Abstract

The study evaluated the residual effect of the known enteric methane inhibitor 3-nitrooxypropanol (3NOP) on anaerobic digestion of cattle feces (feces) in a CH4 potential batch test and two consecutive runs of an anaerobic leach bed reactor at a solids retention time of 40 days. The feces used in this study were collected from beef cattle fed forage- (backgrounding) or grain- (finishing) based diets supplemented with 3NOP in feedlot and metabolism studies. The results showed that CH4 yields were not significantly different from treatments using control feces and feces collected from cattle fed a diet supplemented with 3NOP in both CH4 potential and leach bed studies. Spiking feces with 200 mg 3NOP kg−1 dry matter decreased CH4 production rate by 8.0–18.1% estimated from the Gompertz equation, increased the lag phase time (0.4–3.4 d) in all the treatments, while there was no significant difference in the overall CH4 yield. Results from this study showed that 3NOP can be used as an effective enteric CH4 inhibitor with no residual effect on anaerobic digestion.

STATEMENT OF NOVELTY

The novel enteric methane inhibitor 3-nitrooxypropanol (3NOP) has been demonstrated to have a sustained effect of reducing enteric methane emission in beef cattle. However, its residual effect on subsequent waste treatment such as anaerobic digestion has not yet been evaluated, which will provide information on the fate of the substance after use as a feed supplement and in case of accidental spillage. The results obtained during this study were interesting and showed that there was no significant effect on the overall methane yield but a decrease in the initial methane production rate, increased lag phase time and decreased volatile fatty acid degradation rate during the anaerobic digestion of cattle feces spiked with 3NOP.

INTRODUCTION

Animal agriculture contributes 10–14.5% of global anthropogenic greenhouse gas (GHG) emissions, of which 44–50% of the emissions are in the form of methane (CH4) (Eckard et al. 2010; Garnier et al. 2019). Beef and dairy cattle account for about 74% of agricultural CH4 emissions (Caro et al. 2014). Producers adopt strategies for the mitigation of emissions for economic reasons such as improved production efficiency of beef and dairy products (Beauchemin et al. 2008). In Alberta, reduction of GHG emissions was estimated at 34 million CO2 equivalent, generating $132 million for farmers, and has been the motivation for farmers adopting this strategy (Campbell et al. 2018).

A newly developed CH4 inhibitor, 3-nitrooxypropanol (Duval & Kindermann 2012), was reported to have reduced CH4 emission from beef cattle by 59% (Romero-Perez et al. 2015) and the reduction of DNA copy number of methanogens has also been reported (Romero-Pérez et al. 2017). Other studies with 3NOP have reported reductions in CH4 emissions ranging from 24% in sheep and up to 60% in dairy cattle (Hristov et al. 2015). As cattle lose 3–12% of gross energy they consume by enteric CH4 emission (Kamra et al. 2015), a reduction in CH4 energy loss may improve animal performance (Vyas et al. 2016) in addition to reducing environmental impact. 3NOP is a structural analogue of methyl coenzyme M that binds to the active site of methyl-coenzyme M reductase and oxidizes its active site Ni(I) such that methanogenesis cannot proceed (Duin et al. 2016). Although 3NOP is effective in reducing enteric CH4 production, its residual effect on downstream waste treatment methods such as manure storage, composting and anaerobic digestion has not yet been studied.

Anaerobic digestion is a process whereby organic compounds are degraded into mainly biogas (∼60 CH4 and ∼40% CO2) and a residue that can be used as fertilizer. Industrially, the process is operated under mesophilic temperature of about 35 °C or thermophilic temperature of about 55 °C (Hagos et al. 2017). Using cattle manure as a feedstock for anaerobic digestion by farmers can lead to the generation of extra revenue from the produced CH4, which can be converted and sold as electricity together with the use of the digestate as an organic amendment, reducing the cost of purchasing mineral fertilizers.

The current investigation for the first time evaluated the residual effect of feeding 3NOP to cattle on anaerobic digestion of the feces. Three sets of feces from beef cattle fed 3NOP for the reduction of enteric CH4 emission were evaluated. Anaerobic digestion of the feces was performed in CH4 potential batch tests and in anaerobic leach bed reactors.

MATERIALS AND METHODS

Feces obtained from beef cattle fed 3-nitrooxypropanol

The feces used for evaluating the residual effect of 3NOP on anaerobic digestion were collected from beef cattle that were fed backgrounding and finishing diets in a feedlot (Study 1) and from cattle fed a backgrounding diet in a metabolism study (Study 2). These feeding studies evaluated whether dietary supplementation with 3NOP could mitigate enteric CH4 emissions. Study 1 used 224 heifers housed in 28 pens (eight cattle per pen) within a feedlot in a completely randomized design. The cattle weighed approximately 310 and 460 kg at the start and end of the backgrounding period, respectively, and 510 and 690 kg at the start and end of the finishing period, respectively. During the backgrounding period (day 1 to 105) the cattle received a high forage diet with one of four treatments: control (no supplement), 200 mg 3NOP kg−1 dietary dry matter (DM), 200 mg 3NOP kg−1 dietary DM plus 33 mg monensin kg−1 dietary DM, and 33 mg monensin kg−1 dietary DM. The cattle were then transitioned to a high grain finishing diet over a period of 28 days, and then fed one of four treatments for a period of 105 days: control (no supplement), 125 mg 3NOP kg−1 dietary DM, 125 mg 3NOP kg−1 dietary DM plus 33 mg monensin kg−1 dietary DM, and 33 mg monensin kg−1 dietary DM. 3NOP is manufactured by DSM Nutritional Products AG (Kaiseraugst, Switzerland) and consists of 1,3 propanediol mononitrate (11%) and a carrier (60% silica and 40% propylene glycol). Monensin (Rumensin®, Elanco Animal Health, Guelph, Ontario, Canada) is an approved ionophore in North America that is fed to cattle to improve feed conversion efficiency. The backgrounding (forage-based) diet consisted of (DM basis): 65% barley silage, 5% supplement (providing protein, mineral, and vitamins), and 30% dry rolled barley grain; the finishing (grain-based) diet consisted of: 8% barley silage, 5% supplement and 87% dry rolled barley grain. Cattle feces (5 to 6 kg) were collected from the pen floor of at least three pens per treatment once mid-way through the backgrounding and finishing periods. The collected samples were pooled to obtain one representative sample per treatment for the two different periods. A total of eight samples for the various treatments were collected for the backgrounding (four samples) and finishing (four samples) periods prior to storing at −20 °C until use.

