Preamble
Following the research for and writing in 2008, of, a report for the Climate Change
Department of the Australian Government on the possibility to feed nitrate to livestock to
mitigate enteric methane production, considerable research has been undertaken in
various laboratories. Progress has been made and it now seems feasible that enteric
methane release from ruminants can be considerably lowered by dietary nitrate/sulphate
which could be used in many livestock feeding strategies with out risk. This appears to be
very difficult for scientists involved in the ruminant industry to accept who appear to be
placing their emphasis on feed additives that inhibit methanogens. It is my contention that
inhibition of methanogenesis by specific compounds or groups of compounds is bound to
fail as the microbial fermentative organisms have an enormous capacity to adapt to any
potential substrate. How would the ruminant have evolved if this were not the case?
Provision of alternative hydrogen sinks in the rumen is possibly the only way that methane
production can be persistently lowered. Nitrate and sulphur are high affinity electron
acceptors and both these elements can also be nutritionally beneficial. The major
limitation to commercial application now is the toxicities that are associated with
oversupply of each compound.
Anaerobic nitrate and sulphate metabolism in bacteria and Archae are interactive and
there is a great need to understand these interactions in order to be able to guarantee
lowered methane release and at the same time maintain efficient production by livestock.
Nitrate and sulphate metabolism have a long history of research in diverse anoxic and
anaerobic ecosystems and the interactions that occur in these systems maybe applicable
to the rumen.
The need to overcome any detrimental effects of nitrate on animal performance before
application to commercial animals is an urgent priority.
From 3-4years of exploring the literature a constant message was received in that it is the
interaction of sulphate and nitrate that is the critical issue in the efficient use of dietary
nitrate as a fermentable N source
Without apologies I present these ideas (intuitions) below in the hope that it will stimulate
research aimed at controlling the major objection to nitrate in the role of reducing
methane from digestion in ruminant livestock.
I believe that research scientists have to have a complete as possible knowledge of all
aspects of the background information and that it is only by intuition and knowledge
combined with good scientific approaches that progress will be made. Hypotheses aim to
advance knowledge and certainly no proof can be available until a hypothesis has been
thought out and tested but it is often partially based on intuition. This is the case in
developing the hypothesis put forward here
1
Reducing Enteric Methane Production in Ruminants with Dietary Nitrate; anaerobic nitrate
metabolism in bacteria with special reference to the roles of sulphur and molybdenum.
By
R A Leng
Abstract
Nitrate salts are high affinity electron acceptors that nitrate reducing bacteria (NRB) use
and which allows them to effectively compete with both methanogens and sulphur
reducing bacteria (SRB) in reduction of hydrogen in anaerobic microbial fermentation.
They can potentially limit enteric methane production in herbivorous animals when
included in a diet.
Methane production from herbivorous animals contributes highly significantly to global
warming from the accumulation of atmospheric gases. Animals fed poor quality forages,
particularly in developing countries, produce a large proportion of this methane and they
are fed diets that require supplementation with a fermentable N source. This is often
provided by urea which is converted to the major N source (ammonia) for microbial
growth in the rumen, and therefore production of nutrients for the animal. Nitrate is also
converted to ammonia in the rumen and therefore could potentially play the same role as
urea in these feeding systems. However, accompanying the reduction of nitrate to
ammonia in the rumen is the release of a variable amount of nitrite which is absorbed and
at low concentrations binds haemoglobin to form methaemoglobin restricting the oxygen
carrying capacity of the blood. The result can be severe, from death of the animal to only
slight or no effects depending on the level of nitrite absorbed and other dietary
ingredients.
Recent research has demonstrated that nitrate supplementation of ruminant diets has
been successful and has lowered methane production substantially (up to 40%). Slow
adaptation to nitrate in a feed and higher levels of sulphur in the diet appear to reduce any
toxic effects of nitrate in the feed. However, wide scale acceptance of nitrate
supplementation of ruminant diets is unlikely to occur without the guarantee of low or no
incidences of nitrate poisoning. To achieve this, there is a necessity to determine how and
when nitrite production from dietary nitrate may occur in animals and to avoid or modify
these feeding strategies in order to minimize risk of nitrite production.
For this purpose a review of the relevant literature has been undertaken. An hypothesis is
arrived at that nitrate toxicity is a result of an interaction in the levels of certain minerals in
the diet that effects the microbial populations and the metabolism of sulphur and nitrate in
particular. Nitrite from dietary nitrate is hypothesized to be produced by a minor group of
microorganisms that reduce nitrate and oxidize sulphide (NR-SOB). The amount of
sulphide in the rumen may determine their population density. The hydrogen sulphide
pool in rumen fluid is small and reduced when nitrate is included in a diet, NRB out
compete SRB for electrons and the NR-SOB only partially reduce nitrate and spill nitrite.
The interactions between these three groups of organisms depend on the individual
colonies being favorable positioned in the rumen biofilm associated with particulate
matter. This is well demonstrated in anaerobic biofilm that develop in waste water
treatment systems where the proposed interaction are proven to occur albeit that the
systems have much longer turnover times and are operated at much lower temperatures.
2
The most recent research where nitrate has been fed to ruminants under a number of
different feeding conditions and with a variety of ruminant species indicates that as nitrate
increases in a diet the apparent efficiency of nitrate reduced to ammonia in the rumen
decreases from close to 100% to 59%. This is discussed in relation to the changes that
dietary nitrate could induce in the rumen microbial ecology and the ability of NRB to lower
the partial pressure of hydrogen within the sites of hydrogen production and uptake in the
rumen digesta. Potentially, reduction of nitrate could lower the partial pressure of
hydrogen in the microbial biofilm environment to levels where bacteria oxidizing both
propionate and butyrate (Syntrophomonas species) with the production of hydrogen
could make a significant contribution to total hydrogen production. This may explain the
apparent inefficiency of nitrate in lowering methane production. Replacing urea with
nitrate as a fermentable N source generally decreases the percentages of butyrate and
propionate in rumen fluid and isotope dilution studies have shown that there is direct
synthesis of rumen acetate from both rumen butyrate and propionate in ruminants on
diets based on concentrates or roughages.
The conundrum of the occurrence of nitrate toxicities in ruminants reported in the past
where nitrate is ingested by ruminants is addressed.
High levels of molybdenum in a diet inhibits sulphur reducing organisms with effects on
the availability of hydrogen sulphide and both elevated molybdenum and nitrate may
cause nitrite to accumulate in the rumen.
Molybdenum in pasture dry matter is increased by liming and also soil amendment and
coating of seed with molybdenised fertilizers. These all effect the concentrations of
molybdenum in a feed which can be excessive. An excessive level of molybdenum in
forage reducing sulphide production by SRB causing the NR-SOB inducing nitrite spilling
may explain some of the disastrous experiments in which animals were killed by nitrate
inclusion in a diet.
Introduction
Methane is a greenhouse gas which has a radiative forcing effect of 21 to 72 times
greater then carbon dioxide. The higher level reflecting calculations where the time scale
is rated over 20 rather then a 100years.Methane has a significant role in global warming.
Ruminant animals are a major source of methane by virtue of the fermentative digestion
of their feed. Lowering methane gas in the atmosphere has become a priority area for
research as the world’s ruminant population produces 80 million tonnes of methane
annually. A high percentage of this is produced by ruminants in non industrialized
countries (Steinfeld et al 2006) where animal production is largely managed by small
farmers with limited resources. These farmers usually feed their animals on agro
industrial byproducts, non conventional feeds and native pastures which are of poor
nutritional value. They are often low in true protein and deficient in macro and trace
minerals for the microbes that digest the feed in the rumen and provide an inadequate
balance of minerals in addition to a poor protein to energy in the nutrients absorbed for
even moderate levels of production (see Leng 1991).
Increasing the efficiency of feed utilization and productivity of livestock are the first steps
in mitigation of enteric methane (Leng 1991) particularly where the bulk feed is poor
quality roughage. This is usually accomplished by feeding non protein nitrogen (urea) and
deficient minerals to improve the intake and digestion of feed (Leng 1981). Urea is directly
3
converted to ammonia and synthesized into microbial cells in the rumen improving the
protein nutrition of the animal, increasing productivity and thus lowering methane
production per unit of product produced ( Leng 1991). The anaerobic bacterial reduction
of nitrate also yields ammonia and as it is a high affinity electron acceptor would
significantly reduce methane production if fed to ruminants adapted to it in their diet (Leng
2008a).
In developed countries in low or seasonal rainfall areas and where livestock production
from grazing is a major industry such as the Savannahs of northern Australia and Africa,,
ruminants also depend on native pastures that, depending on seasonal conditions, are
often deficient in crude protein and phosphorus for optimal utilization. In many countries
young cattle are fattened from weaning on grain based diets and for purposes of
balancing the crude protein requirements, a source of fermentable nitrogen is required.
The requirements for fermentable N are mostly met in such diets by supplementation with
urea. Most grain based feedlot diets contain from 1-3% urea and replacing this with
nitrate could have a highly significant effect on methane emissions from animal
agriculture.
