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

The Potential to Feed Nitrates The Potential to Feed Nitrates to Reduce Enteric Methane to Reduce Enteric Methane

Production in RuminantsProduction in Ruminants

ByBy

R A Leng AO, D.Rur.Sc. R A Leng AO, D.Rur.Sc.

Emeritus Professor UNE-Emeritus Professor UNE-ArmidaleArmidale

The issues in a nut shell 1The issues in a nut shell 1 Globally ruminants produce around Globally ruminants produce around

80x1080x1066 tonnes of methane tonnes of methane

Methane production from reduction Methane production from reduction of carbon dioxide allows reduced of carbon dioxide allows reduced cofactors generated in fermentative cofactors generated in fermentative digestion to be re-oxidized allowing digestion to be re-oxidized allowing feed intake and digestion to be feed intake and digestion to be continuous. continuous. An electron sink is An electron sink is essential in rumen digestionessential in rumen digestion. .

The issues in a nut shell 2The issues in a nut shell 2

The rumen milieu can use sulphate and nitrate as The rumen milieu can use sulphate and nitrate as alternative electron acceptors with the production alternative electron acceptors with the production of hydrogen sulphide and ammonia.of hydrogen sulphide and ammonia.

Sulphate is eliminated for this purpose as the end Sulphate is eliminated for this purpose as the end product, product, hydrogen sulphide would be toxic at the hydrogen sulphide would be toxic at the dietary levels requireddietary levels required..

Nitrate would be the choice electron acceptor Nitrate would be the choice electron acceptor since the sink is ammonia the preferred since the sink is ammonia the preferred fermentable –N source for synthesis in cell growth. fermentable –N source for synthesis in cell growth. However, under some nutritional conditions/feed However, under some nutritional conditions/feed management nitrate becomes toxic because of management nitrate becomes toxic because of the accumulation of nitrite in the rumenthe accumulation of nitrite in the rumen

The pathways of nitrate metabolism in the The pathways of nitrate metabolism in the rumen are uncertain but have always been rumen are uncertain but have always been

assumed to be by assumed to be by dissimilatory nitrate dissimilatory nitrate reduction (DNR) to ammonia reduction (DNR) to ammonia

Overall reactionOverall reaction NONO33

--+2H+2H+ + ---------H---------H22O+NOO+NO22-- --------------------1 --------------------1

NONO22--+6H+6H++ ---------H ---------H22O+ NHO+ NH33 -------------------2 -------------------2

Organisms capable DNR of nitrate to nitrite and assimilatory nitrite Organisms capable DNR of nitrate to nitrite and assimilatory nitrite reduction(ANR) to ammonia use formate and hydrogen as the reduction(ANR) to ammonia use formate and hydrogen as the common electron donors common electron donors

3HCO3HCO--22+NO+NO22

--+5H+5H++=3 CO=3 CO22+NH+NH++44+2H+2H22O ---------- 3O ---------- 3

3H3H22+NO+NO22--+2H+2H++ = NH = NH++

44 +2H +2H22O ------------------4O ------------------4

The newly discovered NR-SOB organisms use hydrogen sulphide The newly discovered NR-SOB organisms use hydrogen sulphide as an electron donor for respiratory nitrite ammonificationas an electron donor for respiratory nitrite ammonification

3HS3HS-- + NO + NO22-- +5H +5H++ = 3S = 3S00 + NH + NH++

44 +2H +2H22O --------5O --------5

Pathways for the anaerobic Pathways for the anaerobic fermentation of glucose by gut fermentation of glucose by gut

inhabiting microbes (Nolan 1999inhabiting microbes (Nolan 1999).).

4H+

Glucose

Glucose

Glucose-6-P

2 Pyruvate

2 Acetyl CoA

2 Acetyl-P

2 AcetateAcetate

Butryl-CoA

Butryl-P

ButyrateButyrate

2 Lactate

2 Lactyl-CoA

2 Acrylyl-CoA

2 Propionyl-CoA

2 PropionatePropionate

2 Oxaloacetate

2 Malate

2 Fumarate

2 Succinate

2 Succinyl-CoA

2 Methylmalonyl-CoA

2 Propionyl-CoA

2 PropionatePropionate NAD +

NAD+

H2 MethaneMethane

ATP

2 ATP

ATP

NADH

NADH

NADH NADH

H+

2 NADH

H+

NADH

ATP

NADH

CO2 CO2

The sticking pointThe sticking point

Nitrate has been rejected as a potential N Nitrate has been rejected as a potential N source for ruminants that would also inhibit source for ruminants that would also inhibit methane production because nitrite methane production because nitrite accumulated in rumen fluid when nitrate was accumulated in rumen fluid when nitrate was administered to animals administered to animals without prior without prior adaptation.adaptation.

Nitrite is anti-nutritional because it is absorbed Nitrite is anti-nutritional because it is absorbed and causes mild to extreme and causes mild to extreme methaemoglobinaemia. The symptoms varying methaemoglobinaemia. The symptoms varying from a loss of production to death of the animalfrom 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

Changes in nitrate and nitrite in rumen fluid Changes in nitrate and nitrite in rumen fluid of a cow after ingesting hay over 45 of a cow after ingesting hay over 45

minutes, containing 82 g of nitrate (Kemp et minutes, containing 82 g of nitrate (Kemp et al 1977)al 1977)

0

1

2

3

4

5

6

7

8

9

10

0 0.5 1 1.5 2 2.5 3 3.5

Time after feeding hay (hr)

Nitra

te/nitri

te in r

um

en fl

uid

(m

M)

Nitrate in rumen fluid (mM) Nitrite in rumen fluid (mM)

The changes in methaemoglobin in blood of The changes in methaemoglobin in blood of cattle with increasing amounts of nitrate cattle with increasing amounts of nitrate

entering the rumen entering the rumen (after Crawford et al 1965).

y = 5.09e0.08x

R2 = 0.81

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25 30 35 40

g Nitrate administered per 100kg live weight

% M

etha

emog

lobi

n in

red

blo

od c

ells

Relationship between the peak Relationship between the peak concentration of nitrite in rumen fluid and concentration of nitrite in rumen fluid and

methaemoglobin in the blood of cows (Kemp methaemoglobin in the blood of cows (Kemp et al 1977)et al 1977)

y = 7.78x + 0.64

R2 = 0.86

0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6 7 8 9 10

Peak nitrite in rumen fluid (mM)

% m

eth

aem

oglo

bin

Critical leads suggesting that there is Critical leads suggesting that there is potential for nitrate to be used as a potential for nitrate to be used as a

fermentable N source for ruminants with fermentable N source for ruminants with inhibition of methanogenesisinhibition of methanogenesis

Extreme variability in reported field observations on nitrate toxicosisExtreme variability in reported field observations on nitrate toxicosis LDLD5050 (1-10g nitrate/kg body weight). Effects on death rate, abortions, growth rates (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. and milk yield have all been reported as significant or no effects. Indicating many Indicating many interacting factorsinteracting factors

