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Journal of Dairy Science Vol. 85 No. 8 2015-2022
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Stimulatory and Inhibitory Effects of Protein Amino Acids on Growth Rate and Efficiency of Mixed Ruminal Bacteria

H. Kajikawa, M. Mitsumori and S. Ohmomo1

National Institute of Livestock and Grassland Science, Tsukuba Norindanchi, Ibaraki 305-0901, Japan

Corresponding author:
H. Kajikawa; e-mail:
kajikawa{at}affrc.go.jp.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Mixed ruminal bacteria were incubated in vitro with glucose, xylose, cellobiose, and various protein amino acids replaced isonitrogenously with 25% (i.e., 25 mg of N/L) of ammonia-N, to determine the growth rate and the amount of sugar consumed in the exponential growth phase. The growth rate and efficiency (grams of bacteria per gram of sugars) increased by 46 and 15%, respectively, when a mixture of 20 amino acids was added. On the other hand, neither growth rate nor efficiency increased when any one of these amino acids was added singly, except for Glu and Gln, each of which produced significant but small improvements. The stimulatory effect of the combined amino acids on bacterial growth declined when each of Leu, Trp, Tyr, Glu, Met, Phe, and Val was removed from the original group of 20. When a mixture of only these seven amino acids was used as a supplement, their stimulatory effects on growth rate and efficiency were only 21 and 25%, respectively, of the effects that the mixture of 20 amino acids showed. The effects increased to 76 and 72% on growth rate and efficiency, respectively, when Gly, Cys, and His were supplied in addition to the seven amino acids. The growth rate and efficiency of the ruminal bacteria were inhibited by an addition of each of Ile, Thr, Cys, Phe, Leu, Lys, or Val to ammonia-N, and the effects of the first five of these amino acids were highly significant. Isoleucine, threonine, and phenylalanine were each inhibitory even at a low concentration (1 mg of N/L), while cysteine and leucine showed inhibitory effects at higher concentrations (more than 10 mg of N/L). A higher growth rate of the ruminal bacteria when supplemented with amino acid mixtures was accompanied with a higher growth efficiency, which was attributable to a relatively smaller proportion of energy expended on maintenance according to the Pirt derivation.

Abbreviation key: SAA = seven amino acids

Key Words: rumen bacteria • amino acid • inhibition • stimulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Estimation of microbial synthesis in the rumen is one of the main components in the metabolizable protein system (Fox et al., 1990; AFRC, 1993; National Research Council, 1996). Since the amount of dietary energy available to the microorganisms often restricts the amount of microbial protein synthesized in the rumen, improving the efficiency of microbial growth (i.e., the amount of microbes synthesized per dietary energy consumed) would increase postruminal protein supply. Most ruminal bacteria can grow with nonprotein N, such as ammonia, as their sole N source (Bryant and Robinson, 1962), but supplementation with amino N can improve the growth yield (Maeng et al., 1976; Argyle and Baldwin, 1989), rate (Van Kessel and Russell, 1996), and efficiency (Cotta and Russell, 1982) of ruminal microbes. Several studies showed that certain amino acids or amino acid subgroups stimulated in vitro growth yields of mixed ruminal bacteria, although to a lesser degree than the stimulation provided by whole amino acid mixtures (Maeng et al., 1976; Argyle and Baldwin, 1989). But those reports did not identify the amino acids that were truly essential for improving microbial growth. Several amino acids, on the other hand, are known to inhibit microbial growth (De Felice et al., 1979), and it follows that some mixtures of amino acids might include inhibitory members that diminish the benefits provided by the other members.

The present study was designed to determine 1) which ones among the 20 {alpha}-amino acids that are commonly found in proteins would stimulate, and which would inhibit, microbial growth; 2) which amino acid would be indispensable to the improvement of microbial synthesis that amino acid mixtures are expected to show; and 3) which combination of amino acids would be most efficient for microbial growth in the rumen. These determinations were made by analyzing the growth rate and efficiency of mixed ruminal bacteria during the exponential growth phase in the presence or absence of single or combinations of specific amino acids.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Animals and Diet
Two ruminally fistulated nonlactating Holstein cows (580 kg of average BW) were housed in individual stalls, and were each fed a diet (3.3 kg average DM per meal) consisting of timothy hay (67% on DM), steam-flaked corn grain (21%), and soybean meal (12%) twice a day, at 0900 and 1700 h. This diet satisfied the cows’ energy requirement for maintenance and balanced N degradation and consumption in the rumen based on the level 2 model of the National Research Council (1996). Water was freely given.