For ruminants at pasture or forage based there is a need for either continuous inclusion of
urea in a diet or a seasonal requirement when the pasture matures and senesces or dry
or cold weather causes growth to decline . Thus urea is used through out the livestock
industries in all countries. If nitrate could replace urea in feeds for ruminants a highly
significant reduction in methane release to the atmosphere could be achieved (Leng
2008a). In addition many more feeds low in protein could be promoted for production of
livestock, from non conventional resources; an example being sugar cane and its
byproducts. Already there are clear indications that nitrate can be an effective source of
fermentable N for ruminants on 1) poor quality diets (Trinh Phuc Hao et al 2009; Sangkhom
Inthapanya 2012 2) medium quality forage ( Nolan et al 2010) 3) sugar cane /maize silage(
Hulshof et al 2010) and in sheep and dairy cows on concentrate based diets supporting high
productivity (van Zijderveld et al 2010a,b; van Zijderveld et al 2012). However, there is
resistance to wide scale application of nitrate in ruminant feeds because of the history of
toxicities associated with nitrate accumulation in some forages. Before nitrate can be
used with confidence, the conditions under which nitrate becomes toxic need to be
understood and methodologies developed to circumvent nitrite poisoning. Just as there
has been considerable distrust of feeding urea ( see Coombe and Tribe 1958 ), the same
applies to feeding nitrate. In the following discussion the conditions under which nitrite
poisoning may occur are explored. However, a body of research has now emerged from
SE Asia showing that nitrate can replace urea in the low protein feeds used and without
any signs of ill health in animals (see Leng and Preston 2010). From the out set it appears
that nitrate poisoning has been a condition encountered in live stock fed only high protein
forages or consuming water with excessive nitrate content (Crawford et al 1966). Recent
research where controlled feeding of nitrate to sheep and dairy cows on the higher quality
concentrate feeds have recorded significant decreases (from 30-50%) in methane
production that were persistent over time ( van Zijderveld et al 2010a,b; Nolan et al
2010;Hulshof 2010; van Zijderveld et al 2012)
A considerable amount of research has explored a number of feed additives that
suppress methanogenesis but in most cases the effects are transient and the rumen
organisms adapt (Martin et al., 2010). However with nitrate supplementation the effects
are persistent(van Zijderveld et al 2012) and assuming that ammonia is the end product
of nitrate metabolism and 1 mole of nitrate reduced to ammonia would suppress methane
production by 1 mole per day the efficiency of suppression by nitrate of methane
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production was of the order of 89% (Zijderveld et al 2010a) but this calculation does not
take into account the generation of electrons in propionate conversion to acetate by
sulphur reducing organisms. Assuming an increase in background hydrogen release from
increased acetate production at the expense of propionate, the theoretical calculated
reduction of nitrate to ammonia is close to 100% (see Zjderveld et al 2010a)
The numbers of animals that could be fed low protein forages supplemented with nitrate is
large and could lower methane production per unit of product through the combined
effects of improved feed digestibility, feed conversion efficiency and direct competition
between nitrate reducing bacteria and methanogens for the hydrogen available from
fermentative digestion (Leng 2008a). However, the potential negative aspects of feeding
nitrate are well recognized and the mind set of scientists at present appear to be against
such applications. A similar mind set persisted for some considerable time against urea
supplementation of livestock. However it is now recognized that a slow adaptation to a
non protein nitrogen source removes many problems and slow introduction of nitrate into
a diet has been effective without only small increases in blood methaemoglobin levels(
).
A brief review of nitrate poisoning is presented, including some interaction between nitrate
metabolism in the rumen and the effects of sulphur and or molybdenum content of a diet
on the safety issues of nitrate as a high affinity electron acceptor that reduces per capita
methane production from ruminant animals
Nitrate poisoning
Nitrate toxicity occurs in ruminants mainly where they are grazing fast growing highly
fertilized pastures of high crude protein content and therefore of high nutritional value. It
is usually associated with a sudden increase in nitrate intake from lush green pasture. It is
also associated with green “high quality” pastures that have a temporary impediment to
photosynthesis as might be induced by a lack of soil moisture. It is also a potential
problem in plants that accumulate nitrates and which animals are forced to rely on for
feed when other pasture components have been reduced by selective grazing (see Wright
and Davison 1964) as sometimes occurs in drought. Where nitrate poisoning has been a
problem in cattle grazing temperate pastures, the crude protein content of the feed is
generally or often high between 18 and 38% CP a proportion of which is nitrate.
Nitrate poisoning is a misnomer as the toxic principle is nitrite produced from nitrate in the
rumen under certain conditions. When the absorption rate of nitrite exceeds the capacity
of the red blood cells to bind and oxidize nitrite to nitrate, the per cent methaemoglobin in
red blood cells increases. This in turn reduces the oxygen carrying capacity of the red
blood cells and eventually results in hypoxia of the essential organs and death of the
animal.
The literature on the toxicity of nitrate containing feeds for ruminants is large but
conflicting. Some researchers have reported deaths and also abortions occurred during,
and sometimes following, periods where nitrate was consumed by breeding livestock.
Others report no effects. A comprehensive discussion of the effects of nitrate in feeds
consumed by lactating and breeding stock is presented by Crawford et al (1966). The
adverse effects of nitrate in a diet are associated with sudden increase in nitrate in the
pasture (see later) and the accumulation of nitrite in the rumen.
5
Recent research on nitrate metabolism in a variety of anaerobic ecosystems, for instance,
marine and fresh water anaerobic sediments, rice fields, sewage systems, biodigestors
and oil field brine have revealed some important and detailed aspects of bacterial nitrate
metabolism. In particular the diverse research has demonstrated that there are major
interactions of nitrate and sulphur metabolism in microbial consortia in these ecosystems.
These interactions between sulphur and nitrate metabolism and how they pertain to the
rumen of livestock were raised in a recent report to The Australian Climate Change
Department (Leng 2008a). Since the preparation of that report further published research
has been identified that may help to explain aspects of nitrate poisoning. Reduction in the
risk of nitrate poisoning in livestock is critical before wide scale application of nitrate
feeding will be accepted.
Diets containing nitrate and sulphur have recently been reported from three different
sources to reduce methane production by cattle, goats and sheep by as much as 50%,
37% and 50% respectively (see below). However at the same time information is coming
out of some disastrous effects of feeding nitrate. In this presentation an attempt is made
to rationalize the information and provide an explanation of the contrasting results of
feeding trials. It is hypothesized that sulphur availability and metabolism in the rumen is a
critical issue in direct nitrate ammonification (intra cellular conversion of nitrate through
nitrite to ammonia without release of (termed spilling here) nitrite into the medium.
Nitrate and nitrite metabolism in anaerobic ecosystems
The predominant pathway of nitrate metabolism in the rumen is uncertain but has always
been assumed to be intra-cellular dissimilatory nitrate reduction to ammonia, the overall
two step reduction of nitrate shown below.
NO3- + 2H+  H2O+ NO-2 ------------------------------------------------------------------------Equation 1
NO2- + 6H+  H2O+ NH3 ------------------------------------------------------------------------Equation 2
Organisms capable of nitrite ammonification usually have the ability to reduce nitrate to
nitrite in dissimilatory metabolism (Simon 2002), nitrite being a suitable electron acceptor
for anaerobic respiration. Formate and hydrogen are the common electron donors in
assimilatory nitrite ammonification. Eight electrons are required for the reduction of nitrate
to ammonia.
These substrates are oxidized according to equations 3 and 4 below: One of the special
effects of bacterial anaerobic nitrate metabolism is that a large number of organisms have
the ability or adapt to nitrate as a N and energy source including many of the sulphur
reducing bacteria (Moura et al 2007)Desulfovibrio species are however an exception as
they use nitrite but not nitrate (Mitchell et al 1986). Recently in anoxic ecosystems with
high sulphur content, sulphide has been shown to function as an electron donor for
respiratory nitrite ammonification in nitrate reducing sulphide oxidizing bacteria (NR-SOB)
that oxidize hydrogen sulphide directly (Hubert and Voordouw 2007) (equation 5).
3HCO-2 +NO-2+5H+  3 CO2+NH+4 +2H2O -------------------------------------Equation 3
3H2 + NO-2 +2 H+  NH+4 +2H2O --------------------------------------------------------Equation 4
6
3HS- + NO2- +5H+  3S0 + NH+4 +2H2O -----------------------------------------Equation 5
The reaction in equation 5 indicates that nitrate or nitrite reduction to ammonia could be
stimulated by the availability of sulphide in the medium. As most organisms that reduce
sulphate are also capable of reducing nitrate and/or nitrite, the ratio of nitrate to sulphate
is likely to have an important interaction. .
Sulphate and nitrate taken up by a wide range of organisms in the rumen that have the
capacity to use either or both materials as high affinity electron acceptors (see Leng
2008a) and the substrate of choice in some SRB was actually nitrate when this enters the
medium where-in the SRB were growing( Moura et al 2007).
Anaerobic sulphur metabolism.