Study 2 used eight beef heifers fed four treatments in a 4 × 4 Latin square design with four 28-d periods. The treatments were: control (no additive), 200 mg 3NOP kg−1 dietary DM, 50 g kg−1 dietary DM canola oil, and 200 mg 3NOP kg−1 DM plus 50 g kg−1 DM canola oil. The basal diet was a backgrounding diet similar to that used in Study 1. Monensin was not added to the diets. Canola oil was added in this experiment as it has been reported to mitigate enteric CH4 emissions (Beauchemin & McGinn 2006). The study was conducted in a metabolism barn with cattle housed individually in tie-stalls with rubber mats. Cattle feces was collected from each animal in each period (32 samples), while urine, which was collected separately, was not mixed with the feces because it was acidified. The 32 samples collected over time from the four treatments were stored separately at −20 °C. The samples were defrosted overnight and then pooled and mixed to obtain four representative samples per treatment for the entire study. The samples were then stored again at −20 °C until use.

Inoculum used in the methane potential batch test and anaerobic digestion in leach bed reactors

Methane potential of the cattle feces collected from cattle fed backgrounding and finishing diets of Study 1 and backgrounding diets of Study 2 were performed in three independent runs, using inocula collected at different times from Lethbridge Biogas LP (Lethbridge, Alberta, Canada). The plant digests agro-industrial waste under mesophilic temperature (40 °C) for biogas production and subsequent electricity (2.85 MW) generation. Both CH4 potential batch tests and anaerobic digestion in leach bed reactors were performed simultaneously for each cattle feces using inoculum collected at the same time. Prior to start-up, the inoculum was incubated at 40 °C for 5 days to reduce the background CH4 production.

Methane potential batch tests

The CH4 potential was evaluated using the eight representative samples from Study 1 (feces collected from the pen floor of cattle fed backgrounding and finishing diets in the feedlot study) and from the four representative samples of Study 2 (feces collected from animals fed a backgrounding diet in the metabolism study), with additional samples where 3NOP was added to the feces from animals fed the control treatment. The treatments for the backgrounding diets of Study 1 included: (1) control feces (no 3NOP fed), (2) control feces spiked with 200 mg 3NOP kg−1 DM, (3) control feces spiked with 200 mg 3NOP carrier kg−1 DM, and (4–6) feces collected from the pens wherein diets were supplemented with 200 mg 3NOP kg−1 DM, 200 mg 3NOP kg−1 DM and 33 mg monensin kg−1 DM, and 33 mg monensin kg−1 DM. 3NOP carrier was the same formulation as the 3NOP product without the active ingredient. Prior to 3NOP addition, the required amount was dissolved in an opaque container overnight in the cold room (4 °C), as it was light and heat sensitive. Microcrystalline cellulose (Avicel®, MERCK, Darmstadt, Germany) control was also included in the runs as an internal standard, while 400 mL of the inoculum was digested to subtract background CH4 production from the treatments. The same experimental design was used for the cattle feces collected from the finishing diet in Study 1.

For the feces collected from the backgrounding diet in Study 2, the treatments included: (1) control feces (no 3NOP fed), (2) control feces spiked with 200 mg 3NOP kg−1 DM, (3) control feces spiked with 200 mg 3NOP carrier kg−1 DM, and (4–6) feces collected from animals fed diets supplemented with 200 mg 3NOP kg−1 DM, 50 g kg−1 DM canola oil, and 200 mg 3NOP kg−1 DM plus 50 g kg−1 DM canola oil. Cellulose control and inoculum only were also included in the run as mentioned previously.

Methane potential batch tests were performed in triplicate using 0.5 L Erlenmeyer flasks, which were in turn placed in a heated water bath at 40 °C. The flasks were sealed with rubber stoppers and each flask had an outlet for biogas collection in aluminium gas-tight bags. Approximately 400 mL of inoculum and 24 g wet weight of substrate was added at the beginning and then incubations lasted 40 days. The inoculum substrate ratio (ISR) was set at 2:1 based on g volatile solids (VS). The reaction vessels were manually swirled once a day and biogas volume and composition were analysed every 2 to 3 days for the first two weeks when CH4 production was high and every 3 to 4 days thereafter.

Anaerobic digestions in leach bed reactors

The long term residual effects of 3NOP and adaptability of methanogens when forage-based or grain-based cattle feces were spiked with 3NOP were evaluated in anaerobic leach bed digesters using feces from the feedlot (Study 1) and metabolism studies (Study 2). The experiments were performed in duplicate in leach bed reactors (38 cm high, 6.5 cm internal diameter) at 40 °C. The reactors were sealed with a rubber stopper, which had an outlet for gas sampling and collection using an aluminium gas-tight bag. The set-up was similar to the one reported earlier (Nkemka & Hao 2018). In the experiments, three treatments were performed at a time: control feces, control feces spiked with 200 mg 3NOP kg−1 DM, and feces collected from pens (Study 1) or animals (Study 2) whose diet was supplemented with 200 mg 3NOP kg−1 DM.

At the start of the incubations, 40 g VS of feces, 400 g wet weight inoculum and 100 mL water were mixed externally and then placed in the leach bed reactors. The ISR ratio was set at 0.2:1.0 based on g VS. No internal mixing of the reactor was performed during the incubations and the mixture was allowed to digest for 40 days. Upon completion of the first leach bed digestion (run 1), a second consecutive run (run 2) was performed by adding 40 g VS of feces to 400 mL of the digestate from the first run that served as inoculation for the next run. The CH4 yield of the first run was calculated by dividing the normalized CH4 produced by the amount of VS initially added, while for the second run, the amount of VS carried over from the first batch plus the freshly added substrate were included in the CH4 yield calculations.

During the course of the experiment, the reactors were sampled from the bottom for pH, total chemical oxygen demand (tCOD) and total volatile fatty acid concentration (tVFA). Also, gas samples were taken from the head space of the reactor and the gas volume was measured with a gas-tight 100-mL syringe every 2 to 3 days for the first two weeks when CH4 production was high and every 3 to 4 days thereafter. Samples collected at the start and end of the digestion were analysed for dissolved organic carbon (DOC), water soluble nitrogen (water-N) and NH4+-N.

