J. Anim. Sci. 2005. 83:644-652
© 2005 American Society of Animal Science
Effect of Escherichia coli wild type or its derivative with high nitrite reductase activity on in vitro ruminal methanogenesis and nitrate/nitrite reduction1
C. Sar*,
B. Mwenya*,
B. Santoso*,
K. Takaura*,
R. Morikawa*,
N. Isogai
,
Y. Asakura,
Y. Toride and
J. Takahashi*,2
* Department of Animal Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan; and
and
Fermentation and Biotechnology Laboratories, Global Foods and Amino Acids Company, Ajinomoto Co. Inc., Kawasaki-ku, Kawasaki-shi, 210-8681, Japan
 |
Abstract
|
|---|
The effects of two kinds of Escherichia coli strains, wild-type E. coli W3110 or E. coli nir-Ptac, which has enhanced nitrite reduction activity, on in vitro CH4 production and nitrate and nitrite reduction in cultures of mixed ruminal microorganisms was investigated using continuous incubation systems. Escherichia coli nir-Ptac, a derivative of wild-type E. coli W3110, was constructed by replacing self promoter of nir BD operon encoding subunits of nitrite reductase in E. coli W3110 by tac promoter to make the expression of nir BD higher and constitutive. The nitrite reductase activity of E. coli nir-Ptac was approximately twice as high as E. coli W3110. The culture media consisted of 400 mL of strained ruminal fluid taken from two nonlactating Holstein cows receiving a basal diet of orchardgrass hay at maintenance level (55 g of DM/kg of BW0.75 daily), and 400 mL of autoclaved artificial saliva. Treatments were arranged in two separate 3 x 3 factorials consisting of nitrate (NaNO3; 0, 5, or 10 mM) without E. coli or inoculated with E. coli W3110 or E. coli nir-Ptac, or nitrite (NaNO2; 0, 1 or 2 mM) without E. coli or inoculated with E. coli W3110 or E. coli nir-Ptac. The control culture contained no chemical or microbial additives. Escherichia coli cells were inoculated into in vitro mixed ruminal cultures at approximately 2 x 108 to 109 cells/mL. Methane production by ruminal microorganisms was decreased markedly (P < 0.001) by the addition of nitrate and nitrite, and by the inoculation of cultures with E. coli W3110 or E. coli nir-Ptac (P < 0.01). With mixed nitrite-containing cultures, E. coli nir-Ptac inhibited (P < 0.001) in vitro nitrite accumulation and CH4 production more than E. coli W3110, which may be due to the tac promoter-enhanced nitrite reductase activity of E. coli nir-Ptac accelerating electrons to be consumed for nitrite reduction rather than CH4 biosynthesis. In conclusion, anaerobic cultures of E. coli W3110 or E. coli nir-Ptac may decrease CH4 production in the rumen. The inoculation of E. coli W3110 or, especially, E. coli nir-Ptac to mixed ruminal microorganisms may decrease nitrite toxicity when ruminants consume high-nitrate-containing forages and when nitrite is applied to abate ruminal CH4 production.
Key Words: Escherichia coli nir-Ptac • Escherichia coli W3110 • Methane • Nitrate • Nitrite
|
Introduction
|
|---|
Because methane represents a loss of feed energy to the animal and a significant source of greenhouse gas, policies to decrease methane emissions have been proposed (Johnson and Johnson, 1995
). Alternatively, the addition of nitrate to populations of ruminal microorganisms decreased ruminal methane production in vitro (Anderson and Rasmussen, 1998) and in vivo (Takahashi and Young, 1991; Sar et al., 2002, 2004a). However, its practical use as an alternative electron acceptor is precluded by the toxicity associated with its reduced intermediate, nitrite (McAllister et al., 1996). Toxic nitrite often accumulates in the rumen due to more rapid nitrate reduction to nitrite than the reduction of nitrite to ammonia when ruminants consume an excess of dietary nitrate (Takahashi, 2001). Hence, developing a method to accelerate the rate of ruminal nitrite reduction to ammonia could decrease the occurrence of nitrite toxicity in ruminants.
Wild-type Escherichia coli W3110 is known to have a certain nitrite reductase activity, in which nitrite reductase, consisting of two kinds of subunits encoded by the nirBD operon, is usually involved in the nitrate respiration and induced under oxygen-limited conditions (Gennis and Stewart, 1996). We have constructed the E. coli strain nir-Ptac with higher nitrite reductase activity, in which the nirBD in E. coli W3110 is expressed constitutively by replacing its promoter with a tac promoter, and we found that the nitrite reductase activity of E. coli nir-Ptac was approximately twice as high as E. coli W3110 (Ajinomoto Co., Inc., Tokyo, Japan). However, the effect of this strain on nitrite reduction within the ruminal ecosystem is unknown.