Dissimilatory and assimilatory sulfate reduction is the reduction of sulfate to sulfide by
SRB from which they obtain energy (ATP) for growth and maintenance.( see Figure1
below) Eight electrons are needed for the reduction of one sulfate to one sulfide The SRB
are a diverse group of prokaryotes (Castro et al. 2000), seven phylogenetic lineages
have been identified, five within the bacteria and two within the Archaea (Muyzer and
Stams 2008). The different SRB are able to utilize a wide range of organic electron
donors, including ethanol, formate, lactate, propionate, pyruvate, fatty acids, carbon
monoxide, methanol, methanethiol and sugars (Widdel et al. 2007; Muyzer and Stams
2008). SRB have a higher affinity for hydrogen than methanogens but less then that of
NRB that reduce nitrate to ammonia, and therefore both NRB and SRB inhibit
methanogens at low hydrogen partial pressures in the incubation medium. It is important
to note, however, that sulphur reduction by SRB is only lowered when electron acceptors
with a higher redox potential (e.g. nitrate) are absent..
Sulphur metabolism in the rumen
Sulphur is reduced to hydrogen sulphide in all anaerobic systems by a diversity of SRB. In
the rumen, SRB from both the assimilatory and dissimilatory groups exist; the latter are
responsible for the reduction of sulphur to hydrogen sulphide. Although many bacteria
can produce sulphide, in the rumen, organisms from the Desulfovibrio and
Desulfotomaculum genus are most likely to be dominant (Cumming et al 1995). The
overall reactions are shown in equation 6. The two pathways are given in more detail in
Figure 1. The physiology of SO4- reduction takes place1) in cytoplasm, where carbon
metabolism takes place and 2) at the cell membrane where the energetic reactions occur
SO4- is a stable ion and must be activated before use. Activation takes place by the
enzyme ATP sulphurylase and uses ATP to create adenosine phosphosulphate (APS).
4H2 +SO-4 +H+  4H2O+H2S
Gibbs free energy =-152 kJ mol-1 …………………………… 6
Little is known of the interactions of the SRB, NRB and possibly NR-SOB that develop in
the rumen when nitrate and/or sulphur are elevated in a diet. Just as in other ecosystems
where nitrate is introduced, the populations of organisms that develop will be quite
different to those normally found in the rumen particularly as NRB tend to out-compete
SRB for electron sources and NR-SOB have the capacity to use reduced sulphur and
some SRB will switch to nitrate reduction as the major source of substrate( Moura et al
2007)
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Figure 1. SRB produce two active sulphates , adenosine 5 phosphosulphate (APS) and
phosphoadenosine 5 phosphate ( PAPS) that participate in the dissimilatory or
assimilatory pathways of sulphate reduction ( see Muyzer and Stams 2008)
In the rumen, the extent of dissimilatory sulphate reduction is limited by the amounts of
sulphur containing compounds ingested. The overall sulphur reduction pathway is shown
in equation 6. The predominant sulphide compounds formed in the dissimilatory process
are S2-, So, HS-, or HSO3- (Odom and Singleton, 1993). The pKa for H2S is 7.2 and
therefore the reduced forms of sulphide are readily protonated resulting in most of the
sulphide produced in the rumen entering the rumen gas phase (or the gas cap) as
hydrogen sulphide (H2S). Only small amounts remain in the liquid phase in a variety of
sulphur containing compounds.
Hungate (1965) suggested that, if the rumen gas cap is in equilibrium with rumen fluid
hydrogen sulphide, its concentration would be about 0.1mM which is close to the critical
level for microbial growth of between 1.6 and 3.8 mg sulphide sulphur /liter(Kandylis and
Bray 1987). However the concentration of sulphide in the rumen biofilm can be expected
to be much greater then that in the bulk fluid( see later). The gases in the rumen cap are
largely excreted by eructation, which is followed by inhalation of the gases into the lungs
where a major proportion of the hydrogen sulphide is absorbed, transported to the liver
and converted into sulphate and excreted (Dougherty et al 1965;Kandylis and Bray
1982).The net result is that the pool of hydrogen sulphide in ruminal fluid is small and has
been shown to turnover rapidly. Kandylis and Bray (1987) suggested that the turnover
time could be as short as 10 min and Dewhurst et al (2007) showed that 4 hours after a
meal, the hydrogen sulphide content of the rumen gas phase in dairy cows was below
8
detectable amounts, supporting evidence for the rapid turnover of hydrogen sulphide in
rumen fluid and excretion from the rumen.
Many other factors affect the size of the hydrogen sulphide pool in rumen fluid. Dewhurst
et al (2007) speculated that the much lower hydrogen sulphide levels in the cow’s rumen
fed white clover relative to perennial rye grass resulted from the microbial detoxification of
plant glycosides which are hydrolyzed to cyanide and converted to thiocyanate in the
rumen. A further suggestion from research reviewed later is that potentially higher
molybdenum concentrations in clover compared to rye grass could have inhibited SRB
activity and higher nitrate content in one forage may increase sulphide oxidation. These
are further discussed below.
The sulphur in herbage is largely contained in proteins as methionine and cysteine. Most
of the proteins in fresh plant materials are highly soluble and are rapidly metabolized on
entry into the rumen. In studies with dairy cows on perennial ryegrass pastures, Dewhurst
et al (2007) demonstrated that cysteine-S was more readily converted to hydrogen
sulphide at rates greater than that of methionine-S which, in turn, was faster than the
conversion of sulphate-S to hydrogen sulphide when the same amount of sulphur in these
forms was added to the rumen (see discussion below and Figure 18).
The concentration of hydrogen sulphide in rumen gases increased with increasing dose
rates of sulphur compounds administered into to a cow’s rumen and peaked in rumen
gases 30-40 min after administration of cysteine (Dewhurst et al 2007). Inorganic sulphur
as sulphate is converted to hydrogen sulphide but there is a time lag of up to 10days
between introduction of sulphate into a diet and peak production rate of sulphide in the
rumen, indicating a slow growth and build up of bacterial populations that reduce
elemental sulphur (Gould et al 1997). The slow acclimation to sulphur in a diet may be
more important then the rate of acclimation to nitrate but the two will interact when their
levels are increased in the feed. This is discussed further below.
Substrate versatility of NRB and SRB
Although named after a single electron acceptor, SRB and NRB are metabolically
versatile and therefore able to use other organic or inorganic compounds as terminal
electron acceptors, such as nitrate, nitrite, carbon dioxide, iron(III), fumarate, elemental
sulphur and other sulphur species (Thauer et al 2007). These groups of microorganisms
have a significant environmental impact in several aspects: i) they participate in the
carbon and sulphur cycle by recycling sulphur and nitrogen compounds in the
degradation of organic matter.
In organic rich anoxic situations such as the lower gut of humans and probably the rumen
NRB and SRB appear to be able to utilize a number of organic compounds with the
production of hydrogen providing substrate for methanogens and carbon intermediates for
the synthesis of cells. The growth yield of SRB with nitrate as an alternative electron
acceptor is even higher then those with sulphate (Fauque et al 1991)These electron
donating reactions are shown below
Propionate → Acetate: CH3CH2COO- + 3H2O → CH3COO- + H+ + HCO3- + 3H2
9
Butyrate → Acetate: CH3CH2CH2COO- + 2H2O → 2CH3COO- + H+ + 2H2
Ethanol → Acetate: CH3CH2OH + H2O → CH3COO- + H+ + 2H2
Lactate → Acetate: CH3CHOHCOO- + 2H2O → CH3COO- + HCO3- + H+ + 2H2
Presumably the availability of sulphate and nitrate dictates the extent of utilization of
these substrates when present and could account for the higher proportion of acetate
found in the volatile fatty acids in rumen fluid in nitrate as compared to urea
supplemented diets fed to cattle (Farra and Satter 19 ; Hulshof et al 2011). In this
respect the apparent efficiency of nitrate/sulphate mitigation of methanogenesis would
always be lower then calculated from stoichiometric conversion of nitrate to ammonia or
sulphate reduction to hydrogen sulphide. This is further discussed in the section on
syntrophic reactions.
Sulphur and nitrate metabolism in other anaerobic ecosystems
One of the difficulties of extrapolating research from other anaerobic systems to the
rumen is that the majority of these systems, such as sediments in water, sewage, animal
excreta effluents, biogas digesters and oil field waters, have long turnover times and are
not necessarily continuously fed and some are not as rich in organic matter as a fed
rumen. They generally are at temperatures well below that in the rumen. As there are no
direct removal of organic acids by, for instance absorption across the rumen wall, the
reactions of nitrate in these systems tend to go to completion with dissimilatory reduction
to di-nitrogen as the end product. Never the less there is a growing wealth of information,
from these systems with long –turnover times which may provide leads to nitrate
metabolism in systems with shorter turnover time such as the rumen.
This author’s interest in resource depletion, particularly oil depletion (see Leng 2008b) led
to an examination of the microbiology of sulphur and nitrate in oil fields which together
with research on the same subjects in the rumen of animals appeared to provide leads to
the many unanswered questions that surrounded nitrate metabolism in ruminants. The
recognition of the ability of added nitrate into sewage systems removed the odor of
hydrogen sulphide has also led to the recognition of the role of NR-SOB in removing
hydrogen sulphide (see Mohanankrishnan et al 2009) The potential role of nitrate in the
rumen as a high affinity electron acceptor has the capability to reduce or even eliminate
enteric methane production and make a major contribution to alleviating the threats of
global warming. It is therefore important to explore these leads.