Research demonstrating: Research demonstrating: the substantial capacity of rumen microbes to metabolize nitrate and nitrite to the substantial capacity of rumen microbes to metabolize nitrate and nitrite to

ammoniaammonia with acclimation of sheep to nitrate in their diet the with acclimation of sheep to nitrate in their diet the ability to metabolize nitrate and ability to metabolize nitrate and

nitrite increased 300% and upto 1000%nitrite increased 300% and upto 1000% no nitrite accumulated in rumen fluid no nitrite accumulated in rumen fluid in vivoin vivo or or in vitroin vitro when nitrate fed in the ration when nitrate fed in the ration

of sheep, no of sheep, no ill effects of nitrate was observed in acclimated animalsill 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 nitrate could be used as the sole source of fermentable N in sheep fed a purified diet

high in starch, high in starch, indicating it is a source of N for microbial anabolismindicating it is a source of N for microbial anabolism progressively introducing nitrate into a low protein straw/molasses diets for goats 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 changed a negative N balance to a positive when nitrate provided the sole source of fermentable Nfermentable N

nitrate had no ill effects in sheep when introduced into high protein diets at 3-5% of nitrate had no ill effects in sheep when introduced into high protein diets at 3-5% of the feed intake.the feed intake.

demonstration both demonstration both in vivoin vivo and and in vitroin vitro that methanogenesis was inhibited by that methanogenesis was inhibited by inclusion of nitrate in the diet and /or incubation medium respectively inclusion of nitrate in the diet and /or incubation medium respectively

The research goals largely decided the The research goals largely decided the experimental approach to the study of experimental approach to the study of

nitrate metabolism in ruminantsnitrate metabolism in ruminants. . The toxicological approach has been the fundamental The toxicological approach has been the fundamental

approach used for the last 16 years by the Japanese school approach used for the last 16 years by the Japanese school led by Prof J Takahashiled by Prof J Takahashi 4x4 latin square design where a sub-lethal dose of nitrate was 4x4 latin square design where a sub-lethal dose of nitrate was

administered and four treatments examined. administered and four treatments examined. The objective The objective always to provide an additive (chemical or culture of always to provide an additive (chemical or culture of organisms ) to reduce nitrite accumulation which usually organisms ) to reduce nitrite accumulation which usually exceeded 2mM. exceeded 2mM.

Nutritional approach followed well recognized rules of Nutritional approach followed well recognized rules of thumb thumb Nitrate was introduced over a period of days to weeks to allow Nitrate was introduced over a period of days to weeks to allow

rumen microbial community to adaptrumen microbial community to adapt Nitrate was well mixed through the feed and the feed was Nitrate was well mixed through the feed and the feed was

available two times or more daily. available two times or more daily. Nitrite was undetectable in Nitrite was undetectable in the rumen fluid of sheep at all times the rumen fluid of sheep at all times

Effect of level of K-nitrate fed to sheep on the Effect of level of K-nitrate fed to sheep on the nitrate and nitrite disappearance from strained nitrate and nitrite disappearance from strained

rumen fluid incubated rumen fluid incubated in vitroin vitro (Alaboudi and Jones (Alaboudi and Jones 1985).1985).

0

0.5

1

1.5

2

2.5

3

3.5

4

0 0.5 1 1.5 2 2.5 3

Lvel of nitrate fed (g/ kg live weight/ day)

Nitra

te o

r nitri

te d

isappeara

nce

(m

M/hour

/lite

r of st

rain

ed r

um

en

fluid

)

Nitrate Nitrite

Extrapolation to whole animal indicates that the fermentative nitrogen requirements can be met by nitrate

Comparison of methaemoglobin produced in Comparison of methaemoglobin produced in dairy heifers fed hay containing nitrate or dairy heifers fed hay containing nitrate or

drenched with an aqueous solution of nitratedrenched with an aqueous solution of nitrate(Crawford et al 1965)

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140

Nitrate administered by drench or by feeding it sprayed on hay

% M

eth

aem

oglo

bin

in r

ed b

lood c

ells

This work has been heavily criticized because the animals consumed the feed only slowly- sensible animal response?

Average daily feed intake and N-balance in young Average daily feed intake and N-balance in young goats fed molasses/straw (30/60) with K-nitrate. The goats fed molasses/straw (30/60) with K-nitrate. The % K- nitrate in the feed was adjusted upwards every % K- nitrate in the feed was adjusted upwards every

7th day (Quong Do et al 2008 unpubl.)7th day (Quong Do et al 2008 unpubl.)

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0.3 0.6 1.2 2.4 4.8

N b

alan

ce (

g N

/d

ay)

-100

0

100

200

300

400

500

Fee

d i

ntk

e (g

/d

ay)

N Balance Feed intake

The effects in lambs of adding nitrate and The effects in lambs of adding nitrate and

sulphur to a concentrate based diet.sulphur to a concentrate based diet. SupplementSupplement +0.4%S+0.4%S +3.2% +3.2%

KNOKNO33

+3.2% KNO+3.2% KNO33

+0.4% S+0.4% S+3.2% KNO+3.2% KNO33

+0.8% S+0.8% S

Dietary crude protein (%)Dietary crude protein (%) 8.68.6 10.510.5 10.510.5 10.510.5

Dietary sulphur (%)Dietary sulphur (%) 0.520.52 1.21.2 0.520.52 0.920.92

Daily gain (g/day)Daily gain (g/day) 220220 170170 170170 200200

Feed intake (g/day)Feed intake (g/day) 1.41.4 1.531.53 1.451.45 1.511.51

Feed conversion ratio (g/g)Feed conversion ratio (g/g) 7.07.0 8.88.8 8.68.6 7.57.5

Sulfur balance (gS/day) Sulfur balance (gS/day) 0.670.67 -0.8-0.8 1.221.22 2.862.86

N balance (gN)N balance (gN) 3.95(1.12)*3.95(1.12)* 3.35(1.58)3.35(1.58) 5.60(3.18)5.60(3.18) 3.62(4.94)3.62(4.94)

Wool growth (g per 100cmWool growth (g per 100cm22/day)/day) 2.32.3 2.62.6 2.62.6 2.52.5

Sokolowski et al 1965

* SD

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

et al 1969)

0

1

2

3

4

5

6

7

8

9

N b

alan

ce (

gN

/day

)

Nil Plus 3.2%nitrate

Plus 3.2%nitrate plus0.4% sulfur

Plus 3.2%nitrate plus0.8% sulfur

-1

-0.5

0

0.5

1

1.5

2

2.5

3

Sulp

hur

bal

ance

(g S

/day

)

The effects of dietary molybdenum on the clearance The effects of dietary molybdenum on the clearance of nitrate from rumen fluid of sheep after placing of nitrate from rumen fluid of sheep after placing 500g of feed containing 6.5% K-nitrate and 6.5% 500g of feed containing 6.5% K-nitrate and 6.5% Na-nitrate into the rumen Na-nitrate into the rumen (Tillman et al 1965).

