277 JB1

0-96 b 1"32 h 0"45 h 0"42 b

1'09 h,d 1"38 b.d 0"54h 0"46 b

1-29" 1"67 ' 1'08' 1-29'

1-22 ''a 1'51 d 1"08 '~ 1"24'

"Standard error of the means (n = 2) = + 0'05(A600). ~'-aMeans in same row with different superscripts differ (P < 0"05). cultures of rumen bacterial species. M O D was increased ( P < 0 - 0 5 ) for all species when NH4CI was increased from 1 mM tO 1 1 n ~ , except for Ruminococcusflavefaciens, indicating that all species, except R. flavefaciens, were nitrogen-limited when growing on 1 mM NH4CI. T h e addition of 10 mM methylamine to 1 mM NH4C1 medium reduced M O D in R. amylophilus, P. ruminicola and M. elsdenii strains, but did not change M O D in any other rumen bacterial sepcies tested. T h e combination of 10 mM methylamine and 11 mM NH4C1 did not alter M O D for any species when compared to M O D of cells grown on 11 mM NH4CI alone. Since M O D s of the Ruminobacter amylophilus strains were so drastically reduced in the presence of 10 mM methylamine (62 and 58% reduction, respectively, versus 34% for the next most inhibited species, Megasphaera elsdenii), we chose the more characterized strain H 1 8 to determine the specificity of inhibition by amines. Initial studies with maltose as the carbon source (data not shown) indicated methylamine would not support growth of R. amylophilus when supplied as the sole nitrogen source. W h e t h e r cells were previously adapted to a medium containing either methylamine and NH4CI or a low NH4C1 concentration alone, the cells failed to grow on methylamine. In a series of preliminary studies, the concentration range of NH4Cl-limited growth was established. Optical density response remained linear (R 2= 0-95) from 0"2 to 2"0 mM NH4CI and appeared to reach a plateau at 2.0 mM (data not shown). Cell dry weight increase was linear (R2=0"98) from 0-75 to 4"0 mM NH4C1, then remained constant as NH4C1 was increased from 5 to 12 mM. Therefore, we chose 1 mM NHaCI for all amine inhibition comparisons, since it was well within the range of N-limited growth but still supported sufficient growth for measurable cell density. W h e n R. amylophilus was grown with varying amounts of either methylamine, dimethylamine, trimethylamine, ethylamine or diethylamine in combination with 1 mM

Table 2. Effect of structurally similar alkyl amines on maximum optical density (MOD) of ammonia-limited cultures of R. Amylophilus strain HI 8

Amine-HCl (mM)" Amine

0.25

0"50

1.00

2"00

4.00

6"00

Methyl Dimethyl Trimethyl Ethyl Diethyl

0.59 0-73 0.74 0-70 0.72

0.54 0"71 0-71 0.69 0.73

MOD (A600)a 0.48 0"44 0"68 0.68 0.74 0.78 0.72 0.70 0.73 0.74

0.40 0"66 0.81 0.69 0.75

0.37 0"65 0.77 0.67 0.76

~NH4CI concentration was kept constant at 1 mM across all treatments and maximum optical densities for cultures grown with 1 mM NHaCI alone were 0.76 + 0"01. hSEM = + 0.04(A600).

NH4C1 (see Table 2 for ratios), growth rate (data not shown) was not affected ( P > 0 . 4 3 ) . However, all methylamine concentrations lowered M O D to 4 9 - 7 8 % of control values (Table 2), while dimethylamine was less inhibitory and trimethyl-, ethyl-, and diethylamine were not inhibitory. Methylamine suppression of M O D for R. amylophilus was examined further with a wide range of methylamine to NHaCI ratios (Fig. 1). Both NH4CI concentration and methylamine concentration affected ( P < 0.0001) M O D and there was a NH4C1 x methylamine interaction (P<0"034). Increments in methylamine for all NH4C1 concentrations suppressed M O D ( P < 0 . 0 0 3 ) up to a ratio of 6, above which no further suppression was observed and minimum M O D were 50% of control values. Methylamine concentrations as low as 50 pM (0"2 mM NHnCI ) reduced M O D for R. amylophilus. G r o w t h rate was not affected by methylamine concentration (P>0"23), nor was an NHnCI × methylamine interaction observed ( P > 0"99; data not shown) for growth rate.