Oil field sulphur cycle
A number of Giant oil fields that produce the majority of the world’s oil requirements have
reached their peak production. Peak oil production from an oil province coincides with a
reduced flow of oil that was pumped, initially by harnessing the pressure from within the
oil field. To maintain oil flow rates from these depleting oil provinces, pressure in the well
is maintained by pumping in water, often or mostly sea water which contains considerable
amounts of sulphate ( around 28 mM/L).
10
The organic components of a subsurface oil field (hydrocarbons ,and various products of
microbial digestion including organic acids) are subject to continuing anaerobic
degradation by a consortium of bacteria and archaea producing hydrogen and also the
same volatile fatty acids produced in the rumen ( acetate propionate and butyrate) which
in turn are used by acetogens and methanogens as carbon sources. This maintains the
concentrations of organic acids low and prevents product feed back inhibition of
fermentation. This allows the breakdown of a proportion of organic residues to proceed
continuously (see Zengler et al 1999). As the oil is the remains of organic matter, mostly
algal biomass, the minerals essential to support microbial growth ( as biofilm attached to
inert surfaces)are present. A variety of organisms exist in these microbial consortia and
their microbial mixes are often perturbed and changed when the oil well starts to decline
in production by the addition of sea water, In particularly the sulphur content of sea water
has a major effect since it stimulates growth of SRB.
The SRB in oil fields ecosystems are the most studied since their end product, hydrogen
sulphide, is toxic and an irritant to oil workers. It also reacts with metal elements,
potentially damaging infra structure such as pipe lines. . When sea water is pumped into
an oil well the microbial ecosystem is irreversibly disturbed and large quantities of
hydrogen sulphide are released. Grigoryan and Voordouw (2008) have highlighted the
magnitude of the problem showing that a moderate sized offshore oil field into which
10,000 cubic meters per day of sea water is pumped, provides about 10 tons sulphate
sulphur ( 1g sulphate /L) releasing 1ton of hydrogen sulphide daily ( at 10% conversion).
The oil industry uses amendment with nitrate to reduce or eliminate this hydrogen
sulphide based on a long history of research which began with the recognition that
hydrogen sulphide production in sewage released into sea water could be eliminated by
adding nitrate into the system ( see Smith 2009), a practice still applied to sewage in Paris
and being introduced into city sewers as more understanding of the systems emerge..
The intensive pig industries have also come into prominence as the intensive use of liquid
manures applied to pastures created obnoxious smells ,mainly hydrogen sulphide which
could be eliminated by amendment with nitrates and/or molybdate ( see for example
Moreno 2009)
Research using water from subsurface oil fields in continuous up-flow, packed bed
bioreactors (Hubert et al 2003) that facilitate biofilm formation, has demonstrated the
changes in microbial species and also end products of metabolism in oil field water
following the addition of nitrate. Essentially there is a shift from SRB to heterotrophic NRB
(hNRB) activity and an increasing activity of NR-SOB. Where the turnover times are long,
nitrate-N is converted to di-nitrogen but there is the possibility that in systems with short
turn over times ammonia is the major end product. In short, nitrate is reduced to nitrite
and ammonia or enters the denitrification pathway to form di-nitrogen by hNRB. Sulphate
is reduced to hydrogen sulphide by SRB and the NRB-SOB present oxidize a proportion
of this hydrogen sulphide with the probable production of elemental sulphur and reduce
nitrite to ammonia (see Figure 2)
Figure 2 Impact of nitrate on the oil field sulphur cycle.
(A) Sulphide produced by SRB activity can be recycled to sulphate or sulphur by NR-SOB
reducing nitrate to nitrogen (denitrification) or ammonia (DNRA). (B) hNRB compete with SRB for
organic electron donors, such as lactate, excluding sulphide production by SRB and possibly
lowering their capacity to switch to nitrite reduction. Many SRB and hNRB oxidize lactate
incompletely to acetate and CO2 as shown. The overall reactions in panels A and B are the same:
the oxidation of lactate with nitrate (from Hubert and Voordouw 2007).
11
Hubert et al (2007, 2009) raised the question of relative contribution of hNRB and NRSOB to lowering of hydrogen sulphide production by nitrate addition in these systems
The question was ‘do heterotrophic nitrate-reducing bacteria (hNRB) out-compete SRB or
is there sufficient oxidation of hydrogen sulphide by NR-SOB to reduce hydrogen sulphide
production in these systems’. Highly sophisticated analysis of the ratios of stable isotope
of sulphur and oxygen in the hydrogen sulphide and sulphate in the bioreactor medium
were in excellent agreement; it was the inhibition of SRB activity that largely controlled
hydrogen sulphide release in a nitrate-injected oil well water and NR-SOB oxidation of
hydrogen sulphide played only a minor role in the observed control of hydrogen sulphide
release.
Nitrate –sulphur interactions in waste water treatment systems
The presence of anaerobic conditions in sewer systems results in significant production of sulfide
by SRB from organic matter containing sulphur (see Bentzen et al 1995) Release of hydrogen
sulfide from the liquid to the gas phase causes several detrimental effects including sewer
corrosion, odor nuisance and health hazards. A number of operational strategies
are employed by the wastewater industry to minimize sulfide production in sewers. Addition of a
thermodynamically favorable electron acceptor such as a nitrate salt has been used over the last
70 years to effectively control odors and sulfide production in a number of anaerobic
environments. Long-term addition of nitrate to simulated rising sewage systems, stimulated the
growth and activity of NR-SOB bacteria that appeared to be primarily responsible for the
prevention of sulfide build up in the wastewater in the presence of nitrate
. A short adaptation period of three to four nitrate exposure events (approximately 10 h each) was
required to stimulate biological sulfide oxidation, beyond which no sulfide accumulation was
observed in reactors charged with sewage( Mohanankrishnan et al 2009). Nitrate addition also
effectively lowered methane emmissions. Following adaptation of the sewage system to added
nitrate (after 4 exposures), hydrogen sulphide release to the bulk fluid ceased and nitrite
accumulation began ( see Figure 3 ). Unlike the conclusions from the oil well reservoirs, it was the
sulphide oxidizing activity of NR-SOB that was identified as the predominant mechanism for the
elimination of sulphide release from the biofilm to the bulk fluid. It was only when hydrogen
sulphide concentrations were close to zero that nitrite began to accumulate in the reactor vessels
( Figure 3b) The time course of these changes were relatively slow in reactors simulating the
conditions in rising sewage treatment relative to that in the rumen of animals but it is the first
definite association of nitrite production when sulphide uptake results in no or low levels of
12
hydrogen sulphide in the medium. The end product of sulphide oxidation appears to be elemental
sulphur De Gusseme et al( 2009) also demonstrated rapid utilization of sulphide by NR-
SOB in simulated sewer systems and showed that after a prolonged period of incubation
with nitrate that NR-SOB activity continued without the addition of nitrate suggesting
that an oxidized redox reserve is present in such systems.
Nitrate reducing, sulfide oxidizing processes have been demonstrated in a number of
anaerobic systems amended with nitrate(De Gusseme et al.,2009; Garcia-de-Lomas et
al., 2007). It is likely that as the sewer biofilm adapted to nitrate exposure, the NR-SOB
population, increased and sulphide exceeded sulfide production (by SRB), leading to zero
net sulfide production in the biofilm in the presence of nitrate (Figure 3).These organisms
were possibly already present in the sewer biofilm prior to the addition of nitrate, and
required a relatively short period of time for enzyme activation and for growth. Gevertz et
al. (2000) reported average NR-SOB doubling times of 1.3–2.9 h, which fits well with the
short biofilm adaptation period observed by Mohanankrishnan et al (2009) and also
appears to make it possible for them to exist in situation where the turnover time of the
bulk fluid is relatively rapid such as that in the rumen. Sulfide oxidation occurred in the
outer part of the biofilm, a spatial positioning that would allow NR-SOB access to nitrate
from the bulk fluid, and sulfide from SRB activity within the biofilm. Another possibility is
that SRB switch their activity to nitrate reduction with repeated nitrate exposure events,
resulting in reduced sulfide production rates. Such a switch has been previously
demonstrated in pure culture studies (Mitchell et al., 1986). They do so by inducing the
nitrate reductase and/or nitrite reductase enzymes, responsible for the conversion of
nitrate to nitrite and for the conversion of nitrite to ammonia, respectively (Moura et al.,
1997). It is presumably possible that only when nitrite accumulated would nitrite reductase
be induced or the induction of nitrite reductase takes more time. Such effects in the rumen
could account for the unpredictable accumulation of nitrite rates after nitrate load in the
rumen. Presumably NR-SOB can maintain an energy supply from reducing nitrate to
nitrite when sulphide availability is reduced or zero. A further possibility appears to be the
growth of species of NR-SOB that only convert nitrate to nitrite as demonstrated by
Gevertz et al (2000).
Figure 3 Changes in dissolved sulphur and nitrogen species in a laboratory model rising
sewer during serial exposure events ( over 10hours) tyo nitrate ( after Mohanankrishna et
al 2009)
No or little inhibition of sulphide production by nitrate after 2 exposures of
reactor to nitrate - Zero production of Nitrite .
