Dynamics of in vitro rumen methane production after nitrate addition

Figures & data

Table 1. Dosages of the chemicals used in the experiment.

	Chemical additions [mg/ml rumen fluid]
	NaCl+Urea				NaNO3
Dose^a	0.5	1.0	1.5	2.0	0.5	1.0	1.5	2.0
Chemicals, [mg]
Urea (CH₄ N₂ O)	39	79	118	158	–	–	–	–
Sodium Chloride (NaCl)	76	152	228	305	–	–	–	–
Sodium Nitrate (NaNO3)	–	–	–	–	112	224	336	448
Nitrate (${\mathrm{N}\mathrm{O}}_3^{-}$)^b	–	–	–	–	82	165	247	330

^aThe doses are expressed in mg of $N{O}_3^{-}$ for each ml of rumen fluid. In control bottles, the doses correspond to amounts of $N{O}_3^{-}$ added to the corresponding treated bottles.

^bNitrate amounts calculated from sodium nitrate addition.

Table 2. Main fermentative traits at the end of the fermentative process (48 h) for the different inclusion levels of sodium nitrate (NaNO₃) or other chemicals (NaCl + urea)^a.

	Chemical additions [CA, mg/ml rumen fluid]
	NaCl+Urea				NaNO3
Dose^b	0.5	1.0	1.5	2.0	0.5	1.0	1.5	2.0	CA	Dose	CA* Dose	RMSE
pH	6.71	6.73	6.75	6.76	6.71	6.75	6.76	6.75	NS	**	NS	0.019
NH3 [mg/dl]	67.6	71.1	71.6	76.7	66.9	74.3	74.9	81.4	NS	NS	NS	8.04
Total protozoa, 10^c [cell/ml]	86	82.4	86.8	90	77.2	80.4	95.2	91.6	NS	NS	NS	12.17
Total gas, [ml]	1429	1403	1387^X	1380^X	1413^A	1379^A	1263^YB	1117^YC	**	**	**	40.72
Methane, [%]	20.4	19.1	19.6	20.1^X	19.9^A	17.7^A	16.8^A	13.2^YB	**	**	**	1.
Methane-free gas^c, [ml]	1137	1135	1114	1104^X	1137^A	1133^A	1047^B	968^YC	**	**	**	36.17
Total SCFA, [mmol/l]	79.5	86.6	89.4	91.6	92.4	94.0	86.4	94.9	NS	NS	NS	7.52
Acetate, [%] of SCFA	62.6	62.4^Y	62.9^Y	61.9^Y	63.9^B	67.8^XA	67.9^XA	68.1^XA	**	*	**	1.32
Propionate, [%] of SCFA	17.1	17.4	17.7	17.7	16.2	15.2	16.4	16.9	**	NS	NS	0.88
Iso-butyrate, [%] of SCFA	1.07	0.96	0.95	0.98	1.09	0.91	0.90	0.85	NS	*	NS	0.13
Butyrate, [%] of SCFA	13.9	13.8^X	13.3^X	14.0^X	13.2^A	11.3^YA	9.67^YB	8.98^YB	**	**	**	0.94
Iso-valerate, [%] of SCFA	4.00	4.03	3.90	4.06	4.22	3.68	3.98	3.98	NS	NS	NS	0.21
Valerate, [%] of SCFA	1.19	1.29	1.23	1.23	1.20	1.08	1.10	1.09	**	NS	NS	0.11
Acetate:Propionate	3.76	3.60^Y	3.57^Y	3.53^Y	3.96	4.47^X	4.15^X	4.05^X	**	NS	NS	0.26
Net H2 produced ^d, [mM]	105	113	117	119	124	130	116	114	NS	NS	NS	10.43
[mol/100 mol] of SCFA	133	131	131	131	135	139	135	133	**	NS	NS	2.88

^aMeans with different superscripts are statistically different (^A,B,C,D between dose and within chemical additions (p < 0.01); ^X,Y, between type chemical addition and within dose, p < 0.01).

^bThe doses refer to the final NO3 concentration in fermenters (mg/ml of rumen fluid);

^cTotal gas minus methane.

^din mM = 2[acetate + n-butyrate + iso-butyrate] – [propionate + iso-valerate + valerate].

Table 3. Methane yield at progressive incubation times (6 h intervals) and kinetic parameters of the fitting Gompertz model of the methane yield for the different inclusion levels of sodium nitrate (NaNO₃) or other chemicals (NaCl + urea)^a.

Dose^b	Chemical additions [CA, mg/ml rumen fluid]								CA	Dose	CA* Dose	RMSE
	NaCl+Urea				NaNO3
	0.5	1.0	1.5	2.0	0.5	1.0	1.5	2.0
Methane yield^c, [ml]
6	108	99.2	98.8	99.3^x	92.5	77.3	78.5	73.2^y
12	173	159^x	161^X	163^X	158^A	131^yAB	119^YBC	99.4^YC
18	211	195^x	197^X	201^X	198^A	167^yAB	148^YB	113^YC
24	241	222^x	225^X	228^X	227^A	195^yAB	171^YB	124^YC
30	263	241	244^X	248^X	251^A	216^B	188^YB	132^YC
36	276	252	257^X	260^X	263^A	229^B	200^YB	138^YC
42	282	257	262^X	265^X	270^A	234^B	207^YB	142^YC
48	292	269	273^X	276^X	282^A	245^B	217^YB	149^YC
									**	**	**	18.2
Methane kinetic parameters
B, [ml]	267	245	249^X	253^X	258^A	225^A	197^YA	133^YB	**	**	**	22.5
A, [1/h]	0.21	0.21	0.21	0.21^X	0.19^B	0.18^B	0.21^B	0.31^YA	NS	*	*	0.04
Lag, [h]	2.89	2.87	2.91	2.91^x	3.17^A	3.40^A	3.06^A	2.09^yB	NS	*	*	0.44
MFR^c, [ml/h]	20.8	19.2	19.2	19.6	18.3	15.0	14.4	14.6	**	**	NS	1.32
TMFR^d, [h]	7.64	7.59	7.69	7.68^x	8.38^A	8.97^A	8.08^A	5.52^yB	NS	*	*	1.16

^aMeans with different superscripts are statistically different (^A,B,C,D between dose and within chemical additions (p < 0.01); ^X,Y(p < 0.01) or ^x,y(p < 0.05), between type of chemical addition and within dose).

^bThe doses refer to the final NO3 concentration in fermenters;

^cThe interaction dose*time and dose*time*CA are statistically significant (p < 0.01);

^dCalculated Maximum Fermentation Rate;

^eCalculated Time at Maximum Fermentation Rate.

Figure 1. Methane reduction as a percentage of the relative controls for the different additions of sodium nitrate obtained during fermentation.

alt=“Linear regression of methane yield for different nitrate inclusion level”.

Figure 2. Linear regressions (within fermentation run) at different fermentation times (6,12, 24 and 48 hours) between methane yield (ml) and level of nitrate inclusion (mg).

alt=“Methane percentage reduction for different dosage of nitrate”.