Analytical methods

The pH, VFA, DOC, NH4+-N, water-N, elemental carbon and nitrogen, biogas volume and composition were analysed as described previously (Nkemka & Hao 2018). Biogas volume and methane volumes were corrected to standard conditions (273.15 K and 101 kPa). Total solids (TS) and VS were analyzed according to standard methods (APHA 1998). Analysis of neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent (AD) lignin were determined according to fiber analyses methods (Van Soest et al. 1991). Thermal-stable amylase (Termamyl® 120, Sigma-Aldrich Co. LLC., St Louis, MO) and sodium sulfite (S430-3 sodium sulfite anhydrous, Fisher Scientific Int., Inc., Pittsburgh, PA) were used in the NDF procedure (Mertens et al. 2002).

Modelling of cumulative methane production curve was performed using the Gompertz equation 
formula
(1)
where Mp was cumulative methane production (mL), Pm was ultimate methane production (mL), Rm was methane production rate (mLd−1), X0 was lag phase time (d), and e was exponential1 (2.7183) (Han et al. 2008). Excel Solver was used to minimize the sum of square errors between the experimental and estimated values and the goodness of fit was affirmed by the correlation coefficient R2.

The residual effect of 3NOP in feces and the effect of spiking the feces with 3NOP on means of CH4 yields in the CH4 potential batch test and leach bed reactors were analyzed with SPSS statistical software using Post Hoc Multiple comparisons at a 95% confidence interval. The effects (treatments) were fixed variables, while mean CH4 yields were dependent variable.

RESULTS AND DISCUSSION

Cattle feces and inoculum characteristics

Table 1 presents the initial feedstock (i.e. feces) and inoculum composition. The cattle feces had relatively high fiber (cellulose, hemicellulose and lignin) content as expected, which varied among sources due to differences in the diet composition and digestibility. The C/N ratios of the feces were below 23.8, which has been reported for improved biogas production (Zahan et al. 2018). Digestion of such substrates with low C/N ratio may result in the mineralization of organic nitrogen from mainly proteins, converting it into NH4+-N and NH3, which have been reported to be inhibitory to acetoclastic methanogenesis compared to the tolerant hydrogenotrophic methanogenesis (Amha et al. 2018) and at concentrations of 6 g L−1 of NH4+-N and 0.7 g L−1 of NH3 (Yenigün & Demirel 2013). A common strategy to circumvent potential NH4+-N/NH3 inhibition is to co-digest feces with other feedstocks with high C/N ratios or removal of ammonia in anaerobic digestion (Krakat et al. 2017).

Table 1

Characteristics of feces from beef cattle used for anaerobic digestion

Parameters Sources Forage based fecesFeces from FS 3NOP based forage feces Feces from FS Grain based feces Feces from FS 3NOP grain based feces Feces from FS Forage based feces Feces from MS 3NOP forage based feces Feces from MS Inoculum Lethbridge Biogas LP 
TS (%) 15.7 ± 0.2 16.1 ± 0.5 26.8 ± 0.4 29.8 ± 0.6 16.5 ± 0.0 17.9 ± 0.1 2.8 ± 0.1 
VS (% of TS) 86.5 ± 1.7 83.7 ± 0.0 74.6 ± 0.5 75.0 ± 0.6 86.2 ± 0.0 88.0 ± 0.1 56.6 ± 0.1 
C (% of TS) 46.2 ± 0.2 47.6 ± 2.0 41.2 ± 1.3 40.4 ± 0.7 49.3 ± 0.3 49.8 ± 0.4 36.9 ± 0.3 
N (% of TS) 3.8 ± 0.0 2.7 ± 0.1 2.4 ± 0.1 2.5 ± 0.1 2.6 ± 0.0 2.3 ± 0.0 2.4 ± 0.0 
C/N 12.0 ± 0.1 17.4 ± 0.2 17.1 ± 1.2 16.0 ± 0.6 19.0 ± 0.2 21.6 ± 0.3 15.6 ± 0.3 
Cellulose (% of TS) 15.6 ± 2.1 24.7 ± 1.1 18.1 ± 5.1 20.5 ± 0.1 18.1 ± 0.2 23.1 ± 0.4 – 
Hemicellulose (% of TS) 17.4 ± 3.2 17.7 ± 0.9 17.1 ± 2.2 15.7 ± 0.8 14.9 ± 0.1 15.3 ± 0.8 – 
AD lignin (% of TS) 22.5 ± 3.4 12.7 ± 0.4 13.0 ± 4.2 12.0 ± 5.2 20.1 ± 0.6 13.8 ± 0.8 – 
CP (%TS) 24.0 ± 0.2 17.1 ± 0.6 15.1 ± 0.7 15.8 ± 0.7 16.3 ± 0.2 14.4 ± 0.2 14.7 ± 0.3 
NH4+-N (mg L−1130.2 63.9 14.3 130.8 42.3 89.1 2,727 ± 484 
Water-N (mg L−142.3 32.3 341.1 286.6 263.9 271.5 3,643 ± 179 
DOC (mg L−1974 908 2,226 1,620 1,818 1,848 2,898 ± 211 
Parameters Sources Forage based fecesFeces from FS 3NOP based forage feces Feces from FS Grain based feces Feces from FS 3NOP grain based feces Feces from FS Forage based feces Feces from MS 3NOP forage based feces Feces from MS Inoculum Lethbridge Biogas LP 
TS (%) 15.7 ± 0.2 16.1 ± 0.5 26.8 ± 0.4 29.8 ± 0.6 16.5 ± 0.0 17.9 ± 0.1 2.8 ± 0.1 
VS (% of TS) 86.5 ± 1.7 83.7 ± 0.0 74.6 ± 0.5 75.0 ± 0.6 86.2 ± 0.0 88.0 ± 0.1 56.6 ± 0.1 
C (% of TS) 46.2 ± 0.2 47.6 ± 2.0 41.2 ± 1.3 40.4 ± 0.7 49.3 ± 0.3 49.8 ± 0.4 36.9 ± 0.3 
N (% of TS) 3.8 ± 0.0 2.7 ± 0.1 2.4 ± 0.1 2.5 ± 0.1 2.6 ± 0.0 2.3 ± 0.0 2.4 ± 0.0 
C/N 12.0 ± 0.1 17.4 ± 0.2 17.1 ± 1.2 16.0 ± 0.6 19.0 ± 0.2 21.6 ± 0.3 15.6 ± 0.3 
Cellulose (% of TS) 15.6 ± 2.1 24.7 ± 1.1 18.1 ± 5.1 20.5 ± 0.1 18.1 ± 0.2 23.1 ± 0.4 – 
Hemicellulose (% of TS) 17.4 ± 3.2 17.7 ± 0.9 17.1 ± 2.2 15.7 ± 0.8 14.9 ± 0.1 15.3 ± 0.8 – 
AD lignin (% of TS) 22.5 ± 3.4 12.7 ± 0.4 13.0 ± 4.2 12.0 ± 5.2 20.1 ± 0.6 13.8 ± 0.8 – 
CP (%TS) 24.0 ± 0.2 17.1 ± 0.6 15.1 ± 0.7 15.8 ± 0.7 16.3 ± 0.2 14.4 ± 0.2 14.7 ± 0.3 
NH4+-N (mg L−1130.2 63.9 14.3 130.8 42.3 89.1 2,727 ± 484 
Water-N (mg L−142.3 32.3 341.1 286.6 263.9 271.5 3,643 ± 179 
DOC (mg L−1974 908 2,226 1,620 1,818 1,848 2,898 ± 211 