Conc. of S cmp. (mg/S/L) or N cmp. (mgN/L)
16
14
12
Total dissolved S
Sulfate
10
Nitrate
8
Sulfide
Sulfite
6
Thiosulfate
Poly. (Nitrate)
4
2
0
13
0
0.5
1
1.5
2
Time (hours)
2.5
3
3.5
Suppresion of hydrogen sulphide after 4 exposures of reactor to nitrate
with concomitant production of nitrite
Conc. of S-cmp. ( mg-S/L) or N cmp. ( mgN/L)
20
18
16
Total dissolved S
14
Sulfate
12
Nitrate
ThioS
10
Nitrite
8
Sulfide
Linear (Nitrate)
6
Linear (Sulfide)
4
Linear (Nitrite)
2
0
0
0.5
1
1.5
2
2.5
3
3.5
-2 ecology of anaerobic ecosystems may explain some of the interactions
Microbial
between methanogens, SRB, Time
NRB( Hours
and NR-SOB
In natural or engineered anoxic habitats the largest population of microbes (possibly as
high as 99%) persists attached to surfaces within a structured biofilm and not as freefloating or planktonic organisms (Costerton 2007;Costerton et al 1995a,b ). Moreover,
these natural assemblages of bacteria within the biofilm matrix function as a coordinated
consortium dedicated to the degradation of organic matter (Caldwell 1995; Costerton et al
1995). Although the biofilm microbes have a planktonic existence in fluid medium, in their
sessile lifestyle they function as an integral part of bacterial community within and
attached to a matrix of extracellular polymeric substances(EPS) and their biochemical
actions maybe somewhat different in the two niches ( Davey and O’Toole (2000).
During the past 10-20 years new developments in microscopic and molecular
technologies have been applied to biofilm studies in situ. These technologies have
reveled that biofilms are not simply organism-containing slime layers on surfaces but are
a complex mixture of populations of different species of bacteria/archae and other
microbes with a high level of organization/cooperation and apparently means for
communicating or signaling.(Davey and O’Toole,2000).They form structured, coordinated,
efficient functional communities of syntrophic organisms where digestion products are
readily exchanged between species since a product produced by one species colony is
only a short distance from the organisms that use that end product in its metabolism (see
Stams and Plugge,2009). An additional advantage is that the substrate concentrations
available to the microbial communities are considerably higher and therefore uptake is
faster and greater and more efficient then they would be if the intermediates in energy
metabolism were released to the bulk medium for use by other individual microbes.( see
Wang and Chen 2009)
14
A number of recent articles, have discussed the complex interactions that form the basis
of coexistence of organisms in stable biofilms and the restricted view of microbial
metabolism is modified by the ecological considerations ( see Costerton 2007). Of
immense importance is the ability of biofilm colonies, through syntrophic associations
allowing different combinations of sessile colonies to interact using different terminal
electron acceptors to flourish as the composition of the available substrates change. This
applies particularly where enteric organisms are now recognized to function as biofilm
consortium (McAllister et al 1994;Cheng et al 1995: Edwards et al 2008 ;Firkins 2010;see
also Leng 2011).
The ecology of biofilm will change according to the substrate availability , for instance it
could be expected that the biofilm that associate with particulate matter when the dietary
N source is nitrate as opposed to urea or protein will be differently organized ,may
contain different guilds of microbes and that interactions between these groups will occur.
Biofilm are often referred to as methanogenic or sulphidogenic, which emphasizes the
main terminal acceptor for electrons in the syntrophic associations that develop and
change with the availability of particular electron sinks.
Methanogens are strategically located in biofilms close to fermentative organisms, when
sulphur is available the SRB ,which have a higher affinity for hydrogen (higher Gibbs free
energy change of sulphur reduction) than methanogens may replace them or take up
similar position in the layered structure of the biofilm since they can out compete
methanogens for hydrogen. Since dissimilatory nitrate reduction to ammonia is more
energetically favorable than either methanogenesis or sulphur reduction, NRB will out
compete both SRB and methanogens provided they reside at appropriate sites, close to
hydrogen generation. It is logical that the interactions between the organisms with
different affinities for hydrogen is dependent on the spatial distributed within the biofilm
matrix or they replace their competitors or are closely associated with them allowing the
changes seen where nitrate lowers both sulphur reduction and methanogenesis. The
possibility is also there that some organisms can use two or three routes for disposal of
hydrogen after an adaptation phase. Thus providing a confined localized concentration of
organisms strategically sited at the outer edges of the biofilm matrix would allow the
interactions depicted in Figure 2 and 4 when nitrate is added to the medium containing
both organic matter and sulphur
Rumen biofilm research, however has not advanced to an extent where we can describe
the biofilm that develops under any feeding regimen where sulphur and nitrates are
components of the diet and urgent in situ research is needed to determine the spatial
distribution of the potentially participating organisms in the biofilm matrix
The inference from the studies in better known environments such as water treatment
sewers, is that methanogenic Archae NRB, SRB and NR-SOB are likely to be distributed
through the biofilm, on plant material in the rumen when nitrate is introduced into a diet .
That the distance between species competing for substrate is closely similar and
therefore the substrate with the highest affinity for reducing hydrogen dictates the end
product of hydrogen metabolism.
Roles of rumen protozoa and fungi in methanogenesis in the rumen
Recently reports have indicated that protozoa and fungi may be important in methane
production since they have specialized organelles,hydrogenosomes where hydrogen is
15
produced ( see Yarlett et al 1981;Yarlett et al 1986;Muller 1993). Methanogens closely
associate with the hydrogenosomes and apparently access the hydrogen released in
metabolism ( see Williams 1986) and recently it has been suggested that in an artificial
rumen that the protozoan fraction from whole rumen fluid had significant nitrate reducing
capacity with no nitrite generation over 24hr incubation indicating the end product almost
100% ammonia ( Lin et al 2011). However nothing beyond the paper by Lin et al (2011) is
known about the nitrate reducing capacity of the rumen protozoa, and/ or their
endosymbionts
Nitrate-sulphur interactions in the rumen
The microbiological changes in the rumen when nitrate is introduced either via direct
dosing or continuously in the feed have received little attention other then to measure
rates of nitrate and nitrite disappearance (Aloubadi and Jones 19 ). SRB ( particularly
Desulfovibrio spp that can also reduce nitrate too ammonia ) and NRB( for instance
Selenomonous ruminantium, Viellonella parvula and Wollinella succinogenese, see
Cheng and Phillippe 1986: Cheng et al 1985 ,Iwamoto et al 2002) are usually present in
significant numbers in the rumen and intestinal contents of animals including humans
(Forsberg 1980; Gibson 1990), but their cell numbers will be limited by the sulphur or
nitrate content of a diet. The low hydrogen sulphide content of rumen fluid (around 0.1
mM) (Hungate 1965) would tend to lower the possibility of a NR-SOB being a significant
component of the rumen biofilm unless the animal had been acclimated to high S and
nitrate in the diet).Whilst rumen levels of hydrogen sulphide are low the important issue
will be the concentration in the biofilm where the NR-SOB are likely to be located..
Technically W succinogenese can be regarded as the most likely NR-SOB( see below).
Cysteine-S is rapidly reduced to hydrogen sulphide in the rumen and when cysteine was
injected with a nitrate load; accumulation of nitrite in the rumen of sheep was inhibited
(Takahashi et al 1998).The Desulfovibrio species use electron donors such as formate
propionate or lactate to reduce sulphate to sulphide, and the NR-SOB uses nitrite as the
electron acceptor to re-oxidize the sulphide to sulphate. Because nitrate is reduced
initially to nitrite (Figure 2), nitrite may accumulate, inhibiting dissimilatory sulphate
reduction. This inhibition can be prevented or overcome by the nitrite reductase activity of
the SRB, which reduces nitrite to ammonia. In the rumen the electrons for this are
ultimately provided by reduced cofactors generated in the Embden-Meyerhof pathways of
carbohydrate fermentation. Thus, together an SRB and NR-SOB co-culture could
catalyse the digestion of organic matter with nitrate as a source of fermentable nitrogen.
The critical issue is the availability of sulphide in rumen fluid and the assumption that
NR-SOB also exist in the rumen environment. In the latter case the assumption is
supported in that at least one organism originating from the bovine rumen, Wollinella
succinogenese ,has the ability to oxidize sulphide to sulphur and sulphur to sulphide
(Macey et al 1986) and to reduce both nitrate and nitrite to ammonia (Schroder et al
1985).The hypothesis developed that requires to be tested is shown in the box below.
16
The hypothesis
hNRB reduce nitrate to ammonia using either formate or hydrogen as the electron
source. NRB however out-compete the SRB for the same electron donors and may
lower or inhibit sulphide production The NR-SOB, that are in relative small numbers in
the rumen, reduce nitrate through nitrite and couple reduction of nitrite to the oxidation
of sulphide to sulphate. This can also reduce the availability of hydrogen sulphide.
When nitrate is adequate but sulphide is limiting in the medium the NR-SOB obtain
maintenance energy requirements (or ATP) by converting nitrate to nitrite with 0.25 of
the electron requirements and nitrite is spilled into the medium. Nitrite concentrations
increase in the incubation medium and further inhibit SRB activity. The hypothesized
sequence of events when nitrate is metabolized in an acclimated rumen fed with
adequate sulphur is shown in Figure 4.
