The Potential to Feed Nitrates to Reduce Enteric Methane Production in Ruminants

1. The Potential to Feed Nitrates to Reduce Enteric Methane Production in Ruminants By R A Leng AO, D.Rur.Sc. Emeritus Professor UNE-Armidale

2. The issues in a nut shell 1 • Globally ruminants produce around 80x106 tonnes of methane • Methane production from reduction of carbon dioxide allows reduced cofactors generated in fermentative digestion to be re-oxidized allowing feed intake and digestion to be continuous. An electron sink is essential in rumen digestion.

3. The issues in a nut shell 2 • The rumen milieu can use sulphate and nitrate as alternative electron acceptors with the production of hydrogen sulphide and ammonia. • Sulphate is eliminated for this purpose as the end product, hydrogen sulphide would be toxic at the dietary levels required. • Nitrate would be the choice electron acceptor since the sink is ammonia the preferred fermentable –N source for synthesis in cell growth. However, under some nutritional conditions/feed management nitrate becomes toxic because of the accumulation of nitrite in the rumen

4. The pathways of nitrate metabolism in the rumen are uncertain but have always been assumed to be by dissimilatory nitrate reduction (DNR) to ammonia Overall reaction • NO3-+2H+ ---------H2O+NO2- --------------------1 • NO2-+6H+ ---------H2O+ NH3 -------------------2 Organisms capable DNR of nitrate to nitrite and assimilatory nitrite reduction(ANR) to ammonia use formate and hydrogen as the common electron donors • 3HCO-2+NO2-+5H+=3 CO2+NH+4+2H2O ---------- 3 • 3H2+NO2-+2H+ = NH+4 +2H2O ------------------4 The newly discovered NR-SOB organisms use hydrogen sulphide as an electron donor for respiratory nitrite ammonification • 3HS- + NO2- +5H+ = 3S0 + NH+4 +2H2O --------5

5. Pathways for the anaerobic fermentation of glucose by gut inhabiting microbes (Nolan 1999).

6. The sticking point Nitrate has been rejected as a potential N source for ruminants that would also inhibit methane production because nitrite accumulated in rumen fluid when nitrate was administered to animals without prior adaptation. Nitrite is anti-nutritional because it is absorbed and causes mild to extreme methaemoglobinaemia. The symptoms varying from a loss of production to death of the animal Leng R A (2008) The potential of feeding nitrate to reduce enteric methane production in ruminants A Report to The Department of Climate Change, Commonwealth Government of Australia Canberra ACT Australia

7. Changes in nitrate and nitrite in rumen fluid of a cow after ingesting hay over 45 minutes, containing 82 g of nitrate (Kemp et al 1977)

8. The changes in methaemoglobin in blood of cattle with increasing amounts of nitrate entering the rumen (after Crawford et al 1965).

9. Relationship between the peak concentration of nitrite in rumen fluid and methaemoglobin in the blood of cows (Kemp et al 1977)

10. Critical leads suggesting that there is potential for nitrate to be used as a fermentable N source for ruminants with inhibition of methanogenesis • Extreme variability in reported field observations on nitrate toxicosis • LD50 (1-10g nitrate/kg body weight). Effects on death rate, abortions, growth rates and milk yield have all been reported as significant or no effects. Indicating many interacting factors • Research demonstrating: • the substantial capacity of rumen microbes to metabolize nitrate and nitrite to ammonia • with acclimation of sheep to nitrate in their diet the ability to metabolize nitrate and nitrite increased 300% and upto 1000% • no nitrite accumulated in rumen fluid in vivo or in vitro when nitrate fed in the ration of sheep, no ill effects of nitrate was observed in acclimated animals • nitrate could be used as the sole source of fermentable N in sheep fed a purified diet high in starch, indicating it is a source of N for microbial anabolism • progressively introducing nitrate into a low protein straw/molasses diets for goats changed a negative N balance to a positive when nitrate provided the sole source of fermentable N • nitrate had no ill effects in sheep when introduced into high protein diets at 3-5% of the feed intake. • demonstration both in vivo and in vitro that methanogenesis was inhibited by inclusion of nitrate in the diet and /or incubation medium respectively