FS, feedlot study; MS, metabolism study; AD lignin, acid detergent lignin; TS, total solids or dry matter content; VS, volatile organic matter; CP, crude protein; DOC, dissolved organic carbon; water-N, water soluble nitrogen.

The inoculum was rich in NH4+-N (2,727 ± 484 mg L−1) and water-soluble nitrogen (3,643 ± 179 mg L−1) and also had a high pH of 7.93 and buffering capacity measured as the bicarbonate alkalinity of 15.1 g L−1. This nutrient-rich inoculum provided both macro and micronutrients and also maintained the needed neutral pH by counteracting a decrease in pH due to VFA accumulation as reported elsewhere (Nges et al. 2015).

Residual effect of 3-nitrooxypropanol feces in methane potential batch test

Table 2 shows the cumulative CH4 yields and the estimated Gompertz parameters of the treatments during the anaerobic digestion of cattle feces sets collected from the feedlot (Study1) and metabolism (Study 2) studies. The digestion of feces from cattle fed 3NOP showed similar degradation profile when compared to the control feces. The methane yield (mL g−1 VS) of the control feces was 342.7 ± 6.4 and was comparable to 279.2 ± 51 of 3NOP fed feces in Study 1 that used the backgrounding diet. A similar result was also obtained for Study 2 that used the backgrounding diet. However, the methane yield (mL g−1 VS) of 3NOP fed feces 269.4 ± 2.4 was significantly lower than 298.1 ± 21.7 of the control feces in Study 1 that used a finishing grain diet. Despite this significant difference in the methane yield, the 3NOP fed feces had a higher methane production rate of 28.2 mL d−1 when compared to 16.8 mL d−1, suggesting feeding cattle with 3NOP will not inhibit downstream anaerobic digestion of the organic waste. This observation was also true when digesting cattle feces fed monensin and its combination with 3NOP. The use of the methane potential batch test with an ISR of 2:1 was not suitable for studying the effect of spiking cattle feces with 3NOP, as only one out of the three treatments showed a significant effect on the methane yield. A significantly higher methane yield (mL g−1 VS) of 342.7 ± 6.4 was obtained when compared to 256.2 ± 43.6, while there was no significant difference in the methane yield for the rest of the treatments. For the forage-based feces from the metabolism study, feces from cattle fed both treatments that contained oil had considerably greater CH4 yields (p < 0.05) (oil without 3NOP, 315.2 ± 40.3; 3NOP + oil, 388.4 ± 24.6 mL g−1 VS) compared with the other treatments. The CH4 yield (mL g−1 VS) for the other treatments in the metabolism study were: 210.2 ± 32.8 for control feces, 203.3 ± 26.2 for control feces spiked with 3NOP, 181.4 ± 51.7 for control feces spiked with 3NOP carrier and 206.1 ± 16.3 for 3NOP fed feces. The addition of canola oil (50 g kg−1 DM)) to the diet may have led to an increase in residual fat content in feces, and because 1 g VS of lipid produces 1,014 mL CH4 compared with 1 g VS carbohydrate, which produces 415 mL CH4 and 1 g VS protein, which produces 496 mL CH4 (Møller et al. 2004), CH4 yield for the oil treatments was greater. Our CH4 yields were within the range 155–323 mL g−1 VS reported from the CH4 potential batch test of cattle manure (Kafle & Chen 2016; Ormaechea et al. 2017). A methane yield of 30.8 mL g−1 TS was reported from the rumen of cattle (Vyas et al. 2016) and it is considerable lower than those reported for biogas production. The high ISR of 2:1 used in the current study would have suppressed the inhibitory effect of 3NOP on CH4 production, which was observed in the rumen of beef cattle (Romero-Perez et al. 2015). High ISR, which is based on g VS, is an approximation of the amount of bacteria/methanogenic biomass added to the substrate prior to anaerobic digestion. The use of fresh inoculum resulted in a CH4 yield (393.3 ± 10.9 mL g−1 VS) from cellulose control after 30 days that was 94.8% of the theoretical maximum, indicating that the inoculum was active. The CH4 yield (46.0 ± 7.2 mL g−1 VS) of the pre-incubated inoculum was low. Our results suggest that there was no effect of 3NOP either when fed to cattle or added to feces on CH4 yield in CH4 potential batch tests using an ISR of 2:1.