The reactions shown in Figure 4 require close associations of the microbes that carry out
the individual reactions which can be achieved by the biofilm mode of life.
Support for the hypothesis
When a nitrate load is introduced into the rumen, nitrite production in the rumen is slow
and quantitatively small compared to the nitrate ingested, even though nitrate
disappearance is rapid (see Lewis 1951and Figure 5 ). The thesis is consistent with the
general pattern of suppression or souring ( production of hydrogen sulphide) in the oil
field. In the rumen that has been slowly adjusted to nitrate, hNRB develop together with
NR-SOB ( possibly W succinogenese).The hNRB reduce the activity of SRB and the
sulphide pool declines to an extent where the NR-SOB are limited by sulphide availability
and only partially reduce nitrate to nitrite. The population density of NR-SOB always
remains small in accordance with the generally low intake of sulphur in most diets. Thus
nitrite production in relation to nitrate conversion to ammonia is a small fraction of the
total nitrate uptake by rumen microbes as demonstrated in numerous publications ( Lewis
1951; Takahashi and Young 1994; Tillman et al 1965). In sheep well adapted to nitrate,
methane mitigation indicated that 87-90% of the dietary nitrate was fully reduced to
ammonia (Zijderveld et al 2010a).
Figure 4. A theoretical model of nitrate and sulphate metabolism and interaction in the
rumen
17
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
Figure 5 Changes in nitrate, nitrite and ammonia concentrations in rumen fluid of sheep
fasted for 16 h and injected intra-ruminally with 25g sodium nitrate (Lewis 1951).The zero
time concentrations have been calculated by assuming a rumen fluid volume of 5L
Lewis -Injection 25g NaNitrate
Conc. in rumen fluid (mM)
60
50
40
30
20
10
0
0
2
4
6
8
10
12
Time after injection (hr)
Nitrate-N (mM)
Nitrite -N (mM)
Ammonia N (mM)
Observations leading to the hypothesis that the hydrogen sulphide pool in rumen biofilms
is critical in nitrate metabolism in the rumen
In vitro studies of nitrate metabolism using an artificial rumen
18
Barnett and Bowman (1957) used an artificial rumen to study nitrate metabolism (see
Leng 2008a) and although the research was open to criticism of its methodology, there
are six most interesting observations which add to the concept that nitrite accumulation in
rumen fluid when dietary nitrate is provided is a result of a deficient growth factor (see
Figure 6). The following observations were made by Leng (2008a)
 A considerable amount of nitrate when added to the “rumen” (initial concentration
10.5 mmol/L) disappeared over the first 5hours without reciprocal increases in
either rumen ammonia or nitrite. The nitrate that disappeared rapidly from the
incubation medium in the first 5hours appeared to be released as nitrite over the
next 30h; the nitrite-N concentration at 35hours represented about half the nitrateN at the start of the incubation period.

Powdered cellulose added to the incubation medium had no effect on the pattern of
nitrate disappearance or nitrite accumulation, perhaps indicating a lack of
cellulolytic activity and therefore availability of electron donors.

Addition of glucose increased nitrate clearance, increased the rate of nitrite
accumulation and when nitrate was apparently cleared from the medium, nitrite
disappearance increased. The pattern of nitrate and nitrite change in the
incubation medium with glucose is compatible with an increased availability of
electron donors (high fermentation rate) and increasing activity of nitrate and nitrite
reductase in microbes. It appeared that in this study nitrate may have been first
reduced to nitrite that enters the incubation medium and the nitrite is then reduced
to ammonia by dissimilatory nitrite reductase since ammonia levels increased in
the incubation medium.
Supplying dried grass together with nitrate had a major effect; nitrate
disappearance was extremely rapid (the same as when glucose was added) with
insignificant accumulation of nitrite. Dried grass would have supplied readily
fermentable carbohydrate to generate electron donors for NRB. Nitrate was not
metabolized by a dissimilatory pathway as ammonia in the incubation medium
declined with nitrate disappearance. This could be compatible with a component in
the dried grass stimulating the growth or activity of NRB or NR-SOB that reduced
nitrate to ammonia by an assimilatory pathway. As discussed earlier an NR-SOB
would have oxidized sulphide in nitrite ammonification. The incubation medium
was low in total sulphur and the soluble protein in dried grass could have supplied
readily available sulphur source mainly S-amino acids which are rapidly converted
to sulphide in rumen fluid (see Dewhurst et al 2007

Figure 6. Pattern of change of nitrate, nitrite and ammonia in media contained in an
artificial rumen after adding nitrate 5h after the start of incubation with nil substrate,
powdered cellulose, glucose or dried grass (after Barnett and Bowman 1957).
19
The rate of nitrate disappearance when dried grass was in the incubation medium was the
same as when glucose was added. This may indicate that nitrate metabolism was in both
cases the result of a single species (or group of species) that converted nitrate to nitrite
when sulphide concentrations were low and fermentation of glucose was high (no scarcity
of electron donors) and nitrate to ammonia by assimilatory nitrate reduction providing
ammonia and ATP for microbial growth when dried grass was present. The latter
providing the vital source of electron donors and sulphide. The depression in ammonia
being solely the result of other bacteria growing with the dried grass providing ammonia
from protein and also substrate for fermentation
Feeding trials
20
In ruminants the recommended sulphur content to meet requirements is 0.4% of the dry
matter intake. Sokolowski et al (1969) showed with sheep that adding nitrate to a
concentrate diet given to lambs had little effect on N balance but significantly reduced S
balance suggesting an interaction between nitrate and the sulphur source. Unlike N
balance, that is rather easy to calculate from N excreted in urine plus faeces and the total
intake of N, the S balance has to take into account the additional S that is excreted as
H2S from the rumen gas space.. The negative S balance reported by Sokolowski et al
(1969) in sheep fed nitrate is difficult to reconcile since there was no change in N balance.
Unfortunately the raw data of the sulphur content of faeces and urine was not available.
The negative S balance maybe a result of an increased loss of H2S via breath or an
increased loss of sulphate and sulphur via the urine and faeces. At the time that
Sokolowski was undertaking his research (1959) the H2S pathway was little understood
and would probably not have been considered. However the recognition that at least one
rumen organism namely W. succinogenese can oxidize sulphide to sulphur indicates a
possible reason for the negative S balance since elemental S is only slightly soluble and
may be excreted in faeces .
Supplementing a diet fed to goats consisting of of molasses and mimosa foliage with
urea(2.2% of dietary DM) or calcium nitrate( 3.8% dietary DM) led to a reduction in the
methane/carbon dioxide ratio in the eructed breath of goats compared with control
animals supplemented with urea. Adding 0.8% sulphur as sodium sulphate to the diets
also reduced the methane/carbon dioxide ratio( MC ratio), with the supplements having
additive effects. Calculation of the potential methane lowering effect of replacing urea N
with nitrate N based on the MC ratio( Masden et al 2011) showed that the effects of added
sulphate and nitrate were both effective and additive as was earlier demonstrated by van
Zijderveld et al (2010) ( Figure 7). Supplementary sulphate increased both digestibility of
crude protein and N retention but these were unchanged by the NPN source ( Silvithong
et al. 2011)In both studies the additional sulphur added to the diet would be considered to
be excessive relative to requirements ( 0.93% v 0.8%) but both nitrate and sulphur are
acting as electron sinks. In the research of van Zijderveld et al 2010 , sulphur appeared to
take over the role as an electron sink when nitrate was depleted in the rumen ( see
section )
Figure 7. Effect of added sulphate and nitrate and sulphate plus nitrate on per cent
reduction in methane in the studies by Silvithong et al( 2011) with small goats and that
reported by van Zijderveld et al( 2010a) with sheep..
21
van Zijderveld et al (2010a) have made the most comprehensive study to date of the
effects of nitrate and sulphur on enteric methane production in sheep. In their studies
lambs were gradually introduced to increasing amounts of nitrate, sulphate or nitrate and
sulphate in a maize silage based diet over 4 weeks and CH4-production was
subsequently determined in respiration chambers. CH4-production was significantly
decreased by both supplements (nitrate: - 32%, sulphate: -16% and nitrate + sulphate: 47% relative to the control). The decrease in CH4-production in nitrate fed sheep was
most pronounced in the period directly after feeding and up to 12hours when it returned to
approximately the production rate in animals fed urea as a control treatment, and declined
slowly thereafter. The lowered CH4 production associated with sulphate feeding was
observed during the entire day (Figure 8) although it appeared to increase at the time
nitrate appeared to be depleted at about 12 hours post feeding . The evidence suggests
that hydrogen sulphide production was continuous on this diet in the absence or presence
of nitrate and therefore the pool of sulphur containing metabolites is retained. In these
studies the recommended requirements for sulphur is far exceeded by sulphur in the diet.
No methaemoglobin was apparently produced when nitrate was fed with a high level of S
in the diet. .