Preparation of Mixed Ruminal Bacteria
Liquid and solid portions of the ruminal content, taken by a suction pump and by hand grasp, respectively, were provided through a fistula just before morning feeding. Equal amounts (wt/wt) of these portions were mixed, ground with a homogenizer (model MN-2, Nihon Seiki Co., Tokyo, Japan) at 250 W for 1 min, and squeezed through four-layered gauze. The squeezed fluid was left undisturbed for 30 min at 39°C to separate the feed particles. The fluid obtained from a middle section of the bottle of the undisturbed sample was slowly centrifuged (at 750 x g for 10 min at 10°C) to remove protozoa and then centrifuged again (at 10,000 x g for 15 min at 10°C) to harvest the mixed ruminal bacteria, in which no protozoa was microscopically detected. The mixed ruminal bacteria were washed twice with a buffer (pH 6.8) containing 50 mM Na2HPO4, 10 mM KH2PO4, 4.2 mM Na2S·9H2O, and 4 µM sodium resazurin, and resuspended in the same buffer in a volume equivalent to the original. Anaerobic conditions were maintained throughout the procedure by using an N2 gas stream.

Incubation of Mixed Ruminal Bacteria
The mixed ruminal bacteria were diluted 20 times, thereby attaining an optical density (at 600 nm) of 0.15, into a medium (pH 6.5) containing 2.4 mM K2HPO4, 1.8 mM KH2PO4, 2.1 mM NaCl, 0.1 mM MgSO4·7H2O, 0.01 mM CaCl2·2H2O, 4.2 mM Na2S·9H2O, 4 µM sodium resazurin, 38 mM Na2CO3, and the same amounts of vitamins, VFA, and trace minerals as described by Cotta and Russell (1982). The mixed bacteria were anaerobically incubated at 39°C with 4 mM glucose, 4 mM xylose, and 2 mM cellobiose. For the NH3 only treatment, 3.57 mM (i.e., 100 mg of N/L) of ammonium sulfate was added as a sole N source. In experiments 1 and 3, 25% of ammonia in the NH3 only treatment was isonitrogenously replaced by a single amino acid or by an amino acid mixture in which the same amounts of amino acids were included on isonitrogenous basis. In experiment 2, various amounts of ammonia were isonitrogenously substituted by individual amino acids, each of which had been shown in experiment 1 to inhibit growth. Growth of bacteria was terminated when the optical density (at 600 nm) reached 0.7, which was the middle of the exponential growth phase, by adding formalin (1% in final concentration). Cells were separated by centrifugation (at 15,000 x g for 15 min at 4°C), and the supernatant was stored at –20°C until sugar analysis.

Analysis
Animal feeds were analyzed by the methods of AOAC (Cunniff, 1996) and Van Soest and Wine (1967). The chemical composition of the diet was as follows (% on DM basis): OM, 94.3; CP, 13.1; ether extract, 2.0; NDF 49.5. Sugars in the medium were analyzed by capillary electrophoresis (3DCE, Hewlett Packard, Waldbronn, Germany) with a capillary column (50 µm i.d., 80.5 cm length) using 20 mM 2,6-pyridinedicarboxylic acid and 0.5 mM cetyltrimethylammonium bromide (pH 12.1) as an electrolyte after the sample was filtered through an ultrafiltration unit (Ultrafree-MC, 30,000 NMWL, Millipore Corp., Bedford, MA; Soga and Heiger, 1998). The DM of the cells was identified after drying at 105°C for 16 h. The protein content of the cells was measured by the method of Lowry et al. (1951) after hydrolyzation with 0.2 M NaOH at 100°C for 15 min; protein content was 165 µg/ml at an optical density (at 600 nm) of 1.0 and 44% of cell DM, which was kept constant during the exponential growth phase.

Statistics
Incubations with each N source were performed in triplicate for each cow. Data are described as both the actual values and percentages of the NH3 only treatment for experiments 1 and 2 and percentages of the effect of the 20 amino acids for experiment 3. The statistical model is described below:


Formula

where

Yij= observation,

µ= overall mean,

Ni= mean effect of N source i,

Cj= mean effect of cow j,

eij= residual error.