0

20

40

60

80

100

120

140

0 1 2 3 4 5 6 7 8 9 10

Time after administration of nitrate (hr)

Conce

ntr

ation in r

um

en fl

uid

(m

M)

Nitrate: Basal diet Nitrate: Basal+molybdenum

The difference in nitrate concentration appears to be only due to nitrite production

The effects of dietary Mo on the accumulation of The effects of dietary Mo on the accumulation of nitrite in rumen fluid of sheep after placing 500g of nitrite in rumen fluid of sheep after placing 500g of feed containing 6.5% K-nitrate and 6.5% Na-nitrate feed containing 6.5% K-nitrate and 6.5% Na-nitrate

into the rumen into the rumen (Tillman et al 1965).

0

1

2

3

4

5

6

7

8

9

10

0 2 4 6 8 10

Time after administration of nitrate containing feed (hr)

Conce

ntr

ation o

f ru

men fl

uid

nitri

te

(mM

)

Rumen fluid nitrite Basal-noMo

Rumen fluid nitrite-Basal-plus Mo

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

Changes in nitrate, nitrite and ammonia Changes in nitrate, nitrite and ammonia concentrations in rumen fluid of sheep concentrations in rumen fluid of sheep fasted for 16 hours and injected intra-fasted for 16 hours and injected intra-

ruminally with 25g sodium nitrate (Lewis ruminally with 25g sodium nitrate (Lewis 1951).1951).

05

1015202530

0 5 10

Time after administration of nitrate,hrs

Ru

men

flu

id

co

ncen

trati

on

Nitrate-N (mM) Nitrite -N (mM) Ammonia N (mM)

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

studiesstudies..

0

20

40

60

80

100

120

140

0 2 4 6 8 10 12

Time after administration of nitrate,hrs

Rum

en fl

uid

conce

ntr

ation (

mM

)

Rumen nitrate (mM)-Takahashi and Young(1991)

Rumen nitrate (mM)-Lewis(1951)

Rumen nitrate (mM)-Tillman et al (1965)

Rumen nitrate (mM)-Sar etal (2004)

Appearance of ammonia in rumen fluid Appearance of ammonia in rumen fluid after and intra-ruminal load of urea or after and intra-ruminal load of urea or nitrate (Lewis 1951;Stephenson et al nitrate (Lewis 1951;Stephenson et al

1992)1992)

0

20

40

60

80

100

120

140

0 2 4 6 8 10 12

Time after administration of urea or nitrate (hr)

Rum

en fl

uid

am

monia

conce

ntr

ati

on (

mM

)

Ammonia N (mM)-after 25gnitrate

Ammonia N (mM)-after 10gurea in a solid mix

Ammonia (mM)-after 10g ureain solution

Note: Nitrate was injected into a 60kg sheep, whereas urea was injected into a 35kg Merino

Up to 80% administered nitrate Up to 80% administered nitrate appears to disappear from the rumen appears to disappear from the rumen

within the first hour. Potential within the first hour. Potential explanations for this include:explanations for this include:

Nitrate and nitrite are rapidly absorbed from the rumenNitrate and nitrite are rapidly absorbed from the rumen

Nitrate is sequestered in microbesNitrate is sequestered in microbes

Nitrite and possibly nitrate are bound to protein in a similar Nitrite and possibly nitrate are bound to protein in a similar way as it is bound to haemoglobinway as it is bound to haemoglobin

Nitrite is reduced rapidly to ammonia which is assimilated Nitrite is reduced rapidly to ammonia which is assimilated to inter-cellular amino acids( alanine) which may be to inter-cellular amino acids( alanine) which may be released to the ammonia pool as electron availability is released to the ammonia pool as electron availability is more synchronized with N availabilitymore synchronized with N availability

A nitrate load increases outflow of rumen fluid to the lower A nitrate load increases outflow of rumen fluid to the lower tracttract

1515N in rumen fluid ammonia and nitrate plus N in rumen fluid ammonia and nitrate plus nitrite of a cow injected intra-ruminally with nitrite of a cow injected intra-ruminally with 120g K-nitrate labeled with 120g K-nitrate labeled with 1515N (Wang et al N (Wang et al

1961)1961)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 1 2 3 4 5 6 7 8 9


Ato

ms

% e

xce

ss

Rumen fluid nitrate plus nitrite Rumen fluid ammonia

Nitrate was administered daily for a number of days before the labeled nitrate was given

Mean blood nitrate concentration with time Mean blood nitrate concentration with time after introduction of cattle to diets after introduction of cattle to diets

containing nitrate (containing nitrate (Clarke et al 1970)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Blood nitrate cioncentration

(mM)

0 10 24 49 57 59

Time from commencement of nitrate feeding (days)

Urea with high nitrate Soybean with high nitrate

Soybean with low nitrate Urea with low nitrate

Conclusions from whole animal Conclusions from whole animal researchresearch

Toxicities from nitrite accumulation in and absorption from the rumen appeared to be Toxicities from nitrite accumulation in and absorption from the rumen appeared to be the major constraint to replacing urea with nitrate in low protein dietsthe major constraint to replacing urea with nitrate in low protein diets

Nitrite accumulation in the rumen only occurs where nitrate is introduced abruptly Nitrite accumulation in the rumen only occurs where nitrate is introduced abruptly and in excessive amounts.and in excessive amounts.

The rumen milieu can readily adapt to nitrate as a source of fermentable NThe 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 Sheep on high energy diets supporting high growth rates were unaffected by adding 3.4% nitrate to their diets.3.4% nitrate to their diets.

Dairy cows producing 16-19L milk/day were unaffected by inclusion of 2% nitrate in Dairy cows producing 16-19L milk/day were unaffected by inclusion of 2% nitrate in feed(Farra and Satter 1971)feed(Farra and Satter 1971)

Sulphur balance was effected by nitrate inclusion in sheep fed concentrate diets. 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 Additional Mo in a diet boosted nitrite production from nitrate. Mo suppresses sulphur reducing bacteria and production of hydrogen sulphide in the rumenreducing bacteria and production of hydrogen sulphide in the rumen

Conclusions from whole animal Conclusions from whole animal researchresearch

1.1. Acclimation to nitrate eliminates nitrite Acclimation to nitrate eliminates nitrite accumulation in the rumen removing any accumulation in the rumen removing any detrimental effectsdetrimental effects

2.2. Overall it appeared that nitrate ,at Overall it appeared that nitrate ,at high compared to low concentrations high compared to low concentrations was being metabolized by different was being metabolized by different processes and there appeared to be processes and there appeared to be some interaction with sulphur some interaction with sulphur availability.availability.

3.3. Understanding the pathways of nitrate Understanding the pathways of nitrate and sulphur metabolism offer possible and sulphur metabolism offer possible explanations for nitrite accumulationexplanations for nitrite accumulation. .