Similar results were shown by Nolan et al (2010).In these studies sheep were acclimated
to one of three diets consisting of chaffed oat hay supplemented with 0%, 2% or 4%
potassium nitrate( KNO3) and made iso-nitrogenous by the addition of urea (4
sheep/diet). Nitrate supplementation did not increase blood methaemoglobin, reduce DM
intake or affect whole tract or ruminal (in situ) DM digestibility. Dietary nitrate caused
changes in rumen fermentation consistent with its role as a high-affinity hydrogen
acceptor, i.e. a reduced propionate molar percentage in rumen VFA, increased molar
acetate: propionate and a lower methane yield per kilogram of DM ingested. In sheep fed
diets containing 4%KNO3, methane production was reduced by 23% of the production in
the control group. The data confirm the capacity of dietary nitrate to lower enteric
methane production but these researchers found large between-animal variation in
methane production and better technology or the use of more animals will be required to
quantify the effect with confidence.
Figure 8.The effects over a day on the patterns of methane production in sheep fed maize
silage and concentrate The feed was given at 7 30 am .( Zijderveld et al 2010) and
methane production was measured over hourly period for 24hours.
22
2.00
Methane production ( L/hr)
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
1
2
3
4
5
6
Time of day( Hour)
CON
NO3
SO4
NO3+SO4
More recently research has been reported at the Greenhouse Gases and Animal
Agriculture Conference (2010) showing that both beef cattle and lactating dairy cows may
utilize nitrate as a fermentable nitrogen source to support rumen fermentation of organic
matter and at the same time act as a high-affinity hydrogen acceptor with significant
lowering of enteric methane production (Hulshot et al 2010 Zijderveld et al 2010b).
In the research with sheep (Figure 8) four treatments were compared in terms of the
production of methane (van Ziiderveld et al 2010a) . These were a control diet with urea,
a treatment with additional sulphate added to the control diet and two treatments where
urea was replaced with nitrate with and without added sulphate. In the statistical analyses
of the experiment there was no interaction between the addition of nitrate and sulphate
and this interaction was not significant for any of the time points. This suggests that the
reduction of methane by nitrate is not affected by the presence or absence of sulphate.
However, this seems to be inconsistent with studies from other anaerobic microbial
ecosystems as indicated in the previous section where SRB are inhibited by nitrate and
some SRB adapt and preferentially use nitrate (see above). For these reasons a closer
examination of the date appeared warranted. It should be possible from the data
presented in Figure 8 to asses the 24hr pattern of change in methane production owing to
sulphate addition, by subtracting the methane production in sheep on the nitrate ration
from that on the nitrate plus sulphate supplemented diet. Similarly the reduction in
methane production due to sulphate can be calculated by subtracting the methane
produced in the urea fed sheep from the methane produced in sheep fed urea plus
sulphate. The resultant apparent effects of sulphate on the lowering of methane
production are shown in Figure 9.
Visually, there is a difference between the sulphate supplemented nitrate group and the
nitrate group receiving no extra sulphur. Each of the sheep in the experiment only
received one of the treatment diets and it is therefore not statistically to compare the
different treatments within one sheep, never the less the calculated patterns of change in
the lowering of methane in the presence and absence of nitrate shown in Figure 9 are
23
what had been expected if the hypothesis being argued here is proven. In this respect it is
presented as part of the overall approach taken by the author
The effect of nitrate on methane production apparently ceased between 10 and 12 hours
after the meal had been consumed. At this time there was an apparent upswing in anti
methanogenic activity of sulphate. From Figure 9 it is apparent that sulphate was
increasingly effective in reducing methane production rate up to about 8 or 9 hours post
feeding when urea was the fermentable N source. In contrast nitrate reducing bacteria
appeared to reduce the activity of SRB as there was a steady decrease in methane
production by added sulphate over the same time period in the presence of nitrate as
shown in Figure 9.
Lowered methane production (L/hr) owing to
supplementation with sulphate
Figure 9.The effect of sulphate supplementation in lowering methane production.
(Calculated from data supplied by Zijderveld et al 2010a)
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
7
8
9
10
11
12
13 14
15
16
17
18 19
20
21
22
23 24
1
2
3
4
5
6
Time of day(Hour)
Con minus sulphate
Nitrate minus (nitrate plus sulphate)
The rebound (between 11-and 15hr post feed intake) in the ability of added sulphate (fed
with nitrate) to lower methane production can be explained by a resurgence in SRB
activity and therefore hydrogen sulphide production as nitrate in the rumen was
exhausted.
As 1 mole of methane production from the reduction of carbon dioxide has the same
requirements for electrons as the production of 1 mole hydrogen sulphide, the lowered
methane production rate (liters/hr) equates to the production rate of hydrogen sulphide
owing to the supplementation with sulphate. Thus in the presence of supplemental urea
and sulphate in the rumen over the first 9 hours after feeding hydrogen sulphide
production appears to increase steadily. On the other hand over the same time period
replacing urea with nitrate in the presence of sulphate appears to steadily lower hydrogen
sulphide production. This may be indicative of the suppression of SRB by hNRB as shown
to occur in other anaerobic systems. At 11hours post feeding the hydrogen sulphide
production appears to almost cease and this suggests that substrate for NR-SOB is
depleted and following this nitrite spilling (incomplete nitrate ammonification by NR-SOB)
should occur if the hypothesis proves to be correct.
24
Hydrogen sulphide production (calculated from the reduction in methane generation)
appears to be different when urea is the primary fermentable N source to that when
nitrate is present. With time after feeding, hydrogen sulphide production apparently
increased almost linearly (Figure 11) where as when nitrate was present hydrogen
sulphide production appeared to decrease over a period of 4-5hrs and then increased
over 5 hrs in the same pattern as for the effect in urea fed animals. This is possibly due to
a depletion of nitrate in the medium by bacterial metabolism (compare the nitrate
disappearance of nitrate from the rumen of sheep in Figure 17( after Lewis 1951) with
the apparent change in hydrogen sulphide production in sheep fed nitrate in Figure
9(after Zijderveld et al 2010a) and this is about the same time after feed intake that
methane production in nitrate supplemented animals becomes equal to the rate of
methane production when urea was the NPN source Figure 7 :Zijderveld et al 2010).
The calculated data in Figure 9 and 10 may be explained by sequential use of electron
acceptors in the rumen. It appears that nitrate has a priority for electron capture and early
after feeding reduces methane production by up to 70% but as the concentration of nitrate
in the rumen declines there is a switch to sulphate or sulphur as electron acceptor and a
return to carbon dioxide as the sulphate is also depleted. At the maximum effect of
sulphate in the rumen, methane production rate is lowered (calculated from the apparent
hydrogen sulphide production) by about 30% which is highly significant. Whereas at the
lowest effect in the presence of nitrate, sulphate lowers methane production by a mere
4%
If the unavailability of hydrogen sulphide in the rumen in the presence of nitrate causes
nitrite spilling then any factor that inhibits sulphur metabolism in the rumen will also result
in nitrite spilling following addition of nitrate to the rumen. The concentrations of some
minerals have been demonstrated to lower hydrogen sulphide in rumen contents and also
other anaerobic ecosystems. Of major interest is the effect of dietary molybdenum (Mo).
Reduction in methane production
( liters/hr)
Figure 10. The pattern of effect of dietary sulphate in lowering methane production in the
presence of urea or nitrate in the diet. of sheep between 9 am (time of presentation of
feed 0800hrs) and 2300hrs (from Figure 8 and 9 Zijderveld et al 2010)
0.4
0.35
0.3
y = 0.0141x - 0.0176
R2 = 0.8637
0.25
0.2
0.15
0.1
0.05
0
9
11
13
15
17
19
21
23
Time of day(00hrs)
Methane inhibition by sulphate in urea diet
Methane inhibition by sulphate in a nitrate diet
25
The apparent efficiency of nitrate reduction in the rumen to ammonia and methane
mitigation with increasing dietary nitrate concentrations.
Ruminants maintained on low protein diets, should be able to replace urea with nitrate
and stimulate fermentation rate, however there will always be a balance between amount
of nitrate required to satisfy the fermentable N requirements of the ruminal biota and the
potential reduction in methane production that can be achieved. Thus response curves to
increasing amounts of nitrate replacing other fermentable N sources are required
particularly to provide data for economic analysis. One of the first response curves was
produced with young goats where these were fed a rather exotic diet of rice straw, water
spinch,mimosa leaves and molasses with a mineral supplement. In these goats nitrate
replaced urea and growth rates were measured along with an empirical measure of the
methane reduction using the method of Madsen 2011 which uses marker gas carbon
dioxide together with the ratio of methane to carbon dioxide in breath as an index of the
reduction in methane . The relative rate of methane reduction indicated a positive
curvilinear trend increasing to 60% reduction when all the urea was replaced by nitrate
(Sophae and Preston 2011) The curvilinear nature of the curve suggests that the
apparent efficiency of conversion of nitrate to ammonia is reduced as nitrate in the diet
increases. Without knowing the methane production rate, however, the apparent
stoichiometric efficiency of nitrate reduction in the rumen cannot be calculated.
Figure 11 Methane reduction from adding potassium nitrate to a diet fed to goats
(calculated from ratio of methane to carbon dioxide in gas excreted via mouth) (Sophae
and Preston 2011)
Recent work where nitrate has progressively replaced urea in diets fed to cattle (van
Zijderveld et al 2012) has indicated 2 issues
1. Increasing nitrate at the expense of urea increased the percent reduction in enteric
methane production but the apparent efficiency of nitrate as an electron sink
decreased
2. With increasing nitrate in the fermentable nitrogen of the diet, whilst weight gain
remained constant efficiency of feed utilization increased.