11. The research goals largely decided the experimental approach to the study of nitrate metabolism in ruminants. • The toxicological approach has been the fundamental approach used for the last 16 years by the Japanese school led by Prof J Takahashi • 4x4 latin square design where a sub-lethal dose of nitrate was administered and four treatments examined. The objective always to provide an additive (chemical or culture of organisms ) to reduce nitrite accumulation which usually exceeded 2mM. • Nutritional approach followed well recognized rules of thumb • Nitrate was introduced over a period of days to weeks to allow rumen microbial community to adapt • Nitrate was well mixed through the feed and the feed was available two times or more daily. Nitrite was undetectable in the rumen fluid of sheep at all times

12. Effect of level of K-nitrate fed to sheep on the nitrate and nitrite disappearance from strained rumen fluid incubated in vitro (Alaboudi and Jones 1985). Extrapolation to whole animal indicates that the fermentative nitrogen requirements can be met by nitrate

13. Comparison of methaemoglobin produced in dairy heifers fed hay containing nitrate or drenched with an aqueous solution of nitrate(Crawford et al 1965) This work has been heavily criticized because the animals consumed the feed only slowly- sensible animal response?

14. Average daily feed intake and N-balance in young goats fed molasses/straw (30/60) with K-nitrate. The % K- nitrate in the feed was adjusted upwards every 7th day (Quong Do et al 2008 unpubl.)

15. The effects in lambs of adding nitrate and sulphur to a concentrate based diet. * SD Sokolowski et al 1965

16. N-balance (g/N day) and S-balance (g S/day) in lambs given a high concentrate diet supplemented with nitrate or nitrate plus sulphur (after Sokolowski et al 1969)

17. The effects of dietary molybdenum on the clearance of nitrate from rumen fluid of sheep after placing 500g of feed containing 6.5% K-nitrate and 6.5% Na-nitrate into the rumen (Tillman et al 1965). The difference in nitrate concentration appears to be only due to nitrite production

18. The effects of dietary Mo on the accumulation of nitrite in rumen fluid of sheep after placing 500g of feed containing 6.5% K-nitrate and 6.5% Na-nitrate into the rumen (Tillman et al 1965). In the feeding trial no apparent ill effects in lambs were observed when nitrate was the sole source of fermentable N. Presumably no nitrite was produced

19. Changes in nitrate, nitrite and ammonia concentrations in rumen fluid of sheep fasted for 16 hours and injected intra-ruminally with 25g sodium nitrate (Lewis 1951).

20. Clearance of nitrate from rumen fluid of sheep following administration of a nitrate load in different studies.

21. Appearance of ammonia in rumen fluid after and intra-ruminal load of urea or nitrate (Lewis 1951;Stephenson et al 1992) Note: Nitrate was injected into a 60kg sheep, whereas urea was injected into a 35kg Merino

22. Up to 80% administered nitrate appears to disappear from the rumen within the first hour. Potential explanations for this include: • Nitrate and nitrite are rapidly absorbed from the rumen • Nitrate is sequestered in microbes • Nitrite and possibly nitrate are bound to protein in a similar way as it is bound to haemoglobin • Nitrite is reduced rapidly to ammonia which is assimilated to inter-cellular amino acids( alanine) which may be released to the ammonia pool as electron availability is more synchronized with N availability • A nitrate load increases outflow of rumen fluid to the lower tract

23. 15N in rumen fluid ammonia and nitrate plus nitrite of a cow injected intra-ruminally with 120g K-nitrate labeled with 15N (Wang et al 1961) Nitrate was administered daily for a number of days before the labeled nitrate was given

24. Mean blood nitrate concentration with time after introduction of cattle to diets containing nitrate (Clarke et al 1970)

25. Conclusions from whole animal research • Toxicities from nitrite accumulation in and absorption from the rumen appeared to be the major constraint to replacing urea with nitrate in low protein diets • Nitrite accumulation in the rumen only occurs where nitrate is introduced abruptly and in excessive amounts. • The rumen milieu can readily adapt to nitrate as a source of fermentable N • Sheep on high energy diets supporting high growth rates were unaffected by adding 3.4% nitrate to their diets. • Dairy cows producing 16-19L milk/day were unaffected by inclusion of 2% nitrate in feed(Farra and Satter 1971) • Sulphur balance was effected by nitrate inclusion in sheep fed concentrate diets. • Additional Mo in a diet boosted nitrite production from nitrate. Mo suppresses sulphur reducing bacteria and production of hydrogen sulphide in the rumen

26. Conclusions from whole animal research • Acclimation to nitrate eliminates nitrite accumulation in the rumen removing any detrimental effects • Overall it appeared that nitrate ,at high compared to low concentrations was being metabolized by different processes and there appeared to be some interaction with sulphur availability. • Understanding the pathways of nitrate and sulphur metabolism offer possible explanations for nitrite accumulation.