Treatments with different N sources were analyzed with ANOVA using the GLM procedure of SAS (1988) with cow as block. When an F-test detected a significant difference (P < 0.05), the LSD method was used to determine the significance of the difference between the treatment means (Snedecor and Cochran, 1967).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
Experiment 1
Table 1Go shows the effects of partially replacing either individual or 20 amino acids with ammonia in the NH3 only treatment. Supplementation of the 20 amino acids stimulated the growth rate by 46% and growth efficiency by 15%. Glutamic acid and glutamine also improved the growth rate significantly when described in the percentage values, but did not improve growth efficiency. On the other hand, each of five amino acids (Ile, Thr, Cys, Phe, and Leu) showed more than 10% inhibitions (P < 0.01) of both growth rate and efficiency at the level of 25 mg of N/L (i.e., 1.8 mM), and other two amino acids (Lys and Val) also inhibited bacterial growth significantly when described in the percentage values. The bacteria with a lower growth efficiency showed more consumption in every sugar supplemented during the incubation than those with a higher growth did (data not shown).


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  		Table 1. Effect of addition of individual amino acids on growth rate and efficiency of mixed ruminal bacteria.
 
The effects of removing one amino acid from the 20 amino acids on growth rate and efficiency are shown in Table 2Go. The removal of Leu, Trp, Tyr, Glu, Met, Phe, or Val significantly decreased the stimulatory effects of the 20 amino acids on bacterial growth; especially, the absence of any of the first four amino acids (Leu, Tyr, Trp, Glu) resulted in a disappearance of significant stimulation on the bacterial growth.


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  		Table 2. Effect of removal of individual amino acids on growth rate and efficiency of mixed ruminal bacteria.1
 
Experiment 2
Figure 1Go shows the effect of concentrations of the five amino acids that showed highly inhibitory effect (P < 0.01) on both the growth rate and efficiency in experiment 1. Isoleucine, threonine, and phenylalanine showed inhibitory effects even at low concentrations (1 or 2.5 mg of N/L), whereas Leu significantly inhibited microbial growth at more than 10 mg of N/L. Cysteine showed a stimulatory effect on bacterial growth at low levels (1 and 2.5 mg of N/L), but showed an inhibitory effect at higher levels (more than 10 mg of N/L).


Figure 1
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  		Figure 1. Inhibitory effect of Cys ({blacksquare}), Leu ({circ}, •), Phe ({blacktriangleup}), Thr ({blacktriangleup}), and Ile (x) on growth of the mixed ruminal bacteria. All values with amino acids, except for Leu at 1 and 2.5 mg of N/L (open circles), significantly differ (P < 0.05) from values of the NH3 only treatment (i.e., 0 mM of amino acid). Pooled SEM: 1.7.

 
Experiment 3
The effects of some combinations of amino acids on the growth of the mixed ruminal bacteria are shown in Table 3Go. When the seven amino acids (SAA) whose removal decreased the stimulatory effect of the mixture of 20 amino acids on bacterial growth in experiment 1 were combined and used as a supplement, they produced only 21 and 25% of the stimulatory effects (on growth rate and efficiency, respectively) that the mixture of 20 amino acids did. The addition of mixtures of amino acids in the oxaloacetate and pyruvate family (i.e., Ala, Asn, Asp, and Lys), or in the {alpha}-ketoglutarate family (i.e., Arg, Gln, and Pro) to SAA provided no additional stimulation to the bacterial growth. However, amino acid mixtures in the 3-phosphoglycerate family (i.e., Cys, Gly, and Ser) showed significant improvement in both the growth rate and efficiency when added to SAA; among the acids in this family, Gly was most effective. The addition of Gly, Cys, and His to SAA increased the stimulatory effects to 76 and 72% (on growth rate and efficiency, respectively) of those shown by the mixture of 20 amino acids.


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  		Table 3. Optimal combination of amino acids for improving growth rate and efficiency of mixed ruminal bacteria.
 
Growth rates and efficiencies obtained from the whole experiments were plotted according to the derivation of Pirt (1965) in Figure 2Go. The plot was highly linear, and the maximum growth efficiency (1/intercept) and the maintenance requirement (slope) could be determined.


Figure 2
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  		Figure 2. Relationship between growth rate and efficiency of the mixed ruminal bacteria applied to the Pirt equation. y = 0.467x + 2.83, r2 = 0.745.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
The present study showed that the addition of a mixture of 20 amino acids consisting of proteins improved both the growth rate and efficiency of mixed ruminal bacteria, but when an amino acid was added solely, only Glu and Gln produced significant, albeit small, stimulations of growth; the latter finding concerning Glu was previously reported by Maeng et al. (1976). We postulated from these findings that a certain amino acid, though not having a stimulatory potential by itself, would be indispensable to the improvement of bacterial growth that amino N was expected to produce. The results of the experiment in which specific amino acids were individually removed from the original mixture of 20 amino acids demonstrate that four amino acids, Leu, Tyr, Trp, and Glu, would be essential for improving the bacterial growth, while three other amino acids, Met, Phe, and Val, would be subessential. Fujimaki et al. (1992) reported that no effect was found on the growth yield of mixed ruminal bacteria from a goat when amino acids were individually removed from mixtures of 18 amino acids. Since they used a dense bacterial inoculum at the beginning of incubation (more than 300 µg bacterial protein/ml), some carryover of amino acids from the inoculum may have occurred.