The objectives of this study were, therefore, to evaluate the effects of nitrate, nitrite, and wild-type E. coli W3110 or E. coli nir-Ptac on methane production as well as nitrate and nitrite reduction by mixed ruminal microorganisms.
|
Materials and Methods
|
|---|
The basic instrument consisted of a set of four independent glass fermenter vessels used in parallel. Each vessel had a capacity of 1,000 mL and was equipped with a magnetic stirrer, a buffer input, a solid feed input device, a thermister probe, an inlet to draw sample, and an input for N2 gas. This system was equipped with near-infrared CH4 and CO2 analyzers equipped with an accurate flow meter to measure the rate of gas production (gas production = flow rate of N x gas concentration). Nitrogen gas at a rate of 20 mL/min was continuously bubbled through the fermentation content via a gas input port for maintaining anaerobic conditions and for carrier gas (zero gas). The temperature of each vessel at 39°C was constantly controlled by a circulating water capillary connected to the electronic system, and mixing of the contents was achieved with a magnetic stirrer equipped with aluminum fins appropriately positioned to act as a foam breaker (Takasugi Seisakusho Co., Ltd., Tokyo, Japan). This system was constructed with the advance of continuous measurement of potential CH4 production and with noncontinuous inflow of substrate (feeding only once before measurements).
The in vitro continuous incubation systems described above were used in the present study. Ruminal fluid was obtained from two nonlactating Holstein cows (average of 800 kg BW), each fitted with a ruminal cannula and housed in a tie-stall barn. Cows were fed a basal diet of orchardgrass hay (DM = 87.33%, OM = 98.98%, CP = 14%, ADF = 38.84%, NDF = 73.26%, ADL = 4.10%, and GE = 4.45 Mcal; DM basis) at maintenance (55 g of DM/kg of BW–0.75daily) in two equal portions at 0800 and 1700, and provided with free access to water and trace mineral salt (Fe = 1,232, Cu = 150, Co = 25, Zn = 500, I = 50, Se = 15, and Na = 382 mg/kg). Ruminal fluid was immediately strained through woven nylon cloth into an Erlenmeyer flask with O2-free headspace. Four hundred milliliters of strained ruminal fluid was mixed with 400 mL of autoclaved artificial saliva (McDougall, 1948). All incubations were carried out anaerobically at 39°C for 24 h with only one addition of 10 g of the ground (1-mm screen) hay described earlier as substrate.
To study the effects of nitrate, nitrite, E. coli W3110, and E. coli nir-Ptac on in vitro methanogenesis by ruminal microorganisms, the incubation vessels were supplemented with nitrate (NaNO3; 5 or 10 mM) or nitrite (NaNO2; 1 or 2 mM), and inoculated with cultured E. coli W3110 or E. coli nir-Ptac. To study the effect of E. coli W3110 or E. coli nir-Ptac on in vitro nitrate and nitrite reduction by ruminal microorganisms, cultured E. coli W3110 and E. coli nir-Ptac were inoculated into separate incubation vessels supplemented with nitrate (NaNO3; 5 or 10 mM) or nitrite (NaNO2; 1 or 2 mM). The control culture was incubated without added nitrate and nitrite, and without inoculation of E. coli W3110 or E. coli nir-Ptac.
The wild-type E. coli W3110 and E. coli nir-Ptac were kindly provided by Ajinomoto Co., Inc. Escherichia coli nir-Ptac was constructed by replacing the promoter region upstream of the chromosomal nirBD genes of wild-type E. coli W3110 by the tac promoter. First, a 3-kbp DNA fragment of the nirBD gene was amplified by PCR using E. coli W3110 choromose DNA as the template and oligonucleotides, 5'-AAA AGA ATTCGAGGCAAA AATGAGCAA AGT-3'and 5'-CCCCAA GCT TCATG-CAAA AAGGGGAGGCAT-3' as the primers, and cloned into the EcoRI-HindIII site of an E. coli expression vector pKK223-3 (Amersham Pharmacia Biotech, Piscataway, NJ) containing tac promoter so that nirBD gene was expressed under the regulation of the tac promoter. Then, using this plasmid as the template, a PCR was performed using oligonucleotides, 5'-CGGGGTACCTTC TGGCGTCAGGCAGCCAT-3' and 5'- ACATGCATGCC-GTCTACGCCCAGCAGTTTC-5' as the primers, which gave a 2-kbp DNA fragment with 200 bp of the pKK223-3 vector derived sequence containing tac promoter followed by a 1.8-kbp sequence of the nirBD. The primers were designed so that the amplified DNA fragment had a KpnI site at its 5' end (at the end of the vector derived region) and an SphI site at its 3' end (at the end of the nirBD gene derived region). The amplified DNA fragment was digested with KpnI and SphI and was designated Fragment 1. Another PCR was performed using E. coli W3110 choromose DNA as the template, and oligonucleotides 5'-CGGAATTCGTATGAAGGGCGT-CAGCGCG-3' and 5'-CGGGGTACCTTCTTA AGTCAC-GGA ATTGT-3' as the primers, which gave a 1-kbp DNA fragment with its 3' end 121 bp upstream of the start codon of nirB gene. The primers were designed so that the amplified DNA fragment had an EcoRI site at its 5' end (at the end farther from the nirB gene) and a KpnI site at its 3' end (at the end closer to the nirB gene). The amplified DNA fragment was digested with EcoRI and KpnI and was designated Fragment 2. Fragment 2 and Fragment 1 were ligated into the EcoRI and SphI site of E. coli vector plasmid pHSG299 in the following order: EcoRI—Fragment 2, Fragment 1—SphI. The constructed plasmid was designated pHSG-nir-Ptac. From pHSG-nir-Ptac, the inserted DNA fragment was cut out with HindIII and ligated into HindIII sites of temperature-sensitive vector plasmid, pMAN997, which is a derivative of vector plasmid pMAN031 (Matsuyama and Mizushima, 1985). The resulting plasmid was designated pMAN-nir-Ptac. Wild-type E. coli W3110 was transformed with pMAN-nir-Ptac, cultured at a nonper-missive temperature, and the clones with the promoter upsteam of nirBD replaced by tac promoter were selected. The clone was designated nir-Ptac.