The utilization of nitrate in the The utilization of nitrate in the rumen appears to be different rumen appears to be different

to urea to urea Nitrate rapidly disappears from rumen fluid and unlike urea the major route Nitrate rapidly disappears from rumen fluid and unlike urea the major route

is not by conversion to ammonia is not by conversion to ammonia Nitrate or nitrite was entering the rumen 24 hours after nitrate had been Nitrate or nitrite was entering the rumen 24 hours after nitrate had been

administered. The source of the nitrite/nitrate maybeadministered. The source of the nitrite/nitrate maybe Nitrate cycling between body fluid pools and the rumenNitrate cycling between body fluid pools and the rumen Nitrate/nitrite released from sequestered nitrate/nitrite in the rumenNitrate/nitrite released from sequestered nitrate/nitrite in the rumen Nitrate/nitrite is in equilibrium with the another N pool in the rumen( highly Nitrate/nitrite is in equilibrium with the another N pool in the rumen( highly

unlikely)unlikely)

Nitrate absorbed into blood by humans( 2-4 mM/day) is concentrated up to Nitrate absorbed into blood by humans( 2-4 mM/day) is concentrated up to 10 fold in the saliva. Salivary nitrate is converted to HNO10 fold in the saliva. Salivary nitrate is converted to HNO22 by the acid by the acid stomach which spontaneously breaks down to nitrite. In the ruminant stomach which spontaneously breaks down to nitrite. In the ruminant nitrate would be returned via salivary secretions to the rumen.nitrate would be returned via salivary secretions to the rumen.

Nitrate entering the mouth in monogastric animals is reduced to nitrite by Nitrate entering the mouth in monogastric animals is reduced to nitrite by populations of organisms resident on the tongue. If the entero-salivary populations of organisms resident on the tongue. If the entero-salivary recycling occurred in ruminants it is potentially a significant sources of recycling occurred in ruminants it is potentially a significant sources of rumen nitrite when a nitrate load is given in the rumen.rumen nitrite when a nitrate load is given in the rumen.


assumed to be by assumed to be by dissimilatory nitrate dissimilatory nitrate reduction (DNR) to ammonia reduction (DNR) to ammonia


--+2H+2H+ + ---------H---------H22O+NOO+NO22-- --------------------1 --------------------1

NONO22--+6H+6H++ ---------H ---------H22O+ NHO+ NH33 -------------------2 -------------------2

Organisms capable DNR of nitrate to nitrite and assimilatory nitrite Organisms capable DNR of nitrate to nitrite and assimilatory nitrite reduction(ANR) to ammonia use formate and hydrogen as the reduction(ANR) to ammonia use formate and hydrogen as the common electron donors common electron donors


--+5H+5H++=3 CO=3 CO22+NH+NH++44+2H+2H22O ---------- 3O ---------- 3

3H3H22+NO+NO22--+2H+2H++ = NH = NH++

44 +2H +2H22O ------------------4O ------------------4

The newly discovered NR-SOB organisms use hydrogen sulphide The newly discovered NR-SOB organisms use hydrogen sulphide as an electron donor for respiratory nitrite ammonificationas an electron donor for respiratory nitrite ammonification


44 +2H +2H22O --------5O --------5

At least three types of nitrate At least three types of nitrate reductase catalyze the reduction of reductase catalyze the reduction of

nitrate to nitrite in anaerobic bacterianitrate to nitrite in anaerobic bacteria Included are: Included are:

A soluble, assimilatory nitrate reductase (NAS) A soluble, assimilatory nitrate reductase (NAS) that is present in the cytoplasm.that is present in the cytoplasm.

An energy-conserving nitrate reductase (NAR, An energy-conserving nitrate reductase (NAR, for example,NarG,which is encoded by the first for example,NarG,which is encoded by the first gene of the gene of the narGHJI narGHJI operon) with catalytic sites operon) with catalytic sites located in thelocated in the cytoplasm are associated with the cytoplasm are associated with the cytoplasmiccytoplasmic membrane, from which they receive membrane, from which they receive electronselectrons for nitrate reduction.for nitrate reduction.

A soluble, periplasmic nitrate reductases (NAP) A soluble, periplasmic nitrate reductases (NAP) that are found in many Gram-negative bacteria. that are found in many Gram-negative bacteria.

At least three types of nitrate At least three types of nitrate reductase catalyze the reduction of reductase catalyze the reduction of

nitrate to nitrite in anaerobic bacterianitrate to nitrite in anaerobic bacteria Included are: Included are:

A soluble, assimilatory nitrate A soluble, assimilatory nitrate reductase (NAS) that is reductase (NAS) that is present in the cytoplasm.present in the cytoplasm.

An energy-conserving nitrate An energy-conserving nitrate reductase (NAR, for example, reductase (NAR, for example, NarG,which is encoded by the NarG,which is encoded by the first gene of the first gene of the narGHJI narGHJI operon) with catalytic sites operon) with catalytic sites located in thelocated in the cytoplasm are cytoplasm are associated with the associated with the cytoplasmiccytoplasmic membrane, from membrane, from which they receive electronswhich they receive electrons for nitrate reduction.for nitrate reduction.

A soluble, periplasmic nitrate A soluble, periplasmic nitrate reductase (NAP) that are reductase (NAP) that are found in many Gram-negative found in many Gram-negative bacteria. bacteria.

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.

Production of nitric oxide Production of nitric oxide occurs in the rumen which may occurs in the rumen which may have inhibitory effects on some have inhibitory effects on some

ArchaeArchae Some bacteria that reduce nitrite directly to ammonia generate NO.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 NO is produced in the rumen in small amounts when nitrate is included in a diet/medium diet/medium

Although unproven it is a possibility that this NO is produced by one of the Although unproven it is a possibility that this NO is produced by one of the enzymes that reduce nitrite to ammonia (NrfA or NirBD).enzymes that reduce nitrite to ammonia (NrfA or NirBD).

Nitric oxide and other nitrogen oxides inhibit methanogenesis in some Nitric oxide and other nitrogen oxides inhibit methanogenesis in some ArchaeArchae

W succinogeneseW succinogenese ( a rumen microbe) produces nitric oxide when incubated ( a rumen microbe) produces nitric oxide when incubated with nitratewith nitrate

Nitrate as a component of the diet may have indirect effects on Nitrate as a component of the diet may have indirect effects on methanogenesis other then straight out competition for electrons with methanogenesis other then straight out competition for electrons with carbon dioxide, perhaps providing a competitive edge for NRB.carbon dioxide, perhaps providing a competitive edge for NRB.

Inhibitory effects of N-oxides on Inhibitory effects of N-oxides on Methanosarcina barkeri Methanosarcina barkeri and and Methanobacterium bryantii Methanobacterium bryantii (( aa Klüber and Conrad, 1998 ; Klüber and Conrad, 1998 ;bb

Clarens et al., 1998)Clarens et al., 1998)

N-oxide N-oxide MethanogenicMethanogenic

Species Species N-oxide N-oxide

Concentration Concentration Residual Methanogenic Residual Methanogenic

Activity (%) Activity (%)

Nitrate Nitrate Ms. barkeri Ms. barkeri Mb. bryantii Mb. bryantii

M. mazei M. mazei

30 mM 30 mM 30 mM 30 mM

14.3 mM 14.3 mM

2424aa

4141bb

5454aa

Nitrite Nitrite Ms. barkeri Ms. barkeri Mb. bryantii Mb. bryantii

0.1 mM 0.1 mM 1 mM 1 mM

Complete inhibitionComplete inhibitionaa

5050aa

Nitric oxide Nitric oxide Ms. barkeri Ms. barkeri Mb. bryantii Mb. bryantii

1.7 μM 1.7 μM 0.8-1.7 μM0.8-1.7 μM



Nitrous oxide Nitrous oxide Ms. barkeri Ms. barkeri Mb. bryantii Mb. bryantii

0.95 mM 0.95 mM > 95 μM > 95 μM

1010aa

Complete inhibitionComplete inhibition a a

Some important issues Some important issues concerning sulphur metabolism concerning sulphur metabolism

in the rumen in the rumen The major source of sulphur in grazing ruminants is from the S-The major source of sulphur in grazing ruminants is from the S-

amino acids in proteinamino acids in protein

Sulphur is required for organic S compounds that are synthesized Sulphur is required for organic S compounds that are synthesized in microbial cell growth. in microbial cell growth.