The extents of the decayed improvement (i.e., from 45% for Val to over 100% for Leu, calculated from Table 2Go) by removal of each of the essential and subessential amino acids as noted above from the 20 amino acids were higher than the contents of these amino acids in the ruminal bacteria (i.e., from 1% for Trp to 13% for Glu according to Wallace, 1994). Each of these amino acids is considered to play a more important role as described below in bacterial cells than the average amino acids. Glutamate (and also glutamine) is a key compound used in ammonia assimilation and transamination for the biosynthesis of other amino acids (Reitzer, 1996), which would require considerably high quantities of the amino acid in the cells. Glutamate, in fact, occupies more than half of the intracellular free amino acid pool in both gram-negative and -positive bacteria (Brown and Stanley, 1972). Leucine is known to act as a regulatory signal for Escherichia coli affecting the expression of several operons (Calvo and Matthews, 1994). Adding this amino acid might increase the expression of some genes related to the cell growth of some ruminal bacteria. Ruminal bacteria were reported to synthesize aromatic amino acids from arylacetic acids, showing that they reutilize benzene rings to form carbon skeletons of these amino acids (Kristensen, 1974; Sauer et al., 1975), and supplying aromatic amino acids like Tyr and Trp would be economically beneficial for the growth of the ruminal bacteria. Williams and Moir (1951) showed that bacterial density in the rumen increased with a Met supplementation as compared with a treatment, to which urea was added as a sole N source. Salter et al. (1979) also considered that Met was a limiting amino acid, along with Phe, in the growth of ruminal bacteria. These crucial functions, or some combinations of them, might be concerned with the necessity of these amino acids to improve growth of the ruminal bacteria, although the reason why they showed little effect when added singly has not been made clear in the current study.

The results of experiments 1 and 2 showed that the growth of the ruminal bacteria was inhibited by supplementation with some amino acids, especially Ile, Thr, Phe, Cys, and Leu. The inhibitory effect of amino acids on bacterial growth has been well known for more than 50 yr, but little information on this subject is available for ruminal bacteria. Growth inhibitions by the amino acids mentioned above have been reported for Bacillus anthracis (by Ile, Leu, and Thr; Gladstone, 1939), Streptococcus bovis (by Ile, Leu, and Thr; Washburn, 1948), Escherichia coli (by Cys; Rowly 1953), and for several lactic acid bacteria (by Ile and Leu; Brickson, 1948). Phenylalanine also inhibited the growth of Streptococcus bovis when added with Tyr (Washburn, 1948).

These inhibitions of bacterial growth are supposed to be caused mainly by feedback inhibition of an early enzyme in the synthetic pathway of the amino acid; this inhibition also suppresses the production of other amino acids that use the common enzyme for their syntheses. Threonine inhibited aspartokinase of Escherichia coli, the first enzyme in a multi-branched pathway converting Asp to Lys, Met, and Thr (Stadtman et al., 1961), and it also inhibited homoserine dehydrogenase of Corynebacterium glutamicum, which is used in the Met and Thr biosynthesis (Cremer et al., 1988). Isoleucine caused inhibited {alpha}-acetohydroxy acid synthase, a common enzyme in the synthetic pathways of Ile, Val, and Leu for Escherichia coli (De Felice et al. 1978) and Brevibacterium lactofermentum (Tsuchida and Momose, 1975). This enzyme was also suppressed by leucine for Brevibacterium lactofermentum (Tsuchida and Momose, 1975). Isoleucine, moreover, showed inhibitory effects on homoserine dehydrogenase of Brevibacterium flavum (Miyajima and Shiio 1970), and on threonine deaminase (threonine dehydratase) of Escherichia coli (Eisenstein et al. 1994); the latter inhibition may lead to the accumulation of Thr and then the suppression of aspartokinase. Some toxic compounds may be produced when a large amount of Ile is supplied (Ikeda et al. 1998). Phenylalanine showed an inhibition of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme for the biosynthesis of aromatic amino acids in Escherichia coli (Doy and Brown, 1965). Cysteine does not suppress any activities of the enzymes used for synthesis of other amino acids, but feedback inhibits serine transacetylase, which potentially leads to an accumulation of toxic 3'-phosphoadenosine 5'-phosphosulfate in the cells (Kredich, 1996). Cysteine also acts as a reducing agent, which assures an anoxic condition, but becomes inhibitory when added in high concentrations (Hungate, 1970), which may explain the quadratic pattern of cysteine’s effect shown in experiment 2. Leucine also shows toxicity when it is supplied in high concentrations (more than 10 mg of N/L), although a small amount of this amino acid is considered indispensable for improving bacterial growth.