The nitrite reductase activity of the nir-Ptac was measured. The E. coli nir-Ptac cells were cultured in a 500-mL flask containing 20-mL media (40 g of glucose/L, 1 g of MgSO4•7H2O/L, 24 g of (NH4)2SO4/L, 1 g of KH2PO4/L, 10 mg of MnSO4•7H2O/L, 10 mg of FeSO4•7H2O/L, 2 g of yeast extract/L, 30 g of CaCO3/L, and 10 mM KNO2; pH 7.0) with constant shaking at 37°C for 11 h. The optical density at 660 nm (OD660) and NO2– concentration in the culture media was measured every 1 to 2 h, and the nitrite reductase activity (reduced nitrite, mM•h–1•g–1 dry cells) was calculated by dividing volumetric rates by respective values for cell mass. The dry cell weight was calculated from OD660 by an experimentally obtained formula: dry cell weight = OD660 x 0.67 + 0.002.
Escherichia coli W3110 or E. coli nir-Ptac cells were grown on Luria-Bertani broth agar (Sanko Junyaku Co., Ltd., Tokyo, Japan) at 37°C for 10 h and inoculated into a 500-mL flask containing 50 mL of Luria-Bertani broth (10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl/L) and cultured for 12 h at 37°C with constant shaking (150 rpm/min). Stationary-phase cells were harvested by centrifugation (5,000 x g; 5 min, 4°C), washed in sterile buffer solution (pH 6.8; McDougall, 1948), and resuspended in the sterile buffer solution. The cells of E. coli W3110 or E. coli nir-Ptac were inoculated for in vitro mixed ruminal cultures to provide an initial E. coli cell density of OD660 = 2, which corresponded to approximately 2 x 108 to 109 cells/mL.
Fermenter samples were collected at 0, 0.5, 1, 1.5, 2, 4, 6, 8, 10, 12, and 24 h to immediately determine pH, after which each sample was stored at –20°C for later determination of nitrate, nitrite, ammonia-N, and VFA. The value of pH in the fermenter was measured using a pH meter (HM-21P, TOA Electronics Ltd., Tokyo, Japan). Nitrate and nitrite concentration in the incubation fluids were measured using the nitrate/nitrite Assay Kit-C (Colorimetric, Dojindo, Kumamoto, Japan). The absorption coefficient was measured with a microplate reader (Spectra Max 190, Molecular Devices, Tokyo, Japan). Ammonia-N (NH3-N) concentration in the fermenter was estimated as previously described (Sar et al., 2004b). The concentrations of VFA in the fermenter were analyzed by GLC (Shimadzu GC-14A, Kyoto, Japan) equipped with a flame-ionization detector and a capillary column (Ulbon HR-52, 0.53 mm i.d. x 30 m), with 2-ethyl-n-butyric acid as the internal standard. Values were calculated automatically using a Chromatopac data processing system (C-R 4A, Shimadzu). Continuous CH4 and CO2 production rates were automatically measured using near-infrared CH4 and CO2 analyzer (Takasugi Seisakusho Co., Ltd.), and data were taken and pooled into the computer (Windows XP Professional 1-2 CPU, IMB Corp., Tokyo, Japan) from the analyzer through an interface at 1-min intervals.
In vitro incubations for each treatment were performed on duplicate days with two replicates per day (n = 4), in which two vessels per treatment were assigned randomly. Fermentation vessel was used as the experimental unit for all data. The experiment was designed as a completely randomized design with 3 x 3 presentation of the treatment structure. Main effects were three levels of nitrate (0, 5, or 10 mM) or nitrite (0, 1, or 2 mM) with no inoculation of E. coli or inoculation with 2 x 108 to 109 cells/mL of E. coli W3110 or E. coli nir-Ptac. Two-way interactions between main effects were tested. Cumulative CH4 production was extrapolated by nonlinear regression analyses using the model (CH4, mL = a + b [1 – e–ct]3, where a = first CH4 production; b = second CH4 production; c = constant CH4 production rate; and t = time, min) from the time course of CH4 production for the 24-h incubation (Hartley, 1961). Data for potential values (a + b) of cumulative CH4 production and fermentation data were analyzed using the Mixed procedures of SAS (SAS Inst., Inc., Cary, NC). Nitrite accumulation was analyzed as a completely randomized design with repeated measures over time using the Mixed procedures of SAS; the model included fixed effects of nitrate or nitrite, E. coli, hour, and the two- and three-way interactions. Orthogonal polynomial contrasts were used to test for linear or quadratic effects of nitrate and nitrite treatment. Differences among treatments within incubation were determined using the least squares means procedure (PDIFF option) of SAS. Results are discussed as significant at P < 0.05.