Table 2

Experimental CH4 yield and Gompertz regression parameters during methane potential batch test of forage-based (backgrounding diet) and grain-based (finishing diet) feces collected from a feedlot (Study 1) and metabolism study (Study 2) at an ISR of 2:1

 Experimental Gompertz regression parameters
 
 
CH4 yield (mL g−1 VS) Pm (mL) Rm (mL d−1Xo (d) R2 
Cellulose control 393.3 ± 10.9 415.7 26.2 3.9 0.998 
Study 1 (Backgrounding diets) 
 Control feces 342.7 ± 6.4a 323.6 19.9 0.4 0.993 
 Control feces spiked 3NOP 256.2 ± 43.6b 240.9 16.5 1.4 0.995 
 Control feces spiked 3NOP carrier 298.7 ± 75.8ab 282.1 16.3 3.9 0.999 
 3NOP fed feces 279.2 ± 51.8ab 278.4 15.2 0.5 0.997 
 3NOP/Monensin fed feces 286.2 ± 48.4ab 280.7 15.9 1.2 0.998 
 Monensin fed feces 283.4 ± 46.3ab 265.1 15.7 1.7 0.997 
Study 1 (Finishing diet) 
 Control feces 298.1 ± 21.7a 281.2 16.8 0.6 0.994 
 Control feces spiked 3NOP 274.7 ± 6.8a 272.5 20.0 1.1 0.999 
 Control feces spiked 3NOP carrier 294.8 ± 15.3a 287.8 28.0 1.3 0.998 
 3NOP fed feces 269.4 ± 2.4b 270.7 28.2 1.7 0.999 
 3NOP/Monensin fed feces 234.2 ± 67.2ab 229.7 19.9 0.6 0.995 
 Monensin fed feces 236.1 ± 64.7ab 232.3 17.5 1.0 0.998 
Study 2 (Backgrounding diets) 
 Control feces 210.2 ± 32.8a 206.3 9.1 0.0 0.985 
 Control feces spiked 3NOP 203.3 ± 26.2a 183.8 12.4 0.0 0.977 
 Control feces spiked 3NOP carrier 181.4 ± 51.7a 184.5 6.3 0.0 0.982 
 3NOP fed feces 206.1 ± 16.3a 186.6 15.8 0.0 0.987 
 Oil without 3NOP fed 315.2 ± 40.3b 303.4 14.5 0.1 0.992 
 3NOP + oil fed feces 388.4 ± 24.6c 377.6 17.7 0.4 0.992 
 Experimental Gompertz regression parameters
 
 
CH4 yield (mL g−1 VS) Pm (mL) Rm (mL d−1Xo (d) R2 
Cellulose control 393.3 ± 10.9 415.7 26.2 3.9 0.998 
Study 1 (Backgrounding diets) 
 Control feces 342.7 ± 6.4a 323.6 19.9 0.4 0.993 
 Control feces spiked 3NOP 256.2 ± 43.6b 240.9 16.5 1.4 0.995 
 Control feces spiked 3NOP carrier 298.7 ± 75.8ab 282.1 16.3 3.9 0.999 
 3NOP fed feces 279.2 ± 51.8ab 278.4 15.2 0.5 0.997 
 3NOP/Monensin fed feces 286.2 ± 48.4ab 280.7 15.9 1.2 0.998 
 Monensin fed feces 283.4 ± 46.3ab 265.1 15.7 1.7 0.997 
Study 1 (Finishing diet) 
 Control feces 298.1 ± 21.7a 281.2 16.8 0.6 0.994 
 Control feces spiked 3NOP 274.7 ± 6.8a 272.5 20.0 1.1 0.999 
 Control feces spiked 3NOP carrier 294.8 ± 15.3a 287.8 28.0 1.3 0.998 
 3NOP fed feces 269.4 ± 2.4b 270.7 28.2 1.7 0.999 
 3NOP/Monensin fed feces 234.2 ± 67.2ab 229.7 19.9 0.6 0.995 
 Monensin fed feces 236.1 ± 64.7ab 232.3 17.5 1.0 0.998 
Study 2 (Backgrounding diets) 
 Control feces 210.2 ± 32.8a 206.3 9.1 0.0 0.985 
 Control feces spiked 3NOP 203.3 ± 26.2a 183.8 12.4 0.0 0.977 
 Control feces spiked 3NOP carrier 181.4 ± 51.7a 184.5 6.3 0.0 0.982 
 3NOP fed feces 206.1 ± 16.3a 186.6 15.8 0.0 0.987 
 Oil without 3NOP fed 315.2 ± 40.3b 303.4 14.5 0.1 0.992 
 3NOP + oil fed feces 388.4 ± 24.6c 377.6 17.7 0.4 0.992 

Statistical analysis of CH4 yields was performed using Post Hoc Multiple comparisons for observed means and the data in columns followed by the same lower case letter do not differ significantly at 0.05 probability level.

Table 3

Results summary of anaerobic leach bed digestion of feces from beef cattle fed forage- (Study 1 and 2) and grain-based (Study 1) diets supplemented with 3-nitrooxypropanol (3NOP) or control feces spiked with NOP