S. C. Ricke, D. M. Schaefer, T. S. Chang

256 mM NH~Cl

14 T

1,,~.,,Q4,,08 noe,,1

40 J"

m12 |1.4 nl.6 |1.~ z-

12

.× •

J

•

3O

|

~f~

j j

20 z

•~ 116

10

24

48

72

96

Time (H) 0

0.25

0.5

1

2

4

6

8

• 0

10

Methylamine • 5.0 mM mM )~0.05mM -*-0.5mM

MA:NH4Cl Ratio

Fig. 1. Effect of methylamine chloride :ammonium chloride ratio (MA:NH4CI) on maximum optical density (A6oo) of Ruminobacter amylophilus H 18.

Methylamine was added to in vitro mixed culture fermentations to assess whether methylamine would affect the diverse microbial population present under in vitro plant digestion conditions. Influence on microbial activity was assessed as a function of cell wall disappearance and NH 3 production. The effect of methylamine concentration on digestion of alfalfa cell walls is shown in Fig. 2. There was a significant (P< 0"05) increase in the percent of NDF disappearance over time for all methylamine concentrations. However, methylamine concentration did not affect NDF disappearance, except for slightly less digestion at 96 h for mixed cultures receiving 0"5 mM methylamine. The effect of methylamine concentration on N H 3 production in mixed cultures is shown in Fig. 3. There was a significant (P< 0"05) increase in N H 3 concentration from 24 h to 96 h for all methylamine levels. However, any influence attributable to methylamine concentration was minimal as only a statistical (P< 0"05) decrease in measured N H 3 concentration at 0 h and an increase at 24 h for 0"5 and 5 mM methylamine cultures, respectively, were observed. DISCUSSION The failure of rumen bacteria to increase M O D when methylamine was addded to 1 mM NH4C1 cultures (Table 1) is in agreement with Patterson and Hespell (1979), who reported that methylamine would not support growth of saccharolytic rumen bacteria. The extensive inhibition of M O D in R. amylophilus strains H18 and 70 is apparently peculiar to R. amylophilus, since no other organisms tested were inhibited to the same degree. Such sensitivity to methylamine has been observed in non-rumen bacteria, but at much higher concentrations of methylamine. Servin-Gonzalez et al. (1987) found that E. coli sensitivity to 100 mM methylammonium required glutamine synthetase activity and that resistant mutants possessed an altered glutamine synthetase enzyme. The sensitivity of R. amylophilus to much lower concentrations (50/am) of methylamine than E. coli when cell growth was NH3-N-limited may

Fig. 2.

Effect of methylamine on NDF digestion.

J 4 i /

0

0

24

48

72

96

Time (1t)

[

*0mM

Fig. 3.

-""

10.06mM -)~0.5mM ~-5mM

1

Effect of methylamine on ammonia concentration during NDF digestion.

be due to some unusual property of its NH3-N assimilation physiology. Support for this hypothesis is based on two observations. First of all, R. amylophilus was inhibited by methylamine only when it was nitrogenlimited and not otherwise. Secondly, even when R. amylophilus was nitrogen-limited, it was only inhibited substantially by methylamine and not by any of the other short chain akylamines. Consequently, if amine permeability was the cause of inhibition, akylamines with pKa profiles similar to methylamine, such as ethylamine (Segel, 1976; Ritchie & Gibson, 1987) should cause an equally marked reduction in growth. When methylamine was added to in vitro mixed culture fermentations, there was little discernible change in NDF disappearance and only slight increases in N H 3 production at the higher methylamine concentrations. Based on the inhibition profile of R. amylophilus H18 in Fig. 1, the observed N H 3 concentration and the resulting methylamine:NH 3 ratio were certainly within the inhibitory range for this species. However, since 7 of the 10 rumen bacterial strains in Table 1 were not sensitive to methylamine, there is probably sufficient diversity among resistant rumen bacterial strains to overcome any effects resulting from inhibition of a single species. In conclusion, although methylamine present at physiological concentrations could impose selective pressure on individual members of the rumen microflora, its inhibitory nature does not

Methylamine and rumen bacteria appear to be general enough to cause a detectable change in N D F degradation or N H 3 production during in vitro fermentation of alfalfa. Apparently, the overall rumen population compensates sufficiently to prevent any alteration in rumen fermentation of this substrate, but it is unknown whether this would also be true for substrates that support different rumen microbial populations.

ACKNOWLEDGEMENT This research was supported by Hatch grants 6168 and 2629, which were administered by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, WI 53706. This paper has been approved as technical article n u m b e r T A # 3 1 3 3 5 by the Texas Agricultural Experiment Station.