|
Results
|
|---|
Figure 1 shows potential value (a + b) of cumulative CH4 production in continuous incubation of mixed ruminal microorganisms. There was an interaction (P = 0.05) between the addition of nitrate and E. coli strains on the potential value (a + b) of cumulative CH4 production (Figure 1a). When E. coli W3110 was inoculated with 5 or 10 mM nitrate, a decrease (P < 0.05) in the potential value (a + b) of cumulative CH4 production was observed compared with 5 or 10 mM nitrate alone. In addition, the potential value (a + b) of cumulative CH4 production was drastically decreased (P < 0.05) by the inoculation with E. coli nir-Ptac to 5 or 10 mM nitrate compared with 5 or 10 mM nitrate alone.
View larger version (17K):
[in this window]
[in a new window]
|
Figure 1. Effects of Escherichia coli W3110, E. coli nir-Ptac, nitrate, nitrite, and its combination on potential (a + b) values of cumulative CH4 production in in vitro continuous incubation treated with nitrate (a) and nitrite (b); E. coli effect (P < 0.001), nitrate effect (P < 0.001), nitrite effect (P < 0.001), E. coli x nitrate interaction (P = 0.05, SEM = 24.677), E. coli x nitrite interaction (P < 0.001; SEM = 26.595). Values are mean ± SEM. Potential (a + b) values of cumulative CH4 production were extrapolated by non-linear regression analyses using model (CH4, mL = a + b [1 – e–ct]3, where a = first CH4 production, b = second CH4 production, c = constant CH4 production rate, and t = time, min) from the time course of CH4 production for a 24-h incubation.
|
|
An interaction effect (P < 0.001) between nitrite and E. coli strains was observed on the potential value (a + b) of cumulative CH4 production (Figure 1b
). Compared with cultures containing 1 or 2 mM nitrite alone, the potential value (a + b) of cumulative CH4 production was unaffected by the inoculation with E. coli W3110 to cultures containing 1 or 2 mM nitrite, but markedly decreased (P < 0.01) by the inoculation of E. coli nir-Ptac to cultures containing 1 or 2 mM nitrite, respectively. The potential values (a + b) of cumulative CH4 production was decreased (P < 0.01) in all treatments compared with the control incubation.
Profiles of nitrite accumulation in the in vitro continuous incubation of mixed ruminal microorganism supplemented with nitrate and nitrite are shown in Figures 2a and b. An interaction (P < 0.001) between nitrate and E. coli strains was observed for nitrite accumulation. Nitrite concentration in the inoculation with E. coli W3110 or E. coli nir-Ptac to mixed ruminal population treated with 5 or 10 mM nitrate increased (P < 0.001) the nitrite concentration in the fermenter compared with 5 or 10 mM nitrate alone. There was an interaction (P < 0.001) between the addition of nitrite and E. coli strains for nitrite accumulation. Nitrite concentration in the inoculation with E. coli W3110 to cultures containing 1 mM nitrite decreased (P < 0.001) compared with cultures containing 1 mM nitrite alone. The inoculation with E. coli nir-Ptac to cultures containing 1 mM nitrite inhibited (P < 0.001) nitrite concentration compared with the addition of E. coli W3110 with cultures containing 1 mM nitrite. Compared with cultures containing 2 mM nitrite alone, nitrite concentration in the fermenter was decreased (P < 0.001) by the addition of E. coli W3110 to 2 mM nitrite-containing cultures, with the greatest (P < 0.001) decrease observed with the addition of E. coli nir-Ptac. Nitrite was not detectable in the control incubation or incubation inoculated with E. coli W3110 or E. coli nir-Ptac.
View larger version (22K):
[in this window]
[in a new window]
|
Figure 2. Effect of Escherichia coli W3110 or E. coli nir-Ptac on nitrite accumulation in in vitro continuous incubation treated with nitrate (a) and nitrite (b); E. coli effect (P < 0.001), nitrate effect (P < 0.001), nitrite effect (P < 0.001), time effect (P < 0.001), nitrate x time interaction (P < 0.001; SEM = 0.195), nitrite x time interaction (P < 0.001; SEM = 0.011), E. coli x nitrate interaction (P < 0.001; SEM = 0.102), E. coli x nitrite interaction, (P < 0.001; SEM = 0.006), E. coli x nitrate x time interaction (P < 0.001; SEM = 0.339), E. coli x nitrite x time interaction (P < 0.001; SEM = 0.018).