Excess sulphur in the rumen is reduced to hydrogen sulphide Excess sulphur in the rumen is reduced to hydrogen sulphide which has limited solubility and quickly enters the gas head space. which has limited solubility and quickly enters the gas head space. The head space gases are eructated and inhaled into the lungs The head space gases are eructated and inhaled into the lungs where a proportion is absorbed and converted to sulphate in the where a proportion is absorbed and converted to sulphate in the liver.liver.

Sulphide levels in rumen fluid are low often about 0.1 mMSulphide levels in rumen fluid are low often about 0.1 mM

Sulphide in the rumen is rapidly cleared and in cows fasting for 4 Sulphide in the rumen is rapidly cleared and in cows fasting for 4 hours following a period of grazing, hydrogen sulphide hours following a period of grazing, hydrogen sulphide concentrations in the gas space is zero.concentrations in the gas space is zero.

Sulphur reducing (SRB) and sulphide Sulphur reducing (SRB) and sulphide oxidizing bacteria (NR-SOB) exist in a oxidizing bacteria (NR-SOB) exist in a

number of anaerobic ecosystemsnumber of anaerobic ecosystems The SRB in the rumen are grouped by the mechanism used to reduce The SRB in the rumen are grouped by the mechanism used to reduce

sulphates:sulphates:

assimilatory process, assimilatory process, dissimilatory process. dissimilatory process.

In general, the dissimilatory reduction of sulphur compounds is used for the In general, the dissimilatory reduction of sulphur compounds is used for the generation of ATP, while the assimilatory process reduces sulphur compounds generation of ATP, while the assimilatory process reduces sulphur compounds for incorporation into other organic compounds necessary for cell survival. The for incorporation into other organic compounds necessary for cell survival. The dissimilatory pathways are responsible for the reduction of sulphur to hydrogen dissimilatory pathways are responsible for the reduction of sulphur to hydrogen sulphite and hydrogen sulphide (Desulfovibrio and Desulfotomaculum spp).sulphite and hydrogen sulphide (Desulfovibrio and Desulfotomaculum spp).

Most SRB are also NRB since they can actively reduce Most SRB are also NRB since they can actively reduce nitritenitrite but not but not nitrate nitrate to ammoniato ammonia

Nitrate reducing, sulphide oxidizing bacteria (NR-SOB) convert nitrate to Nitrate reducing, sulphide oxidizing bacteria (NR-SOB) convert nitrate to nitrite and utilize nitrite to oxidize hydrogen sulphide to poly S or sulphate nitrite and utilize nitrite to oxidize hydrogen sulphide to poly S or sulphate and reduce nitrite to ammonia with the generation of ATP and reduce nitrite to ammonia with the generation of ATP ((Wolinella succinogenese, Desulfovibrio spp)Wolinella succinogenese, Desulfovibrio spp)

Impact of nitrate on sulphur Impact of nitrate on sulphur cycle in the anaerobic cycle in the anaerobic

ecosystems found in oil wellsecosystems found in oil wells Sulphide produced by SRB activity can be recycled to sulphate or sulphur Sulphide produced by SRB activity can be recycled to sulphate or sulphur

by NR-SOB reducing nitrate to ammonia (DNRA) or in static anaerobic by NR-SOB reducing nitrate to ammonia (DNRA) or in static anaerobic ecosystems denitrification occurs via nitrogen oxides to nitrogen gas.ecosystems denitrification occurs via nitrogen oxides to nitrogen gas.

Most SRB can use nitrite as an alternative electron acceptorMost SRB can use nitrite as an alternative electron acceptor

Introduction of nitrate increases NRBIntroduction of nitrate increases NRB

hNRB compete with SRB for organic electron donors, such as lactate, hNRB compete with SRB for organic electron donors, such as lactate, excluding sulphide production and nitrite reduction(?) by SRB. The extent excluding sulphide production and nitrite reduction(?) by SRB. The extent of the effect depending on the fermentation rate (availability of electron of the effect depending on the fermentation rate (availability of electron donors)donors)

Many SRB and hNRB oxidize lactate incompletely to acetate and COMany SRB and hNRB oxidize lactate incompletely to acetate and CO22

Lowered sulphide availability prevents nitrite ammonification (respiration) Lowered sulphide availability prevents nitrite ammonification (respiration) by NR-SOB and stimulates nitrate reduction to nitrite which may by NR-SOB and stimulates nitrate reduction to nitrite which may accumulate accumulate

Sulfurospirillum sp switch from nitrite switch from nitrite production to ammonia production production to ammonia production

according to the relative availability of according to the relative availability of electron donors in the medium- is this a electron donors in the medium- is this a

model for the rumen?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


44 +2H +2H22O --------5O --------5


assumed to be by assumed to be by dissimilatory nitrate dissimilatory nitrate reduction (DNR) to ammoniareduction (DNR) to ammonia


--+2H+2H+ + ---------H---------H22O+NOO+NO22-- ----------------1 ----------------1

NONO22--+6H+6H++ ---------H ---------H22O+ NHO+ NH33 ---------------2 ---------------2

Organisms capable DNR of nitrate to nitrite and Organisms capable DNR of nitrate to nitrite and assimilatory nitrite reduction(ANR) to ammonia assimilatory nitrite reduction(ANR) to ammonia use formate and hydrogen as the common use formate and hydrogen as the common electron donors electron donors


--+5H+5H++=3 CO=3 CO22+NH+NH++44+2H+2H22O ------3O ------3

3H3H22+NO+NO22--+2H+2H++ = NH = NH++

44 +2H +2H22O -------------4O -------------4

The newly discovered NR-SOB organisms use The newly discovered NR-SOB organisms use hydrogen sulphide as an electron donor for hydrogen sulphide as an electron donor for respiratory nitrite ammonificationrespiratory nitrite ammonification

3HS + NO3HS + NO22-- +5H +5H++ = 3S = 3S00 + NH + NH++

44 +2H +2H22O ----5O ----5

Fermentable OM

CO2 + VFA

NO3-

SO42-

H2S

NO3-

NH3

NH4+

Fermentable OM

CO2 + VFA

H2S

SO42-

Competitive Exclusion of SRB by NRB

Sulphide removal by NR-SOB.When sulphide limiting NR-SOB reduce nitrate to nitrite