27. The utilization of nitrate in the rumen appears to be different to urea • Nitrate rapidly disappears from rumen fluid and unlike urea the major route is not by conversion to ammonia • Nitrate or nitrite was entering the rumen 24 hours after nitrate had been administered. The source of the nitrite/nitrate maybe • Nitrate cycling between body fluid pools and the rumen • Nitrate/nitrite released from sequestered nitrate/nitrite in the rumen • Nitrate/nitrite is in equilibrium with the another N pool in the rumen( highly unlikely) • Nitrate absorbed into blood by humans( 2-4 mM/day) is concentrated up to 10 fold in the saliva. Salivary nitrate is converted to HNO2 by the acid stomach which spontaneously breaks down to nitrite. In the ruminant nitrate would be returned via salivary secretions to the rumen. • Nitrate entering the mouth in monogastric animals is reduced to nitrite by populations of organisms resident on the tongue. If the entero-salivary recycling occurred in ruminants it is potentially a significant sources of rumen nitrite when a nitrate load is given in the rumen.

28. The pathways of nitrate metabolism in the rumen are uncertain but have always been assumed to be by dissimilatory nitrate reduction (DNR) to ammonia Overall reaction • NO3-+2H+ ---------H2O+NO2- --------------------1 • NO2-+6H+ ---------H2O+ NH3 -------------------2 Organisms capable DNR of nitrate to nitrite and assimilatory nitrite reduction(ANR) to ammonia use formate and hydrogen as the common electron donors • 3HCO-2+NO2-+5H+=3 CO2+NH+4+2H2O ---------- 3 • 3H2+NO2-+2H+ = NH+4 +2H2O ------------------4 The newly discovered NR-SOB organisms use hydrogen sulphide as an electron donor for respiratory nitrite ammonification • 3HS- + NO2- +5H+ = 3S0 + NH+4 +2H2O --------5

29. At least three types of nitrate reductase catalyze the reduction of nitrate to nitrite in anaerobic bacteria • Included are: • A soluble, assimilatory nitrate reductase (NAS) that is present in the cytoplasm. • An energy-conserving nitrate reductase (NAR, for example,NarG,which is encoded by the first gene of the narGHJI operon) with catalytic sites located in thecytoplasm are associated with the cytoplasmicmembrane, from which they receive electronsfor nitrate reduction. • A soluble, periplasmic nitrate reductases (NAP) that are found in many Gram-negative bacteria.

30. At least three types of nitrate reductase catalyze the reduction of nitrate to nitrite in anaerobic bacteria • Included are: • A soluble, assimilatory nitrate reductase (NAS) that is present in the cytoplasm. • An energy-conserving nitrate reductase (NAR, for example, NarG,which is encoded by the first gene of the narGHJI operon) with catalytic sites located in thecytoplasm are associated with the cytoplasmicmembrane, from which they receive electronsfor nitrate reduction. • A soluble, periplasmic nitrate reductase (NAP) that are found in many Gram-negative bacteria.

31. Two biochemically distinct nitrite reductases catalyse the reduction of nitrite to ammonia • The NADH-dependent NirBD nitrite reductase reduces nitrite directly to ammonia in the cytoplasm of some bacteria. NirBD’s role is to detoxify nitrite generated by NarG (the membrane-associated nitrate reductase) during anaerobic growth in the presence of excess nitrate. • More widely distributed is the cytochrome c nitrite reductase Nrf, which catalyses the reduction of nitrite to ammonia in the periplasm of Gram-negative bacteria. This enzyme is the terminal component of an electron-transfer pathway in which electrons are transferred from physiological substrates, especially formate (hence Nrf). Nap, the periplasmic nitrate reductase, and Nrf, the periplasmic nitrite reductase, are coordinately regulated to provide a pathway for the reduction of nitrate to ammonia in the periplasm.