|
|
Fermentation characteristics in the in vitro continuous incubation are shown in Tables 1 and 2. There was an interaction (P < 0.05) between the addition of nitrate and E. coli strains on pH and total VFA in the fermenter (Table 1). Compared with cultures containing 5 or 10 mM nitrate alone, the tendency for a decrease (P < 0.10) in pH and an increase (P < 0.10) total VFA by the inoculation with E. coli W3110 to 5 or 10 mM nitrate-containing cultures, respectively, was observed. In contrast, the inoculation with E. coli nir-Ptac to 5 or 10 mM nitrate-containing cultures did not affect pH and total VFA in the fermenter, respectively, compared with cultures containing 5 or 10 mM nitrate alone. Also, an interaction (P < 0.01) between nitrite and E. coli strains was observed for pH and total VFA in the fermenter (Table 2). Compared with cultures containing 1 mM nitrite alone, pH and total VFA in the fermenter were unaffected by the addition of E. coli W3110 to 1 mM nitrite cultures, but total VFA was increased (P = 0.046) by the addition of E. coli W3110 to 2 mM nitrite cultures compared with cultures containing 2 mM nitrite alone. Although pH and total VFA in the fermenter were unaffected by the addition of E. coli nir-Ptac to 1 mM nitrite-containing cultures compared with 1 mM nitrite-containing cultures, the decrease (P = 0.038) in pH and the increase (P < 0.001) in total VFA were observed in the inoculation with E. coli nir-Ptac to 2 mM nitrite-containing cultures compared with cultures containing 2 mM nitrite alone. Compared with the control incubation, the tendency for an increase (P > 0.10) in pH and decrease (P < 0.10) in total VFA were observed by the addition of 5 or 10 mM nitrate (Table 1). The addition of 1 or 2 mM nitrite increased (P < 0.01) pH and decreased (P < 0.01) total VFA compared with the control incubation (Table 2). Compared with the control incubation, total VFA was increased (P < 0.05) by the inoculation with E. coli W3110. The inoculation with E. coli W3110 or E. coli nir-Ptac to mixed ruminal populations did not affect the acetate-to-propionate ratios compared with control cultures. There was an interaction (P = 0.006) between nitrite and E. coli strains on the NH3-N concentration (Table 2). The NH3-N concentration was unaffected by the inoculation with E. coli W3110 to 1 mM nitrite-containing cultures, but tended (P = 0.09) to increase by the inoculation with E. coli W3110 to 2 mM nitrite-containing cultures compared with cultures containing 1 or 2 mM nitrite alone, respectively. Compared with cultures containing 1 or 2 mM nitrite alone, the NH3-N concentration was decreased (P = 0.003) by the inoculation with E. coli nir-Ptac to 1 mM nitrite-containing cultures and tended (P = 0.07) to decrease by the inoculation with E. coli nir-Ptac with 2 mM nitrite-containing cultures. Compared with the control cultures, the NH3-N concentration was increased (P < 0.01) by the inoculation with E. coli W3110, but it tended (P < 0.10) to decrease by the inoculation with E. coli nir-Ptac.
View this table:
[in this window]
[in a new window]
|
Table 1. Effects of Escherichia coli W3110, E. coli nir-Ptac, nitrate, and its combination on fermentation characteristics in in vitro continuous incubationa
|
|
View this table:
[in this window]
[in a new window]
|
Table 2. Effects of Escherichia coli W3110, E. coli nir-Ptac, nitrite, and its combination on fermentation characteristics in in vitro continuous incubationa
|
|
|
Discussion
|
|---|
Our studies clearly show that CH4 production by mixed populations of ruminal microorganisms was inhibited by the administration of nitrate, which is consistent with previous reports (Takahashi and Young, 1991; Anderson and Rasmussen, 1998; Sar et al., 2004a). Allison and Reddy (1990) suggested that this inhibition was attributed to the ability of nitrate to act as the electron acceptor and thereby decrease the availability of electrons for the reduction of CO2 to CH4. Additionally, Iwamoto et al. (2002) reported that during the reduction of 1 mol of nitrate to ammonia, 4 mol of reducing equivalents are used. This implies that hydrogen production decreased by 4 mol, which is equivalent to 1 mol (22.4 L) of methane. An inoculation of E. coli W3110 or E. coli nir-Ptac to such a population drastically changed the effect of nitrate on CH4 production in the fermenter. However, a decrease in nitrite accumulation was not observed, which does not support the hypothesis that inoculation of nitrate-containing cultures with E. coli W3110 or E. coli nir-Ptac will stimulate nitrate and nitrite reduction, and consequently decrease nitrite accumulation. As nitrate reduction in the fermenter has been shown to be accelerated by the inoculation of E. coli W3110 or E. coli nir-Ptac (our unpublished data), it is assumed that nitrite reduction in E. coli W3110 or E. coli nir-Ptac in fermenters was limited by insufficient electron supply in the present experiment. It has been shown that nitrite reduction in E. coli strains was promoted by electron donors such as formate (Abou-Jaoude et al., 1977) and especially pyruvate when strains of E. coli were deficient in NADH-nitrite reductase activity and unable to produce formate from pyruvate due to a lack of pyruvate formatelyase activity (acetyl-CoA: formate C-acetyltransferase; EC 2.3.1.54; Pascal et al., 1981).
A decrease in CH4 production was observed when ruminal populations were incubated with increasing levels of nitrite. Methane production in the fermenter was decreased by 29 and 84% by 1 and 2 mM nitrite, respectively. Iwamoto et al. (2002) reported that with in vitro cultures of mixed methanogens, CH4 production was decreased to half even by the addition of 0.5 mM nitrite, and it was completely inhibited with 3 mM nitrite. These results indicate that methanogens are especially sensitive to nitrite, and the inhibition of methanogenesis by nitrite may be attributed to its toxic effect on growth of methanogens rather than its ability in competition with methanogenesis for available electrons. Marais et al. (1988) showed that nitrite inhibited bacteria that produce ATP via electron transport systems, but had no effect on microbes that lack cytochromes and rely on glycolysis for ATP generation. As methanogens do not contain menaquinone or appreciable amounts of b- or c- cytochromes, and obtain energy exclusively by the electron transport phosphorylation system (Thauer et al., 1977), inhibition of methanogens by nitrite at the electron-carrier system is suggested.