NRB

SRB

NR-SOB

ADP

ATP

Fermentable OM

Fermentable OM

CO2 + VFA

CO2 + VFA

SO42-

SO42-NO3

-

NO3-

NH3

NH3

H2S

H2S

NRB SRB

SRB NR-SOB

Heterotrophic nitrate reducing bacteria (NRB) out compete SRB

Nitrate reducing sulphide oxidizing bacteria (NR-SOB) lower H2S

Three processes may lower rumen fluid hydrogen Three processes may lower rumen fluid hydrogen sulphide sulphide

1)loss to the gas space, 2)competition between NRB and 1)loss to the gas space, 2)competition between NRB and SRB SRB

for electron donors and 3)oxidation to sulphate by NR-for electron donors and 3)oxidation to sulphate by NR-SOBSOB

Nitrate poisoning is a result of the Nitrate poisoning is a result of the establishment of a single group of establishment of a single group of

microorganisms the NR-SOB ?microorganisms the NR-SOB ? When fermentable organic matter is high NRB and When fermentable organic matter is high NRB and

SRB can co exist maintaining rumen fluid HSRB can co exist maintaining rumen fluid H22S S When electron donors are in short supply NRB out When electron donors are in short supply NRB out

compete SRB and Hcompete SRB and H22S in rumen fluid declinesS in rumen fluid declines When rumen degradable protein is high the When rumen degradable protein is high the

population density of NR –SOB depends on nitrate population density of NR –SOB depends on nitrate and Hand H22S in rumen fluid S in rumen fluid

When HWhen H22S and nitrate adequate NR-SOB convert S and nitrate adequate NR-SOB convert nitrate to ammonia but convert nitrate to nitrite in nitrate to ammonia but convert nitrate to nitrite in

the absence of Hthe absence of H22S S

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

The effects of adding The effects of adding nitrate into an nitrate into an artificial rumen artificial rumen inoculated with inoculated with

rumen fluidrumen fluid Nitrate was cleared rapidly in all Nitrate was cleared rapidly in all

incubations but nitrate N could incubations but nitrate N could not be accounted for in either not be accounted for in either ammonia or nitrite, particularly in ammonia or nitrite, particularly in first 5hr of the incubation.first 5hr of the incubation.

Nitrite accumulated when nil, Nitrite accumulated when nil, glucose of cellulose was included glucose of cellulose was included suggesting nitrite reduction to suggesting nitrite reduction to ammonia is inhibited.ammonia is inhibited.

Nitrite accumulation was Nitrite accumulation was negligible when dried grass was negligible when dried grass was included in the medium.included in the medium.

0

2

4

6

8

10

12

0 10 20 30 40

Time from addition of nitrate to artificial rumen (h)

Nitr

ate

N (

mg

N/1

00m

l)

0

1

2

3

4

5

6

0 10 20 30 40


Nitr

ite -

N (

mgN

/100

ml

0

10

20

30

40

50

60

70

80

0 10 20 30 40


Am

mon

ia -

N (

mgN

/100

ml)

Nil Cellulose Glucose Dried grass

Conclusion protein (sulphide) and electrondonors preserve nitrite assimilatory conversion to ammonia in NR-SOB (source Barnett and Bowman 1957)

The effects of protein intake on the The effects of protein intake on the accumulation of nitrite in rumen fluid after accumulation of nitrite in rumen fluid after administration of a nitrate load (30g Na-administration of a nitrate load (30g Na-nitrate) in sheep (Takahashi et al 1980)nitrate) in sheep (Takahashi et al 1980)

Feed Feed intake intake (g/day)(g/day)

CP CP

(%DM(%DM))

N N intake intake (g/day(g/day

))

Peak Peak nitrite nitrite in RF in RF (mM)(mM)

Peak Peak nitrate nitrate in RF in RF (mM)*(mM)*

843843 12.312.3 2121 0.70.7 2525

488488 30.730.7 20.420.4 3.33.3 2828

784784 6.96.9 11.811.8 0.90.9 2929

490490 15.415.4 11.811.8 1.41.4 2828

y = 0.11x - 0.25R2 = 0.92

00.5

11.5

22.5

33.5

0 10 20 30 40

% Crude protein in dietPeak n

itri

te (

mM

) fo

llow

ing a

nit

rate

load

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

* First measurement of nitrate concentration after nitrate load given

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

al 2004).

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 1 2 3 4 5 6 7 8 9


Meth

ane p

roduct

ion

(mg/kg0

.75/m

in)

Methane production-nitrate administered Methane production-control

Changes in rumen fluid nitrate and nitrite Changes in rumen fluid nitrate and nitrite with time after administration of Na-nitrate with time after administration of Na-nitrate

alone or a combination of Na-nitrate and alone or a combination of Na-nitrate and cysteine to sheep cysteine to sheep (Takahashi et al 1998).

0

10

20

30

40

50

60

70

0 2 4 6

Time after administration (hr)

Con

cent

ration

in r

umen

flui

d (m

M)

Rumen nitrate (mM) followingnitrate/ cysteine load

Rumen nitrite (mM) followingnitrate/ cysteine load

rumen nitrate (mM) followingnitrate load

rumen nitrite (mM) followingnitrate load

Effects of iso-S supplements on the concentration of hydrogenEffects of iso-S supplements on the concentration of hydrogen sulphide in the rumen head-space gas of dairy cows. The cows sulphide in the rumen head-space gas of dairy cows. The cows had been without feed for 4 hours prior to administration of S-had been without feed for 4 hours prior to administration of S-

source (Dewhurst et al 2007).source (Dewhurst et al 2007).

0

100

200

300

400

500

600

0 20 40 60 80 100 120 140 160

Time after administration into the rumen of a 4hr fasted cow

ppm

hydro

gen s

ulp

hid

e in r

um

en

gas

space

Control Cysteine Methionine Sodium sulphate

Effects of increasingEffects of increasing quantities of L-cysteine injected into the quantities of L-cysteine injected into the rumen on hydrogen sulphide concentration in rumen head rumen on hydrogen sulphide concentration in rumen head

space gas in dairy cows (Dewhurst 2007).space gas in dairy cows (Dewhurst 2007).

0

200

400

600

800

1000

1200

1400

0 20 40 60 80 100 120 140 160

Time after injection into the rumen of a 4hr fasted cow(min)

ppm

hydro

gen s

ulp

hid

e in g

as

in

rum

en g

as

space

Nil cysteine 4g cysteine 8g cysteine 12gcysteine

The relationship between hydrogen sulphide The relationship between hydrogen sulphide in rumen fluid and in the gas space in the in rumen fluid and in the gas space in the

rumen (After Gould rumen (After Gould et alet al 1997) 1997)

y = 0.025xR2 = 0.53

0

2

4

6

8

10

0 50 100 150 200 250 300

Sulphide conc. in rumen fluid (micro-mole/ L)

Conc.

of su

lphid

e in

rum

en

gas

spac

e (p

pm

x103

)

Nitrate poisoning Nitrate poisoning preconditions preconditions

The population densities of NRB,SRB and NR-The population densities of NRB,SRB and NR-SOB in the rumen depend on the degradable SOB in the rumen depend on the degradable protein content of the grazed forageprotein content of the grazed forage

Fertilizer application controls growth rate of Fertilizer application controls growth rate of pasture soluble sugars, protein and nitrate in pasture soluble sugars, protein and nitrate in forage,forage,

Lowering photosynthesis rate of the plant Lowering photosynthesis rate of the plant from environmental reasons may increase the from environmental reasons may increase the accumulation of nitrate up to 10 fold.accumulation of nitrate up to 10 fold.