32. Production of nitric oxide occurs in the rumen which may have inhibitory effects on some Archae • Some bacteria that reduce nitrite directly to ammonia generate NO. • NO is produced in the rumen in small amounts when nitrate is included in a diet/medium • Although unproven it is a possibility that this NO is produced by one of the enzymes that reduce nitrite to ammonia (NrfA or NirBD). • Nitric oxide and other nitrogen oxides inhibit methanogenesis in some Archae • W succinogenese ( a rumen microbe) produces nitric oxide when incubated with nitrate • Nitrate as a component of the diet may have indirect effects on methanogenesis other then straight out competition for electrons with carbon dioxide, perhaps providing a competitive edge for NRB.

33. Inhibitory effects of N-oxides on Methanosarcina barkeri and Methanobacterium bryantii (a Klüber and Conrad, 1998 ;b Clarens et al., 1998)

34. Some important issues concerning sulphur metabolism in the rumen • The major source of sulphur in grazing ruminants is from the S-amino acids in protein • Sulphur is required for organic S compounds that are synthesized in microbial cell growth. • Excess sulphur in the rumen is reduced to hydrogen sulphide which has limited solubility and quickly enters the gas head space. The head space gases are eructated and inhaled into the lungs where a proportion is absorbed and converted to sulphate in the liver. • Sulphide levels in rumen fluid are low often about 0.1 mM • Sulphide in the rumen is rapidly cleared and in cows fasting for 4 hours following a period of grazing, hydrogen sulphide concentrations in the gas space is zero.

35. Sulphur reducing (SRB) and sulphide oxidizing bacteria (NR-SOB) exist in a number of anaerobic ecosystems • The SRB in the rumen are grouped by the mechanism used to reduce sulphates: • assimilatory process, • dissimilatory process. In general, the dissimilatory reduction of sulphur compounds is used for the generation of ATP, while the assimilatory process reduces sulphur compounds for incorporation into other organic compounds necessary for cell survival. The dissimilatory pathways are responsible for the reduction of sulphur to hydrogen sulphite and hydrogen sulphide (Desulfovibrio and Desulfotomaculum spp). Most SRB are also NRB since they can actively reduce nitrite but not nitrate to ammonia • Nitrate reducing, sulphide oxidizing bacteria (NR-SOB) convert nitrate to nitrite and utilize nitrite to oxidize hydrogen sulphide to poly S or sulphate and reduce nitrite to ammonia with the generation of ATP (Wolinella succinogenese, Desulfovibrio spp)

36. Impact of nitrate on sulphur cycle in the anaerobic ecosystems found in oil wells • Sulphide produced by SRB activity can be recycled to sulphate or sulphur by NR-SOB reducing nitrate to ammonia (DNRA) or in static anaerobic ecosystems denitrification occurs via nitrogen oxides to nitrogen gas. • Most SRB can use nitrite as an alternative electron acceptor • Introduction of nitrate increases NRB • hNRB compete with SRB for organic electron donors, such as lactate, excluding sulphide production and nitrite reduction(?) by SRB. The extent of the effect depending on the fermentation rate (availability of electron donors) Many SRB and hNRB oxidize lactate incompletely to acetate and CO2 • Lowered sulphide availability prevents nitrite ammonification (respiration) by NR-SOB and stimulates nitrate reduction to nitrite which may accumulate

37. Sulfurospirillum sp switch from nitrite production to ammonia production according to the relative availability of electron donors in the medium- is this a model for the rumen? • Lactate-to-nitrate ratios in medium containing fermentable organic matter determine whether nitrate reduction by Sulfurospirillum sp. strain KW yields nitrite or ammonia. • When this ratio is high (nitrate limiting), ammonia is the major end product • When it is low (fermentable organic matter limiting), nitrite is the major end product • The hypothesis In the rumen a sudden load of nitrate creates the conditions for a NR-SOB to favor nitrite production. As the nitrate levels decline these conditions reverse, where ammonia production is favored. • The overall control appears to be the suppression of sulphate reduction and lowered hydrogen sulphide concentrations accentuating production of nitrite by inhibiting the NR-SOB further metabolizing nitrite to ammonia coupled to oxidation of sulphide • 3HS- + NO2- +5H+ = 3S0 + NH+4 +2H2O --------5

38. The pathways of nitrate metabolism in the rumen are uncertain but have always been assumed to be by dissimilatory nitrate reduction (DNR) to ammonia Overall reaction • NO3-+2H+ ---------H2O+NO2- ----------------1 • NO2-+6H+ ---------H2O+ NH3 ---------------2 Organisms capable DNR of nitrate to nitrite and assimilatory nitrite reduction(ANR) to ammonia use formate and hydrogen as the common electron donors • 3HCO-2+NO2-+5H+=3 CO2+NH+4+2H2O ------3 • 3H2+NO2-+2H+ = NH+4 +2H2O -------------4 The newly discovered NR-SOB organisms use hydrogen sulphide as an electron donor for respiratory nitrite ammonification • 3HS + NO2- +5H+ = 3S0 + NH+4 +2H2O ----5