Feces Control feces
 
Control feces spiked with 3NOP
 
3NOP fed feces
 
Run 1 Run 2 Run 1 Run 2 Run 1 Run 2 
Study 1 (Backgrounding diets) 
ISR 0.2 0.5 0.2 0.30 0.2 0.3 
Total CH4 (L) 8.4 ± 0.0 13.1 ± 1.8 8.3 ± 1.3 12.9 ± 1.1 8.1 ± 0.2 12.9 ± 0.1 
CH4 yield (mL g−1 VS) 210.6 ±0.6a 233.3 ± 3.0b 207.3 ± 31.7ab 249.2 ± 93.6ab 202.5 ± 6.2a 251.9 ± 1.0bc 
KRm (mL d−18.9 16.6 8.1 13.6 11.8 12.2 
Lag phase (d) 8.3 3.2 11.7 2.8 10.1 2.0 
CH4 content (%) 52.5 ± 1.5 49.2 ± 2.5 51.3 ± 5.5 52.4 ± 1.1 52.0 ± 3.2 50.6 ± 1.5 
VS reduction (%) 46.3 ± 0.5 55.9 ± 1.3 41.9 ± 0.8 77.0 ± 23.0 44.3 ± 1.4 65.7 ± 2.6 
DOC (mg L−12,800 ± 375 1,078 ± 55 2,908 ± 23 1,115 ± 1 2,880 ± 636 1,041 ± 52 
NH4+-N (mg L−1736 ± 21 1,169 ± 116 817 ± 27 1,162 ± 66 825 ± 86 997 ± 14 
Water-N (mg L−12,158 ± 98 1,444 ± 81 2,277 ± 283 1,475 ± 77 2,177 ± 110 1,377 ± 218 
Study 1 (Finishing diets) 
ISR 0.2 0.5 0.2 0.4 0.2 0.5 
Total CH4 (L) 8.7 ± 0.9 8.3 ± 0.6 7.0 ± 0.4 9.2 ± 0.1 8.7 ± 0.5 9.2 ± 0.5 
CH4 yield (mL g−1 VS) 218.1 ± 22.0a 139.9 ± 4.2b 174.8 ± 8.8c 160.9 ± 9.1c 218.3 ± 12.1a 149.6 ± 12.3bc 
Rm (mL d−18.7 7.3 7.0 9.2 9.9 8.8 
Lag phase (d) 3.7 0.0 5.3 0.7 4.8 0.7 
CH4 content (%) 47.9 ± 4.4 45.0 ± 3.6 41.1 ± 1.6 46.3 ± 0.4 54.5 ± 1.9 45.6 ± 1.4 
VS reduction 52.1 ± 5.0 35.7 ± 1.7 41.0 ± 1.2 37.7 ± 2.6 45.6 ± 0.3 28.8 ± 8.2 
DOC (mg L−11,078 ± 55 994 ± 167 1,041 ± 52 1,026 ± 144 1,115 ± 1 1,359 ± 21 
NH4+-N (mg L−11361 ± 19 620 ± 6 1308 ± 132 624 ± 20 1395 ± 75 666 ± 2 
Water-N (mg L−11444 ± 81 1200 ± 77 1377 ± 218 1150 ± 12 1475 ± 77 1309 ± 59 
Study 2 (Backgrounding diets) 
Feces       
ISR 0.2 0.4 0.2 0.4 0.2 0.5 
Total CH4 (L) 6.5 ± 0.6 6.5 ± 1.2 5.6 ± 1.3 7.1 ± 0.4 6.0 ± 0.3 9.2 ± 1.0 
CH4 yield (mL g−1 VS) 162.7 ± 15.2a 111.6 ± 18.8b 138.8 ± 32.9ab 123.9 ± 6.8b 150.5 ± 7.9a 157.2 ± 15.6a 
Rm (mL d−15.5 6.8 5.5 7.9 6.2 8.1 
Lag phase (d) 2.4 2.2 2.3 3.6 2.2 4.0 
CH4 content (%) 52.5 ± 1.5 52.1 ± 1.7 54.9 ± 0.2 48.0 ± 4.2 55.1 ± 1.2 47.2 ± 0.2 
VS reduction 34.9 ± 4.8 26.1 ± 4.9 31.2 ± 0.6 39.1 ± 3.6 28.8 ± 7.5 24.3 ± 4.7 
DOC (mg L−11224 ± 19 1165 ± 52 1093 ± 4 977 ± 146 1,234 ± 137  992 
NH4+-N (mg L−11218 ± 2 902 ± 76 1087 ± 100 642 ± 105 1017 ± 11 664 
Water-N (mg L−11281 ± 26 960 ± 124 997 ± 78 656 ± 136 1144 ± 112 663 
Feces Control feces
 
Control feces spiked with 3NOP
 
3NOP fed feces
 
Run 1 Run 2 Run 1 Run 2 Run 1 Run 2 
Study 1 (Backgrounding diets) 
ISR 0.2 0.5 0.2 0.30 0.2 0.3 
Total CH4 (L) 8.4 ± 0.0 13.1 ± 1.8 8.3 ± 1.3 12.9 ± 1.1 8.1 ± 0.2 12.9 ± 0.1 
CH4 yield (mL g−1 VS) 210.6 ±0.6a 233.3 ± 3.0b 207.3 ± 31.7ab 249.2 ± 93.6ab 202.5 ± 6.2a 251.9 ± 1.0bc 
KRm (mL d−18.9 16.6 8.1 13.6 11.8 12.2 
Lag phase (d) 8.3 3.2 11.7 2.8 10.1 2.0 
CH4 content (%) 52.5 ± 1.5 49.2 ± 2.5 51.3 ± 5.5 52.4 ± 1.1 52.0 ± 3.2 50.6 ± 1.5 
VS reduction (%) 46.3 ± 0.5 55.9 ± 1.3 41.9 ± 0.8 77.0 ± 23.0 44.3 ± 1.4 65.7 ± 2.6 
DOC (mg L−12,800 ± 375 1,078 ± 55 2,908 ± 23 1,115 ± 1 2,880 ± 636 1,041 ± 52 
NH4+-N (mg L−1736 ± 21 1,169 ± 116 817 ± 27 1,162 ± 66 825 ± 86 997 ± 14 
Water-N (mg L−12,158 ± 98 1,444 ± 81 2,277 ± 283 1,475 ± 77 2,177 ± 110 1,377 ± 218 
Study 1 (Finishing diets) 
ISR 0.2 0.5 0.2 0.4 0.2 0.5 
Total CH4 (L) 8.7 ± 0.9 8.3 ± 0.6 7.0 ± 0.4 9.2 ± 0.1 8.7 ± 0.5 9.2 ± 0.5 
CH4 yield (mL g−1 VS) 218.1 ± 22.0a 139.9 ± 4.2b 174.8 ± 8.8c 160.9 ± 9.1c 218.3 ± 12.1a 149.6 ± 12.3bc 
Rm (mL d−18.7 7.3 7.0 9.2 9.9 8.8 
Lag phase (d) 3.7 0.0 5.3 0.7 4.8 0.7 
CH4 content (%) 47.9 ± 4.4 45.0 ± 3.6 41.1 ± 1.6 46.3 ± 0.4 54.5 ± 1.9 45.6 ± 1.4 
VS reduction 52.1 ± 5.0 35.7 ± 1.7 41.0 ± 1.2 37.7 ± 2.6 45.6 ± 0.3 28.8 ± 8.2 
DOC (mg L−11,078 ± 55 994 ± 167 1,041 ± 52 1,026 ± 144 1,115 ± 1 1,359 ± 21 
NH4+-N (mg L−11361 ± 19 620 ± 6 1308 ± 132 624 ± 20 1395 ± 75 666 ± 2 
Water-N (mg L−11444 ± 81 1200 ± 77 1377 ± 218 1150 ± 12 1475 ± 77 1309 ± 59 
Study 2 (Backgrounding diets) 
Feces       
ISR 0.2 0.4 0.2 0.4 0.2 0.5 
Total CH4 (L) 6.5 ± 0.6 6.5 ± 1.2 5.6 ± 1.3 7.1 ± 0.4 6.0 ± 0.3 9.2 ± 1.0 
CH4 yield (mL g−1 VS) 162.7 ± 15.2a 111.6 ± 18.8b 138.8 ± 32.9ab 123.9 ± 6.8b 150.5 ± 7.9a 157.2 ± 15.6a 
Rm (mL d−15.5 6.8 5.5 7.9 6.2 8.1 
Lag phase (d) 2.4 2.2 2.3 3.6 2.2 4.0 
CH4 content (%) 52.5 ± 1.5 52.1 ± 1.7 54.9 ± 0.2 48.0 ± 4.2 55.1 ± 1.2 47.2 ± 0.2 
VS reduction 34.9 ± 4.8 26.1 ± 4.9 31.2 ± 0.6 39.1 ± 3.6 28.8 ± 7.5 24.3 ± 4.7 
DOC (mg L−11224 ± 19 1165 ± 52 1093 ± 4 977 ± 146 1,234 ± 137  992 
NH4+-N (mg L−11218 ± 2 902 ± 76 1087 ± 100 642 ± 105 1017 ± 11 664 
Water-N (mg L−11281 ± 26 960 ± 124 997 ± 78 656 ± 136 1144 ± 112 663 