N-fertilization of perennial rye grass N-fertilization of perennial rye grass (PRG) increases both crude protein (PRG) increases both crude protein

content of forage and there is a linear content of forage and there is a linear increase in nitrate content (Lovett increase in nitrate content (Lovett

2004)2004)y = 0.032x - 4.1

R2 = 0.94

00.5

11.5

22.5

33.5

4

100 150 200 250

Crude protein in PRG (g/ kg DM)

Nit

rate

conte

nt

of PR

G

(g/k

g D

M)

N-fertilization of perennial rye grass (PRG) N-fertilization of perennial rye grass (PRG) (Lovett 2004) or Kikuyu grass (Marais 2008) (Lovett 2004) or Kikuyu grass (Marais 2008)

increases crude protein and nitrate increases crude protein and nitrate

0

1

2

3

4

5

6

100 150 200 250 300 350

Crude protein in PRG (g/ kg DM)

Nit

rate

conte

nt

of PR

G

(g/k

g D

M)

Dewhurst et al 2004

Marais 2008

N-fertilization of alfalfa increases both crude N-fertilization of alfalfa increases both crude protein content of forage and there is a protein content of forage and there is a

linear increase in nitrate content (Cherney et linear increase in nitrate content (Cherney et al 1994)al 1994)

y = 0.077x - 15

R2 = 0.86

012345678

100 150 200 250 300

Crude protein in alfalfa (g/ kg DM )

Nit

rate

-N (

g/k

gD

M)

Nitrate poisoning--The sudden Nitrate poisoning--The sudden introduction of nitrate into the rumen introduction of nitrate into the rumen causes a series of flow on effects as causes a series of flow on effects as

followsfollows nNRB are provided with additional substrate and inhibit SRB activity for both nNRB are provided with additional substrate and inhibit SRB activity for both

sulphur and nitrite reduction by competition for organic electron donorssulphur and nitrite reduction by competition for organic electron donors

Reduced activity of SRB reduces concentrations of hydrogen sulphide in rumen Reduced activity of SRB reduces concentrations of hydrogen sulphide in rumen liquor and the rumen gas head spaceliquor and the rumen gas head space

As hydrogen sulphide levels in rumen fluid decrease the growth capacity of NR-As hydrogen sulphide levels in rumen fluid decrease the growth capacity of NR-SOB is lowered (nitrite ammonification is normally synchronised with hydrogen SOB is lowered (nitrite ammonification is normally synchronised with hydrogen sulphide oxidation), uncoupling growth and substrate metabolism and directing sulphide oxidation), uncoupling growth and substrate metabolism and directing electrons to nitrite formation from nitrate at an enhanced rateelectrons to nitrite formation from nitrate at an enhanced rate

SRB are competitively hampered in using nitrite by the availability of electron SRB are competitively hampered in using nitrite by the availability of electron donors which are usurped by NRB. This is only reversed when nitrate levels fall donors which are usurped by NRB. This is only reversed when nitrate levels fall below a critical concentration.below a critical concentration.

Nitrite accumulates in rumen fluid and is absorbed and binds to haemoglobin.Nitrite accumulates in rumen fluid and is absorbed and binds to haemoglobin.

As nitrate levels are lowered in rumen fluid and, dependent on the availability of As nitrate levels are lowered in rumen fluid and, dependent on the availability of electron donors (fermentation potential), the effects of electron starvation on electron donors (fermentation potential), the effects of electron starvation on SRB cease at a critical level and nitrite uptake by SRB and NR-SOB returns to SRB cease at a critical level and nitrite uptake by SRB and NR-SOB returns to normal. normal.

Effects of a meal(9.5 kgEffects of a meal(9.5 kg wet weight) of white clover or wet weight) of white clover or perennial rye grass on the concentration of hydrogen sulphide perennial rye grass on the concentration of hydrogen sulphide in the rumen head-space of dairy cows (Dewhurst et al 2007).in the rumen head-space of dairy cows (Dewhurst et al 2007).

0

100

200

300

400

500

600

700

800

0 50 100 150 200

Time after consumption of forage in a cow fasted for 4hours (min)

ppm hydrogen sulphide in gas

space of the rumen

white clover Ryegrass

Clover had almost double the protein and therefore cysteine content compared to rye grass

Effect of high sulfur and various compounds on in Effect of high sulfur and various compounds on in vitro ruminal hydrogen sulfide production. Low vitro ruminal hydrogen sulfide production. Low

sulfur = 0.29% S; High sulfur = 1.09% S. AQ = 10 sulfur = 0.29% S; High sulfur = 1.09% S. AQ = 10 ppm (fluid basis) of 9,10 anthraquinone; Molybdate ppm (fluid basis) of 9,10 anthraquinone; Molybdate = 25 ppm of molybdate; Monensin = 5 ppm. (Data = 25 ppm of molybdate; Monensin = 5 ppm. (Data

from Bracht and Kung, 1997.)from Bracht and Kung, 1997.)

Does monensin inhibit NR-SOB and if it does what are the effects on nitrite production in the rumen during a nitrate load.

Major conclusions 1Major conclusions 1

Urea can be replaced by nitrate as a source of Urea can be replaced by nitrate as a source of fermentable nitrogen in diets where fermentable nitrogen in diets where the the nitrogen source is mixed in the feednitrogen source is mixed in the feed with the with the expectations of reduced enteric methane expectations of reduced enteric methane production.production. Diets include grain based feedlot dietsDiets include grain based feedlot diets Hand feeding in drought with grain, hay and straw/other Hand feeding in drought with grain, hay and straw/other

crop residuescrop residues Feedlot based feeding of alkali treated crop residues (35 Feedlot based feeding of alkali treated crop residues (35

million tonnes of straw)million tonnes of straw) Sugar cane based dietsSugar cane based diets Molasses, cassava, sugar beet based dietsMolasses, cassava, sugar beet based diets


Nitrate as a supplement has wide scale Nitrate as a supplement has wide scale application in developing countries where application in developing countries where ruminant production is largely based on using ruminant production is largely based on using crop residuescrop residues

Urea maybe replaced by nitrate in dry season Urea maybe replaced by nitrate in dry season supplements fed through out Australia (requires supplements fed through out Australia (requires research to develop delivery systems)research to develop delivery systems)

For each 1% inclusion of K-nitrate in a diet For each 1% inclusion of K-nitrate in a diet methane production will be reduced by 10%methane production will be reduced by 10%

As an example in grain based feed lot rations 1% As an example in grain based feed lot rations 1% urea is usually required to optimise rumen urea is usually required to optimise rumen efficiency. This would be replaced with 3.3% K-efficiency. This would be replaced with 3.3% K-nitrate reducing methane production by 33%? nitrate reducing methane production by 33%?