The decay of growth due to feedback inhibition by an amino acid could be prevented by supplementing another amino acid whose synthesis is also inhibited. This means, in other words, when an amino acid shows an inhibitory effect on rumen bacterial growth, a new requirement for some additional amino acid would arise. Although various kinds of amino acid are usually found in ruminal fluid (Volden et al., 2001), supplementing some antagonistic amino acid may become necessary in order to maintain a satisfactory microbial growth when an animal is fed a diet rich in the inhibitory amino acid. Further qualitative and quantitative studies are needed to clarify the antagonistic relationships among amino acids in relation to effects on microbial growth in the rumen.

To determine the optimal combinations of amino acid for improving microbial growth in the rumen, the seven amino acids whose removal decayed the stimulatory effect should be included because any combinations of amino acids without any of them could not reach the potential the 20 amino acid mixture exhibits. Since a mixture of the SAA only demonstrated 20 to 25% of stimulation that the mixture of 20 amino acids did, these SAA seem necessary but not sufficient for the exhibition of the maximum effect. Among the other amino acids, some additional effects on growth were shown in experiment 3 only when the 3-phosphoglycerate families were added. This was especially evident with Gly, which might be beneficial because of its function as a precursor of purines (Pitts et al., 1961). However, even adding Gly to the SAA along with Cys and His, which also produced some additional effects on the growth rate, did not completely recover the stimulatory effect that the 20 amino acids showed. These findings demonstrate that considering the deficiency or excess of specific amino acids, rather than the optimal combination of amino acids, would be a more practical way to stimulate microbial growth in the rumen.

In the present study, the addition of amino acids affected both the rate and efficiency of bacterial growth in the rumen. A higher efficiency of bacterial growth unlikely is due to their preference to a specific sugar having a higher potential to support bacterial growth because bacteria with a higher efficiency showed less consumption in every supplemented sugar during the same growth yield than those with a lower growth. There is a wide variation in growth efficiency of ruminal microbes according to microbial species and their growth conditions, showing no specific tendency suggesting superiority or inferiority for the growth among sugars (Russell and Wallace, 1997). The relation between rate and efficiency seem to differ according to the kind of metabolic reaction. In the case of sugar utilization, ruminal bacteria shift their transport system of sugar to slower but more efficient ones when environmental concentrations of the sugars decline (Kajikawa et al. 1997/1999). The plot shown in Figure 2Go, however, suggests that a higher growth rate of the ruminal bacteria, when supplemented with amino acid mixtures, would be accompanied by a higher growth efficiency, which would be attributable to a relatively smaller expenditure of total energy on maintenance. Balancing energy and N available for the ruminal microbes is likely important to the stimulatory effect of amino acids, because a higher growth rate with less energy spilling was suggested only when amino N was provided to an energy-excess culture (Van Kessel and Russell, 1996). Moreover, the balance should be considered between the growth rate and the outflow rate from the rumen. When the microbial growth rate is much faster than the outflow rate, the higher growth of the microbes may lead to a more wasteful use of feed components, because more autolysis of the microbial cells likely occurs.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
Seven amino acids, Leu, Trp, Tyr, Glu, Met, Phe, and Val, were indispensable to the stimulatory effect of amino N on ruminal bacterial growth, but using these seven as the sole supplement did not produce sufficient effects. Certain amino acids, especially Ile, Thr, Cys, Phe, and Leu, inhibited bacterial growth, although some other amino acids may antagonistically prevent inhibition. These results suggest that deficiencies or excesses of specific amino acids, rather than the best combination of amino acids, should be considered for more practical exhibitions of the stimulatory effect that amino N is expected to show on microbial growth in the rumen.


   FOOTNOTES
 
1 Current address: Japan International Research Center for Agricultural Sciences, Tsukuba, Japan. Back

Received for publication November 9, 2001. Accepted for publication February 14, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
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