REFERENCES Aii, T., Yonaga, M. & Tanaka, H. (1982). Trimethylamine oxide concentration in the rumen of cattle and its origins. Japan. J. Zootech. Sci., 53, 565-73. Barnes, R. F. (1967). Collaborative in vitro rumen fermentation studies on forage substrates. J. Anita. Sci., 26, 1120-30. Broderick, G. A. & Kang, J. H. (1980). Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media. J. Dairy Sci., 63, 64-75. Carmer, S. C. & Swanson, M. R. (1973). An evaluation of ten pairwise multiple comparison procedures by Monte Carlo methods. J. Am. Statist. Assoc., 68, 66-74. Church, D. C. (1969). Digestive Physiology and Metabolism of Ruminants. O&B Books, Corvallis, Washington. Goering, H. K. & Van Soest, P. J. (1975). Forage fiber analyses. Agriculture Handbook, No. 379, 1-20. Hespell, R. B. (1984). Influence of ammonia assimilation pathways and survival strategy on ruminal growth. In Herbivore Nutrition in the Subtropics and Tropics, ed. F. M. C. Gilchrist & R. I. Mackie. The Sciences Press, South Africa, pp. 346-58. Hill, K. J. & Mangan, J. L. (1964). The formation and distribution of methylamine in the ruminant digestive tract. Biochem. J., 93, 39-45. Hippe, H., Caspari, D., Fiebig, K. & Gottschalk, G. (1979). Utilization of trimethylamine and other N-methyl compounds for growth and methane formation by Methanosarcina barkeri. Proc. Nat. Acad. Sci. USA, 76, 494-8.

257

Hume, I. D., Moir, R. J. & Somers, M. (1970). Synthesis of microbial protein in the rumen. I. Influence of the level of nitrogen intake. Australian J. Agric. Res., 21,283-96. Itabashi, H. & Kandatsu, M. (1978). Formation of methylamine by rumen microorganisms. Japan. J. Zootech. Sci., 49, 110-18. Kleiner, D. (1981). The transport of NH 3 and N H ; across biological membranes. Biochim. Biophys. Acta, 639, 41-52. Leibholz, J. & Kellaway, R. C. (1980). The nitrogen requirement of steers fed alkali-treated straw ad libitum. Proc. Australian Soc. Anim. Prod., 13,481. McDougall, E. I. (1948). Studies on ruminant saliva. I. The composition and output of sheep saliva. Biochem. J., 43, 99-109. Neill, A. R., Grime, D. W. & Dawson, R. M. C. (1978). Conversion of choline methyl groups through trimethylamine into methane in the rumen. Biochem. J., 170, 529-35. Patterson, J. A. & Hespell, R. B. (1979). Trimethylamine and methylamine as growth substrates for rumen bacteria and Methanosarcina barkeri. Curr. Microbiol., 3, 79-83. Pisulewski, P. M., Okorie, A. U., Buttery, P. J., Haresign, W. & Lewis, D. (1981 ). Ammonia concentration and protein synthesis in the rumen. J. Sci. FoodAgric., 32,759-66. Ricke, S. C., Schaefer, D. M., Cook, M. E. & Kang, K. H. (1988). Differentiation of ruminal bacterial species by enzyme-linked immunosorbent assay using egg yolk antibodies from immunized chicken hens. Appl. Environ. Microbiol., 54, 596-9. Ricke, S. C. & Schaefer, D. M. (1991). Growth inhibition of the rumen bacterium Selenomonas ruminantium by ammonium salts. Appl. Microbiol. Biotech., 36, 394-9. Ritchie, R. J. & Gibson, J. (1987). Permeability of ammonia, methylamine and ethylamine in the cyanobacterium, Synechococcus R-2 (Anacystis nidulans) PCC 7942. J. Membrane Biol., 95, 131-42. Robertson, J. B. & Van Soest, P. J. (1977). Dietary fiber estimation in concentrate feedstuffs. J. Anim. Sci., 45 (Suppl. 1), 254. Schaefer, D. M., Davis, C. L. & Bryant, M. P. (1980). Ammonia saturation constants for predominant species of rumen bacteria. J. Dairy Sci., 63, 1248-63. Segel, I. H. (1976). Biochemical Calculations. John Wiley & Sons, New York, NY. Servin-Gonzalez, L. O., Gonzalez, A. & Bastarrachea, F. (1987). glnA Mutations conferring resistance to methylammonium in Escherichia coli K12. J. Gen. Microbiol., 133, 1631-9. Wallace, R. J. (1979). Effect of ammonia concentration on the composition, hydrolytic activity and nitrogen metabolism of the microbial flora of the rumen. J. Appl. Bacteriol., 47,443-55. Wohlt, J. E., Clark, J. H. & Blaisdell, E S. (1976). Effect of sampling location, time, and method on concentration of ammonia nitrogen in rumen fluid. J. Dairy Sci., 59, 459-64.