ISR, Inoculum substrate ratio; VS, volatile organic matter; DOC, dissolved organic carbon; Water-N, water soluble nitrogen. R2 for the determined parameters of the Gompertz equation ranged from 0.98 to 0.99. Statistical analysis of CH4 yields was performed using Post Hoc Multiple comparisons for observed means and the data in rows followed by the same lower case letter do not differ significantly at 0.05 probability level.

Residual effect of the anaerobic digestion of 3NOP feces on methane production in a leach bed reactor

Figure 1 shows the daily CH4 production rate and the cumulative CH4 yields during the anaerobic digestion in leach bed reactors of the control feces, 3NOP spiked feces and the 3NOP feces for all three cattle feces sets collected from the feedlot (Study 1) and metabolism (Study 2) studies. The results show an increase in the maximum daily CH4 production rate for all three feces sets tested (Figure 1). For instance, the maximum daily CH4 production rate was 719 mL d−1 on day 15 for run 1 which increased to 1,290 mL d−1 on day 47 (7 days into run 2) for run 2, during the anaerobic digestion of the control forage-based feces from the feedlot study. There was higher volumetric methane production in run 2 than in run 1 for all tested feces sets (Table 3). For example, the total CH4 produced after 40 days of digestion in run 1 was 8.4 ± 0.0 L, increasing to 13.1 ± 1.8 L in the second run of the anaerobic digestion of the control feces from forage-based feces of the feedlot study. The increase in the daily CH4 production rate and total CH4 produced was likely due to an increase in the ISR ratio from about 0.2 to 0.5, an increase in the amount of VS added from the use of partially digested material from run 1 as inoculation for run 2 and the adaptation of the methanogens to the substrate.

Figure 1

Daily methane production rate and cumulative methane yield during the anaerobic digestion in a leach bed reactor of forage- (a and b) and (c and d) grain-based feces collected from a feedlot (Study 1) and forage-based (e and f) feces from a metabolism study (Study 2). Day 0 to 40 represented run 1 and Day 40 to 80 represented run 2.

Figure 1

Daily methane production rate and cumulative methane yield during the anaerobic digestion in a leach bed reactor of forage- (a and b) and (c and d) grain-based feces collected from a feedlot (Study 1) and forage-based (e and f) feces from a metabolism study (Study 2). Day 0 to 40 represented run 1 and Day 40 to 80 represented run 2.

There was no residual effect on anaerobic digestion when 3NOP was used as a feed supplement in beef cattle diets for all three feces sets tested in both runs 1 and 2. This was evidenced by comparable (p > 0.05) CH4 yields of 210.6 ± 0.6 mL g−1 VS in run 1 of the control feces and 202.5 ± 6.2 mL g−1 VS for the 3NOP in Study 1 for the backgrounding forage-based feces of the feedlot. This similarity in CH4 yield was also observed for the other two treatments and also mentioned for the CH4 potential batch test.

Spiking the control feces with 3NOP did not result in a significant decrease in the overall CH4 yields for all three feces sets tested in both runs 1 and 2. Similar (p > 0.05) CH4 yields of 210.6 ± 0.6 mL g−1 VS for the control feces and 207.3 ± 31.7 mL g−1 VS for the 3NOP spiked feces were obtained in run 1 from the forage-based feces of the feedlot study. However, spiking 3NOP resulted in a decrease in the initial CH4 production rate, and the rate constant Rm decreased from 8.9 to 8.1 mLd−1 (9.0%) in run 1 and from 16.6 to 13.6 mL d−1 (18.1%) in run 2 for the forage-based (backgrounding diet) feedlot feces in Study 1. A similar effect was also observed for the grain-based (finishing diet) feedlot feces in Study 1. However, the rates were rather constant for run 1 in Study 2 but then decreased by 8.8% in run 2. Spiking 3NOP also resulted in an increase in the lag phase time from 0.4 to 3.4 d, which was observed in all three treatments. The CH4 yields of the control feces were 210.6 ± 0.6, 218.1 ± 22.0 and 162.7 ± 15.2 mL g−1 VS for the forage and grain- based feces from the feedlot study and the forage based feces from the metabolism study, respectively.