Using nitrate to decrease enteric methane production Using nitrate to decrease enteric methane production when consumed by grazing ruminants when consumed by grazing ruminants on high protein on high protein dietsdiets has major barriers has major barriers Accessing animalsAccessing animals Incidence of nitrate poisoningIncidence of nitrate poisoning Increased NOIncreased NOxx from excreta (surplus N in diet). from excreta (surplus N in diet).

A paradigm shift in research and pasture/animal A paradigm shift in research and pasture/animal management would be needed to:management would be needed to: Produce lower protein highly digestible forages( similar to Produce lower protein highly digestible forages( similar to

sugar cane)sugar cane) Produce pasture/forage with a higher percentage of the protein Produce pasture/forage with a higher percentage of the protein

as bypass protein (tanniniferous legumes- e g sulla)as bypass protein (tanniniferous legumes- e g sulla) Develop energy efficient feed processing of forages to optimize Develop energy efficient feed processing of forages to optimize

digestibility of crop residues and mature forages (this already digestibility of crop residues and mature forages (this already underway ) underway )

Develop strategies for providing nitrate under grazing.Develop strategies for providing nitrate under grazing.

Nitrite is not normally an intermediate Nitrite is not normally an intermediate released into the medium( rumen fluid) released into the medium( rumen fluid)

by NRB -1by NRB -1 Rumen NRB convert nitrate to ammonia without Rumen NRB convert nitrate to ammonia without

production of nitrite and mainly by assimilatory production of nitrite and mainly by assimilatory pathways.pathways.

A small specialized population of NR-SOB are A small specialized population of NR-SOB are universally present in the rumen and population universally present in the rumen and population density is controlled by the extent and pattern of density is controlled by the extent and pattern of availability of nitrate and sulphide. availability of nitrate and sulphide.

NR-SOB are versatile organisms with a wide spectrum NR-SOB are versatile organisms with a wide spectrum of sources of nutrients but when nitrate is available of sources of nutrients but when nitrate is available and hydrogen sulphide concentration is adequate they and hydrogen sulphide concentration is adequate they reduce nitrate to ammonia without the intracellular reduce nitrate to ammonia without the intracellular intermediate, nitrite, being released to the medium.intermediate, nitrite, being released to the medium.

Nitrite is not normally an intermediate Nitrite is not normally an intermediate released into the medium( rumen fluid) released into the medium( rumen fluid)

by NRB -2by NRB -2 The rapid reduction of nitrate by both NRB and NR-SOB effectively The rapid reduction of nitrate by both NRB and NR-SOB effectively

compete with SRB for the electrons generated in fermentation, compete with SRB for the electrons generated in fermentation, lowering sulphur reduction to sulphide.lowering sulphur reduction to sulphide.

Decreased sulphur reduction and increased sulphide oxidation lower Decreased sulphur reduction and increased sulphide oxidation lower sulphide concentrations in the rumen which then inhibits sulphide concentrations in the rumen which then inhibits ammonification of nitrite in NR-SOB and nitrite accumulates in the ammonification of nitrite in NR-SOB and nitrite accumulates in the medium.medium.

This then suggests that nitrite is not normally produced by one group This then suggests that nitrite is not normally produced by one group ofof organisms to be used by a second group in the rumen. organisms to be used by a second group in the rumen.

This then suggests that in the rumen nitrite is only produced in small This then suggests that in the rumen nitrite is only produced in small quantities and only by NR-SOB when sulphide availability is curtailed.quantities and only by NR-SOB when sulphide availability is curtailed.

NR-SOB population densities are controlled by sulphide NR-SOB population densities are controlled by sulphide concentrations (and indirectly RDP)concentrations (and indirectly RDP)

Is it worthwhile investing in Is it worthwhile investing in major research programs 1?major research programs 1?

I believe that the answer is a whole hearted YES I believe that the answer is a whole hearted YES for the following reasonsfor the following reasons Considerable research to attain this goal to date by Considerable research to attain this goal to date by

other processes has been unrewarding.other processes has been unrewarding. Application of nitrate to replace urea in low protein Application of nitrate to replace urea in low protein

pasture/ forage and high energy feeds (grain based or pasture/ forage and high energy feeds (grain based or sugar/starch based) has been proven albeit in only a sugar/starch based) has been proven albeit in only a limited number of studies.limited number of studies.

The majority of the world populations of domestic The majority of the world populations of domestic ruminants are maintained on crop residues for all or a ruminants are maintained on crop residues for all or a substantial part of their lives and are hand fed and substantial part of their lives and are hand fed and because they produce at rates 10-20% of their genetic because they produce at rates 10-20% of their genetic capacity contribute disproportionately to total enteric capacity contribute disproportionately to total enteric methane production particularly when methane is methane production particularly when methane is calculated on the basis of liters per unit of production.calculated on the basis of liters per unit of production.

Is it worthwhile investing in Is it worthwhile investing in major research programs 2?major research programs 2?

I believe that the answer is a whole hearted YES for the I believe that the answer is a whole hearted YES for the following reasonsfollowing reasons

The depletion of critical resources and the high energy costs The depletion of critical resources and the high energy costs associated with future agriculture suggests that ruminant animals associated with future agriculture suggests that ruminant animals will become increasingly the preferred meat, milk, fiber and will become increasingly the preferred meat, milk, fiber and maybe energy providers in the future increasing world numbers maybe energy providers in the future increasing world numbers substantiallysubstantially

Already industries are gearing up to apply high technology to Already industries are gearing up to apply high technology to improve the utilization of crop residues which appears to have improve the utilization of crop residues which appears to have been highly successful as in some cases straw fed ruminants with been highly successful as in some cases straw fed ruminants with good supplement management are achieving levels of production good supplement management are achieving levels of production approaching the animals genetic potential. approaching the animals genetic potential.

Successful utilization of nitrate, with high levels of production on Successful utilization of nitrate, with high levels of production on low protein crop residues such as treated straw would provide the low protein crop residues such as treated straw would provide the stimulus for paradigm shifts in pasture management and forage stimulus for paradigm shifts in pasture management and forage breeding that would optimize biomass production but with lower breeding that would optimize biomass production but with lower protein content in the biomass. protein content in the biomass.

The Optimistic View Point The Optimistic View Point The ENDThe END

The ruminant population of the world is estimated to The ruminant population of the world is estimated to produce 80 million MT enteric methaneproduce 80 million MT enteric methane

Most of this is produced by animals on low quality foragesMost of this is produced by animals on low quality forages There appears to be no problems feeding nitrate to animals There appears to be no problems feeding nitrate to animals

so long as the following principles are applied so long as the following principles are applied N in the feed is kept within stoichiometric requirements for efficient N in the feed is kept within stoichiometric requirements for efficient

fermentative digestionfermentative digestion Animals are adjusted to nitrate slowly.Animals are adjusted to nitrate slowly. Previous diet and the production diet are low in readily Previous diet and the production diet are low in readily

fermentable( or rumen degradable) protein.fermentable( or rumen degradable) protein. Potentially 50% enteric methane production could be Potentially 50% enteric methane production could be

prevented by wide scale application of nitrate and a change prevented by wide scale application of nitrate and a change in production management to low protein diets in production management to low protein diets

Documents

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