When mixed ruminal cultures were inoculated with cells of E. coli W3110 containing 1 or 2 mM nitrite, rate of nitrite reduction was accelerated, consequently causing an inhibition in nitrite accumulation by 48 and 29% in the 1 mM nitrite treatment or by 29 and 35% in the 2 mM nitrite treatment after 0.5 and 2 h of incubation, respectively. These results agree with previous reports that E. coli W3110 has an effect on nitrite reduction activity in anaerobic cultures (Gennis and Stewart, 1996). It is apparent that E. coli W3110 has an enhanced ability to reduce nitrite accumulation in the rumen; however, the inoculation of E. coli W3110 to mixed ruminal populations did not change the effect of nitrite on CH4 production. Interestingly, when mixed with nitrite-containing cultures, E. coli nir-Ptac had a greater ability than E. coli W3110 to inhibit nitrite accumulation and CH4 production in the fermenter. These findings may suggest that the tac promoter-enhanced nitrite reductase activity of E. coli nir-Ptac accelerated electrons to be consumed for nitrite reduction rather than CH4 bio-synthesis. The tac promoter has been known to be one of the most popular promoters used for gene expression studies in E. coli (Xue et al., 1996). The inhibition of nitrite accumulation by the inoculation of E. coli W3110 or E. coli nir-Ptac throughout the time course of mixed ruminal incubation containing nitrite (Figure 2b) indicate that nitrite reductase derived from the added E. coli W3110 or E. coli nir-Ptac cells were active and effective for several hours of incubation. Hence, E. coli W3110 or E. coli nir-Ptac may survive for long periods of time even in the ruminal environment. Thus, it is suggested that enhanced reduction of nitrite in the rumen may have practical implications because E. coli W3110 or E. coli nir-Ptac may be useful as inoculants to prevent livestock poisoning of nitrite by high nitrate-containing forages.
Results of the present work showed that when E. coli W3110 was added to the nitrate-containing incubation, a decrease in pH and increase in total VFA concentrations were observed, and these are in conformity with reports that low ruminal pH can be caused by an accumulation of VFA in the rumen (Burrin and Britton, 1986). However, the cause of ruminal VFA accumulation is not entirely clear (Lana et al., 1998). In contrast, the addition of E. coli nir-Ptac did not change the effect of nitrate on pH and total VFA concentrations. The administration of nitrate resulted in an increase in pH and decrease in total VFA concentrations in the fermenter, which is in agreement with other reports of in vitro experiments (Takahashi, 1989). The increase in pH has normally resulted in a shift in ruminal fermentation toward more reduced VFA, such as propionate and butyrate. Additionally, Farra and Satter (1971) reported that nitrate is effective as an electron sink in ruminal fermentation, thereby lowering the amount of relatively reduced acids, such as propionate and butyrate, and increasing acetate. When E. coli W3110 or E. coli nir-Ptac was inoculated into 1 mM nitrite-containing cultures, pH and total VFA concentrations seemed to be unaffected. However, pH and total VFA concentrations were altered when E. coli W3110 was added to cultures containing 2 mM nitrite compared with 2 mM nitrite treatment. Increases in total VFA concentrations have been linked to the presence of protozoa in the rumen (Broudiscou et al., 1994). As no inhibition of CH4 production by the inoculation with E. coli W3110 to 2 mM nitrite-containing incubation was observed, the increase in total VFA may be attributed to survival and cell growth of protozoa caused by the inhibition of nitrite accumulation by E. coli W3110. In addition, the inoculation of E. coli nir-Ptac to 2 mM nitrite-containing cultures increased total VFA concentrations adversely affected by the administration of 2 mM nitrite, while strongly decreasing CH4 production as described above. These may be attributed to reducing electrons, accelerated by the tac promoter contained in E. coli nir-Ptac, diverting from CH4 production toward increased production of reduced VFA (Table 2). Increasing nitrite resulted in a significant increase in pH and a significant decrease in total VFA concentrations, which might suggest that nitrite inhibits activities of bacteria producing reduced VFA. It has been reported that Selenomonas ruminantium contributes to the production of reduced VFA (Ogimoto and Sochi, 1981), but is inhibited by the addition of nitrite in in vitro experiments (Marais et al., 1988). Based on the relationship between the energy content of the hexose fermented and the VFA produced during carbohydrate fermentation by ruminal bacteria, Ørskov (1977) produced the following equation: hexose = 2 acetate + 2 propionate + butyrate. Stimulation of total VFA concentrations caused by the inoculation of E. coli W3110 to mixed ruminal populations in our results may be due to E. coli W3110 enhancing the fermentation of hexose source from substrate toward yielding more reduced VFA such as acetate, propionate, and butyrate. This conclusion is further supported by the fact that production of acetate, propionate, and butyrate were increased by E. coli W3110 (Table 1 and 2). With the inoculation of E. coli W3110 or E. coli nir-Ptac to mixed ruminal populations, the acetate-to-propionate ratio did not decrease, although the decrease in CH4 production by ruminal microbes was observed. Moss and Givens (2002) reported that ruminal stoichiometry could not explain the change in CH4 production. When E. coli W3110 was added to 1 mM nitrite-containing cultures, NH3-N concentration in the fermenter seemed to be un-affected, but an increase in NH3-N concentration was observed by the inoculation of E. coli W3110 to 2 mM nitrite-containing cultures. This supports our hypothesis that when nitrite reduction in the fermenter was stimulated by the addition of E. coli W3110, NH3-N concentration should be high. In contrast, the inoculation of E. coli nir-Ptac to nitrite-containing incubation resulted in a decrease in NH3-N concentration in the fermenter compared with incubation containing nitrite alone. As the addition of cells of E. coli nir-Ptac alone to the mixed ruminal population decreased NH3-N concentration, it might be assumed that E. coli nir-Ptac per se decreased the concentration and/or activities of ammonia-producing bacteria in the rumen.