The CH4 yields obtained from the leach bed reactors were lower (by 61.5–77.4%) than the yields obtained in the CH4 potential batch test, which were 342.7 ± 6.4, 298.1 ± 21.7 and 210.2 ± 32.8 mL g−1 VS for the forage-based feedlot study feces, grain-based feedlot study feces and the forage-based metabolism study feces, respectively. Methane potential batch tests are generally optimized by using active and nutrient-rich inoculum source, high ISR, efficient mixing, substrate with reduced particle size, optimum substrate concentration and supplementation with nutrients, micronutrient and vitamins in case of a nutrient-deficient inoculum for the determination of the ultimate CH4 potential from a feedstock (Angelidaki et al. 2009). Our anaerobic digestion in the leach bed reactor simulated dry digestion systems for feedstock with high total solids content (TS 15–40%), which has the benefits of both high organic loading rates (5–12 g VS L−3 d−1) and biogas yields (0.622 L CH4 g VS−1), reduced reactor volumes and heating demand, low supervision/maintenance cost, digestate with high TS and nutrient content, less need for feedstock pretreatment and generally more stability than wet digestion (TS < 15%) processes (Karthikeyan & Visvanathan 2013; Massé & Saady 2015). However, dry digestion systems have the disadvantage of inefficient mixing, which results in mass transfer limitations and poor contact between bacteria, enzymes and substrate. A CH4 yield of 280 mL g−1 VS was obtained in a previous study of dry anaerobic digestion of dairy feces under thermophilic temperature (55 °C). In another study of dry (27% TS) anaerobic digestion of dairy feces, a CH4 yield of 146.0 ± 9.9 mL g−1 VS was obtained under psychrophilic temperature (20 °C) (Massé & Saady 2015). A CH4 yield of 227 mL g−1 VS was reported in a previous study of the anaerobic digestion of beef cattle manure and grass silage (85:15) (Massé & Saady 2015). The variability in the CH4 yields could be due to the differences in feedstock chemical composition and operational conditions.

The performance of the leach bed anaerobic digestion of all three feces sets was also evaluated based on VS reduction, DOC, and the mineralization of organic nitrogen into NH4+-N (Table 2). VS reduction is the amount of organic matter converted into mainly CH4 and CO2 during the anaerobic digestion and expressed as a percentage of the amount of VS initially added. The results show that high VS reduction values correspond to batches with high CH4 yields and vice versa. The digestate obtained after 40 days of digestion from mainly the second run of all feces sets were stable with DOC <1,500 mg L, which has been reported as a criterion for a stable digestate (Astals et al. 2013). Other criteria that are also used to assess digestate stability include thermal analysis, biochemical oxygen demand (BOD5day), DOC/TKN (total Kjeldahl nitrogen) and VFA/TIC (total inorganic carbon) (Huang et al. 2016). Most water-N was in the form of NH4+-N, which was generally higher when compared to the amount present in feedstock prior to anaerobic digestion.

A slower VFA degradation profile was observed from days 10 to 40 for all three treatments spiked with 3NOP when compared with the control feces and 3NOP feces (Figure 2). This indicates that spiking the cattle feces with 3NOP will reduce methanogenic activity by limiting the conversion of VFA into biogas. A sharp drop in pH was also observed in run 1 when compared to run 2 for the anaerobic digestion of the three sets of feces. This drop in pH corresponded to the periods with high tCOD (results not shown) and VFA concentrations. The solubilisation of polymeric organic compounds into soluble organics (tCOD, VFA) was lower during the anaerobic digestion of forage-based feces from the metabolic study when compared to the digestion of the feces sets from the feedlot study. The low organic matter solubilisation was also reflected in the low CH4 yields. The reason for the low CH4 yield could be slowly degrading lignocellulose fibers (cellulose, hemicellulose and lignin) or poor digestibility in the leach bed reactor, as the batch CH4 yields were higher. The CH4 yields of the slowly degrading feces fraction could be improved using physical, chemical, or thermal pretreatments, or a combination thereof (Jönsson & Martín 2016).

Figure 2

Profile of pH and tVFA during anaerobic leach bed digestion of forage (a1-3) and (b1-3) grain-based feces from a feedlot (Study 1) and forage-based feces from a metabolism study (Study 2). Day 0 to 40 represented run 1 and Day 40 to 80 represented run 2.

Figure 2

Profile of pH and tVFA during anaerobic leach bed digestion of forage (a1-3) and (b1-3) grain-based feces from a feedlot (Study 1) and forage-based feces from a metabolism study (Study 2). Day 0 to 40 represented run 1 and Day 40 to 80 represented run 2.

The results of the present study showed no residual effect of 3NOP on CH4 production, as CH4 yields and CH4 production profiles were comparable. In a previous study, the supplementation of beef cattle feed with 3NOP for 112 days did not result in a significant residual effect on enteric CH4 production when 3NOP was removed from the diet in a proceeding 16-day recovery period (Romero-Perez et al. 2015). Another study on the use of 3NOP as an enteric CH4 inhibitor speculated that the compound is possibly absorbed, rapidly metabolized or washed out of the rumen and consequently repeated supplementation to animals is necessary for sustained reduction in enteric CH4 production (Hristov et al. 2013). Supplementation of higher concentration of 10 g L−1 monensin in the continuous anaerobic digestion of manure resulted in 75% less methane when compared to no significant effect when 1 g L−1 was supplemented (Arikan et al. 2018). Based on the results from our study, it appears that the use of 3NOP in animal diets has no or minimal effect on anaerobic digestion of the feces for biogas production. These results need to be confirmed in other beef cattle studies using a variety of diets and with dairy cows, together with its effect on other waste treatment methods such as composting and manure stock piling.

CONCLUSIONS

The study is the first to examine the potential residual effects of using the manure from beef cattle fed forage- and grain-based diets supplemented with the novel enteric CH4 inhibitor, 3-nitrooxypropanol (3NOP), for biogas potential. Comparable CH4 yields were obtained in both batch and leach bed digesters from the control feces when compared to feces from cattle fed 3NOP. Spiking 3NOP to the control feces did not result in a significant decrease in CH4 yield but resulted in a decrease in CH4 production rate, increased the lag phase time and decreased the degradation of volatile acids. Our results showed that 3NOP, which has been reported to reduce enteric CH4 emissions in dairy and beef cattle, has no residual effect on downstream anaerobic digestion when used as a waste management method.

ACKNOWLEDGEMENTS

We acknowledge funding from Alberta Livestock and Meat Agency and Agriculture and Agri-Food Canada Growing Forward 2 program. Technical support was provided by Jessica Stoeckli, Kui Lui and Darrel Vedres. We thank DSM Nutritional Products for supplying the experimental methane inhibitor 3NOP.

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