39. Fermentable OM NO3- NRB CO2 + VFA NH4+ Competitive Exclusion of SRB by NRB SO42- Fermentable OM SRB CO2 + VFA H2S H2S ADP NO3- NR-SOB SO42- NH3 ATP Sulphide removal by NR-SOB.When sulphide limiting NR-SOB reduce nitrate to nitrite

40. Three processes may lower rumen fluid hydrogen sulphide 1)loss to the gas space, 2)competition between NRB and SRB for electron donors and 3)oxidation to sulphate by NR-SOB Heterotrophic nitrate reducing bacteria (NRB) out compete SRB SO42- NO3- Fermentable OM NRB SRB NH3 CO2 + VFA H2S Nitrate reducing sulphide oxidizing bacteria (NR-SOB) lower H2S SO42- NH3 Fermentable OM NR-SOB SRB NO3- H2S CO2 + VFA

41. Nitrate poisoning is a result of the establishment of a single group of microorganisms the NR-SOB ? • When fermentable organic matter is high NRB and SRB can co exist maintaining rumen fluid H2S • When electron donors are in short supply NRB out compete SRB and H2S in rumen fluid declines • When rumen degradable protein is high the population density of NR –SOB depends on nitrate and H2S in rumen fluid • When H2S and nitrate adequate NR-SOB convert nitrate to ammonia but convert nitrate to nitrite in the absence of H2S

42. Percentage of nitrite and ammonia formed as a functionof the lactate-to-nitrate ratio in cultures of oil field NR-SOB (Sulfurospirillum spp.) (Hubbert and Voordouw 2007) % N as ammonia % N as nitrite

43. The effects of adding nitrate into an artificial rumen inoculated with rumen fluid • Nitrate was cleared rapidly in all incubations but nitrate N could not be accounted for in either ammonia or nitrite, particularly in first 5hr of the incubation. • Nitrite accumulated when nil, glucose of cellulose was included suggesting nitrite reduction to ammonia is inhibited. • Nitrite accumulation was negligible when dried grass was included in the medium. Conclusion protein (sulphide) and electron donors preserve nitrite assimilatory conversion to ammonia in NR-SOB (source Barnett and Bowman 1957)

44. The effects of protein intake on the accumulation of nitrite in rumen fluid after administration of a nitrate load (30g Na-nitrate) in sheep (Takahashi et al 1980) * First measurement of nitrate concentration after nitrate load given The hypothesis that arises is that at high concentrations of nitrate in the rumen, the fermentable biomass in the rumen determines the rate of availability of electrons which in turn dictates the proportion of nitrate converted to nitrite or ammonia. The extent to which this happens is determined by the population density of the NR-SOB which in turn depends on hydrogen sulphide generation largely from organic sulphur containing compounds and is mainly dependent on the amount of protein degraded in the rumen. This is consistent with the accumulation of nitrite being higher at higher protein intake( Table

45. The effects on methane production of administration of a nitrate load in fed sheep un-acclimated to nitrate in their diet (Sar et al 2004).

46. Changes in rumen fluid nitrate and nitrite with time after administration of Na-nitrate alone or a combination of Na-nitrate and cysteine to sheep (Takahashi et al 1998).

47. Effects of iso-S supplements on the concentration of hydrogensulphide in the rumen head-space gas of dairy cows. The cows had been without feed for 4 hours prior to administration of S-source (Dewhurst et al 2007).

48. Effects of increasingquantities of L-cysteine injected into the rumen on hydrogen sulphide concentration in rumen head space gas in dairy cows (Dewhurst 2007).

49. The relationship between hydrogen sulphide in rumen fluid and in the gas space in the rumen (After Gould et al 1997)

50. Nitrate poisoning preconditions • The population densities of NRB,SRB and NR-SOB in the rumen depend on the degradable protein content of the grazed forage • Fertilizer application controls growth rate of pasture soluble sugars, protein and nitrate in forage, • Lowering photosynthesis rate of the plant from environmental reasons may increase the accumulation of nitrate up to 10 fold.