Our data clearly show that CH4 production by ruminal microorganisms in vitro can be inhibited by the administration of nitrate and nitrite, although mechanisms of inhibition seem different. Nitrate, via its reduction, directed reductant away from methane biosynthesis whereas nitrite seemed to inhibit methanogens at the electron-carrier system. Also, the inoculation of E. coli W3110 or E. coli nir-Ptac to mixed ruminal microbes decreased CH4 production although its mechanism has not yet been determined. When mixed in nitrate-containing cultures, the inoculation of E. coli W3110 or E. coli nir-Ptac did not inhibit ruminal nitrite accumulation, but decreased ruminal CH4 production. When mixed with nitrite-containing cultures, the inoculation of E. coli W3110 had an enhanced ability to decrease nitrite accumulation while keeping CH4 production low, the greatest values corresponding to the inoculation of E. coli nir-Ptac. Thus, it is reasonable to expect that an enrichment of strain E. coli W3110 or E. coli nir-Ptac could be maintained in the rumen as the results of dietary manipulation, and may provide the host with a detoxification capacity that could decrease the risk of nitrite poisoning.
|
Implications
|
|---|
Evidence herein indicates that nitrate, nitrite, and inoculated Escherichia coli W3110 or Escherichia coli nir-Ptac decreased methane production by ruminal microorganisms. Escherichia coli nir-Ptac inhibited in vitro ruminal nitrite accumulation and CH4 production more than Escherichia coli W3110 in nitrite-containing cultures. Thus, anaerobic cultures of Escherichia coli W3110 or Escherichia coli nir-Ptac may decrease CH4 production in the rumen. The inoculation of Escherichia coli W3110 or, especially, Escherichia coli nir-Ptac to mixed ruminal microorganisms may decrease nitrite toxicity when ruminants consume high-nitrate-containing forages and when nitrite is applied to abate CH4 production. More research are needed, however, to assess the effect of Escherichia coli W3110 or Escherichia coli nir-Ptac on in vitro mixed ruminal cultures containing nitrate with the addition of an electron donor such as formate or pyruvate, and on nitrate-nitrite toxicity when nitrate or nitrite is applied to abate methane emission from ruminants.
|
Footnotes
|
|---|
1 The authors thank Ajinomoto Co. Inc. (Tokyo, Japan) for kindly providing E. coli W3110 and E. coli nir-Ptac and analyzing nitrate and nitrite concentration in the rumen. 
2 Correspondence—phone: +81-155-49-5421; fax: +81-155-49-5421; e-mail: junichi{at}obihiro.ac.jp.
Received for publication May 14, 2004.
Accepted for publication December 2, 2004.
|
Literature Cited
|
|---|
Abou-Jaoude, A., M. Chippaux, M. C. Pascal, and F. Casse. 1977. Formate: A new electron donor for nitrite reduction in Escherichia coli K12. Biochem. Biophys. Res. Comm. 78:579–583.
Allison, M. J., and C. A. Reddy. 1990. Adaptations of gastrointestinal bacteria in response to changes in dietary oxalate and nitrate. Vet. Hum. Toxicol. 32:248–256.
Anderson, R. C., and M. A. Rasmussen. 1998. Use of a novel nitrotoxin-metabolizing bacterium to reduce ruminal methane production. Bioresour. Technol. 64:89–95.
Broudiscou, L., S. Pochet, and C. Poncet. 1994. Effect of linseed oil supplementation on feed degradation and microbial synthesis in the rumen of ciliated-free and refaunated sheep. Anim. Feed Sci. Technol. 49:189–202.
Burrin, D. G., and R. A. Britton. 1986. Response to monensin in cattle during subacute acidosis. J. Anim. Sci. 63:888–893.
Farra, P. A., and L. D. Satter. 1971. Manipulation of the ruminal fermentation. III. Effect of nitrate on ruminal volatile fatty acid production and milk composition. J. Dairy. Sci. 54:1018–1024.[Abstract/Free Full Text]
Gennis, R. B., and V. Stewart. 1996. Respiration. Pages 217–261 in Escherichia coli and Salmonella: Typhimurium cellular and Molecular Biology. Vol. 1. 2nd ed. F. C. Neidhardt, R. Curtiss, J. L. Ingramham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger, ed. ASM Press, Washington, DC.
Hartley, H. O. 1961. The modified Gauss-Newton method for the fitting of non-linear regression functions by least squares. Technometrics 3:269–280.
Iwamoto, M., N. Asanuma, and T. Hino. 2002. Ability of Selenomonas ruminantium, Veillonella parvula, and Wolinella succinogenes to reduce nitrate and nitrite with special reference to the suppression of ruminal methanogenesis. Anaerobe 8:209–215.
Johnson, K. A., and D. E. Johnson. 1995. Methane emission from cattle. J. Anim. Sci. 73:2483–2492.[Abstract]
Lana, R. P., J. B. Rusell, and M. E. Van Amburgh. 1998. The role of pH in regulating ruminal methane and ammonia production. J. Anim. Sci. 76:2190–2196.[Abstract/Free Full Text]
Marais, J. P., J. J. Therion, R. I. Mackie, A. Kistner, and C. Dennison. 1988. Effect of nitrate and its reduction products on the growth and activity of the rumen microbial population. Br. J. Nutr. 59:301–313.[Medline]
Matsuyama, S., and S. Mizushima. 1985. Construction and characterization of a deletion mutant lacking micF, a proposed regulatory gene for ompF synthesis in Escherichia coli. J. Bacteriol. 162:1196–1202.[Abstract/Free Full Text]
McAllister, T. A., E. K. Okine, G. W. Mathison, and K. J. Cheng. 1996. Dietary, environmental and microbiological aspects of methane production in ruminants. Can. J. Anim. Sci. 76:231–243.
McDougall, E. I. 1948. Studies on ruminant saliva. I. The composition and output of sheep’s saliva. Biochem. J. 43:99–109.
Moss, A. R., and D. I. Givens. 2002. The effect of supplementing grass silage with soya bean meal on digestibility, in sacco degradability, rumen fermentation and methane production in sheep. Anim. Feed Sci. Technol. 97:127–143.
Ogimoto, K. J., and I. M. Sochi, 1981. Rumen bacteria. Page 90 in Atlas of Rumen Microbiology. Jpn. Sci. Soc. Press, Tokyo.
Ørskov, E. R. 1977. Relative importance of ruminal and postruminal digestion with respect to protein and energy nutrition in ruminants. Trop. Anim. Prod. 3:91–103.
Pascal, M. C., M. Chippaux, A. Abou-Jaoude, H. P. Blaschkowski, and J. Knappe. 1981. Mutants of Escherichia coli K12 with defects in anaerobic pyruvate metabolism. J. Gen. Microbiol. 124:35–42.[Medline]
Sar, C., B. Santoso, X. G. Zhou, Y. Gamo, A. Koyama, T. Kobayashi, S. Shiozaki, and J. Takahashi. 2002. Effects of ß1-4 galacto-oligosaccharide (GOS) and Candida kefyr on nitrate-induced methaemoglobinemia and methane emission in sheep. Page 179–183 in Greenhouse Gases and Animal Agriculture. 1st ed. J. Takahashi and B. A. Young, ed. Elsevier, Amsterdam, The Netherlands.
Sar, C., B. Santoso, Y. Gamo, T. Kobayashi, S. Shiozaki, K. Kimura, H. Mizukoshi, I. Arai, and J. Takahashi, 2004a. Effects of combination of nitrate with ß1-4 galacto-oligosaccharides and yeast (Candida kefyr) on methane emission from sheep. Asia-Aust. J. Anim. Sci. 17:73–79.
Sar, C., B. Santoso, B. Mwenya, Y. Gamo, T. Kobayashi, R. Morikawa, K. Kimura, H. Mizukoshi, and J. Takahashi, 2004b. Manipulation of rumen methanogenesis by the combination of nitrate with ß1-4 galacto-oligosaccharides or nisin in sheep. Anim. Feed Sci. Technol. 115:129–142.
Takahashi, J. 1989. Effect of nitrate content of forage on the production of volatile fatty acids by sheep rumen microbes in in vitro. Jpn. J. Zootech. Sci. 60:476–483.
Takahashi, J., 2001. Nutritional manipulation of methanogenesis in ruminants. Asian-Aust. J. Anim. Sci. 14(Special issue):131–135.
Takahashi, J., and B. A. Young. 1991. Prophylactic effect of L-cysteine on nitrate-induced alteration in respiratory exchange and metabolic rate in sheep. Anim. Feed Sci. Technol. 35:105–113.
Thauer, R. K., K. Jungermann, and K. Decker. 1977. Energy conservation in chemotrophic anaerobic bacteria. Bacteriol. Rev. 41:100–180.[Free Full Text]
Xue, G. P., J.S. Johnson, D. J. Smyth, L. M. Dierens, X. Wang, G. D. Simpson, K. S. Gobius, and J. H. Aylward. 1996. Temperature-regulated expression of the tac/Lacl system for overproduction of a fungal xylanase in Escherichia coli. Appl. Microbiol. Biotechnol. 45:120–126.[Medline]
Top
Abstract
Introduction