In this study 36 Holstein steers (288 ± 25 kg BW) were fed increasing levels of dietary
nitrate (0 to 2.7% of DM; 6 levels)replacing urea on an iso-nitrogenous basis in corn
26
silage based, total mixed ration and methane production was subsequently determined in
respiration chambers. Diets were fed restricted to maintain similar feed intake among all
treatments. Methane production by cattle decreased linearly with increasing nitrate dose.
Lowered enteric methane production by inclusion of nitrate in diets given to beef cattle
have been recently reported (Hulshof et al.2011). However, the apparent efficiency with
which dietary nitrate lowered methane production decreased with increasing dose of
nitrate in this experiment (Zijderveld et al 2012) These results together with previously
reported research indicates there is a negative correlation between amount of nitrate
given per kg metabolic body weight and the apparent efficiency with which nitrate lowers
methane production, based on the assumption that all nitrate is reduced to ammonia in
the rumen ( Figure 12 ).
Stoichiometrically, 100 g of dietary nitrate reduced to ammonia in the rumen should lower
methane emissions by 25.8 g in ruminants (Van Zijderveld et al., 2010) unless the added
nitrate alters the quantity or the pathways by which substrates are degraded and in this
way stimulates additional hydrogen generation. The inefficient use of nitrate has been
hypothesized to originate from species differences or incomplete nitrate reduction in the
rumen (van Zijderveld et al., 2011). This difference in methane mitigation by dietary
nitrate was hypothesized to originate from the pattern of fermentation in which more
propionate was produced in the rumen in dairy cows fed high concentrate rations relative
to sheep and beef cattle on much higher fibrous feeds (van Zijderveld et al., 2011).
However, in these cattle with the same feed intake the efficiency of methane mitigation
apparently decreased with increasing nitrate dose while animals were of the same breed
and of similar weight. Thus, differences in the efficiency of methane mitigation of nitrate
could only be related to the different doses of nitrate used in the study.
Figure 12 Apparent efficiency of methane reduction (observed methane reduction/
theoretical maximum methane reduction) with increasing doses of nitrate fed to various
species of ruminant animals.( van Zijderveld et al 2012d)
27
Potential explanations for the apparent poor efficiency of methane mitigation based on
the change in metabolism of VFA in the rumen amended with nitrate.
From the data in Figure 12 it appears that of the efficiency of nitrate in reducing methane
production was negatively related to the amount of nitrate included in a diet and at the
highest reported level of intake of nitrate was only 59%. There appear to be only 3
reasons why this could occur
 Nitrate or nitrite is absorbed and lost in the urine
 Nitrate changes the microbial ecology of the rumen and stimulates additional
hydrogen production when compared to urea as the fermentable N source.
 Both of the above are involved
If the first is correct then nitrate excretion at the high intakes could potentially produce
more GHG(NOx) then that saved by the reduction in methane production. However
unlikely, this would be a major impediment to the acceptance of nitrate at the higher
levels for the purpose of mitigation of methane production.
The second appears more likely since in the research quoted, if 40% of the nitrate had not
been reduced to ammonia, an N deficiency for efficient fermentative digestion could have
been expected which would have translated into a reduced digestibility and possibly feed
intake, which did not occur. A more likely explanation is associated with the change in the
relative production of the VFAs in the rumen, with significant increased production of
acetate relative to propionate and butyrate which is accompanied by an increased
hydrogen production. There also appears to be an increased microbial cell yield (which is
in itself an electron sink)( Nolan et al 2010). An increased acetate to propionate
production may result from a channeling of more carbohydrate through pyruvate and
acetyl CoA in the fermentative pathways or it could be a result of acetogenic oxidation of
butyrate and propionate by the boosted nitrate reducing capacity of rumen contents and
therefore increased populations of NRB ( Alaboudi and Jones 19 ) and SRB organisms
that results with increasing nitrate in the diet.. SRB from animal and human large intestine
have been shown to oxidatively convert propionate and butyrate to acetate( see Gibson
1990).
The observed increase in ruminal acetate:propionate ratio that accompanies
the increased nitrate content in a diet is also consistent with the selective increase of
Gram-positive Ruminococci, which produce primarily acetate, and the potential
suppression of the Gram-negative bacteria, such as Fibrobacter succinogenes, which
produces primarily succinate, and Selenomonas ruminantium, which converts succinate
to propionate.
A further possibility is that nitrate inclusion in a diet stimulates the efficient growth of
syntrophic organisms through changes in the partial pressures of hydrogen within the
biofilm consortia associated with feed particles( Leng 2014).
Syntrophic oxidation of butyrate and propionate
Syntrophy is an essential interaction in methane production, which involves the
interaction between hydrogen- and formate-producing microbes with hydrogen- and
formate-using partners. The Gibbs free energy changes involved in syntrophic
metabolism are very low, close to the minimum free energy change needed to sustain
microbial growth. Alone, the oxidation of butyrate to acetate and hydrogen is energetically
unfavorable. However, when a methanogens are co-cultured with bacteria capable of
28
butyrate oxidation , methanogenesis significantly lowers the concentration of hydrogen
(down to 10−5 atm) and thereby shifts the equilibrium of the butyrate oxidation reaction
under standard conditions (ΔGº’) to non-standard conditions (ΔG’). Because the
concentration of one product is lowered, the reaction is "pulled" towards the products and
shifted towards net energetically favorable conditions (for butyrate oxidation: ΔGº’= +48.2
kJ/mol, but ΔG' = -8.9 kJ/mol at 10−5 atm hydrogen )Because of their higher affinity for
hydrogen, sulphur or nitrate reducing bacteria can lower the partial pressure of hydrogen
at the site of butyrate oxidation more rapidly and to a greater extent in the biofilm matrix
( see Lovely and Godwin 1988) increasing the potential growth rates of butyrate oxidising
bacteria. Methanogens appear to be unable to consume hydrogen below partial
pressures of 6.5 Pa ( Loveley 1985).The threshold partial pressures of a number of
methanogens has recently been measured and varied from a Pa of 1to 4.7 ( Kim 2012).
The most dominant methanogen in the rumen ( Methanobrevibacter spp )were reported to
have a H2 threshold of Pa 4.7
In support for this suggestion that butyrate oxidizing organisms similar to
Syntrophomonas wolfi have been isolated from the rumen (McInerney et al 1981 )and
have been shown to oxidize butyrate to acetate when hydrogen partial pressures are
maintained extremely low by co-culture with methanogens( Lovely and Godwin 1988).
The same organism is known to also oxidize propionate to acetate The situation is
complicated further by the fact that syntrophic propionate-oxidizing bacteria appear to be
able to reduce sulfate also (see Schink 1997) and maybe nitrate( Moura et al 2007).
Nitrate having a much higher affinity for electron capture then carbon dioxide could
maintain the hydrogen partial pressure low enough to allow these organisms to proliferate
and explain the lowered net production of butyrate when nitrate replaces urea in these
diets. These organisms also obtain ATP for growth in these reactions. (Schink 1997)
It has been calculated that the rate of organic substrate degradation in H2-syntrophic cocultures is dependent on the efficiency of the hydrogen consumer to use low
concentrations of hydrogen (see Corde-Ruwisch et al 1988). In light of these observations
the terminal electron acceptor in anaerobic systems may be the limiting factor for the rate
of substrate oxidation. This is in accordance with the observation that syntrophic
cocultures grow more rapidly with sulfate reducers than with methanogens as
H2,scavengers (Boone and Bryant 1980; McInerney et al. 1981) and therefore growth of
S wolffi with nitrate should be greater then with sulphate or carbon dioxide as terminal
hydrogen acceptors . In support of this concept the H2 concentrations associated with the
specified predominant terminal electron-accepting reactions in bottom sediments of a
variety of surface water environments were: methanogenesis 7 to 10 nM; sulfate
reduction, 1 to1.5 nM and for nitrate reduction, less than 0.05 nM ( Lovely and Godwin
1988)
The successful hydrogenotrophic organism in any anaerobic system is the one that keeps
the hydrogen partial pressure below the level that is necessary to allow hydrogen uptake
by competitors. Sulfate-reducing bacteria should therefore have lower H2 threshold levels
than methanogenic bacteria. In fact, it has been demonstrated that thresholds of
hydrogen oxidation were about one order of magnitude lower in sediments which
contained sulfate besides bicarbonate as electron acceptor (Lovley et al. 1982). And
threshold levels for nitrate are extremely low (Cord-Ruwisch et al 1988; Kim 2012)
indicating that nitrate would establish the most favorable growth conditions for syntrophic
metabolism of butyrate to acetate.
Calculating the potential additional hydrogen load through syntrophic metabolism in the
rumen
29
The NRB ( which are probably also capable of reducing sulphur)are able to utilize a
number of organic compounds with the production of hydrogen providing substrate for
methanogens and carbon intermediates for the synthesis of cells. These electron
donating reactions are shown below and provide an explanation for the changes in VFA
production patterns brought about when nitrate is introduced into a diet( Farra and Satter
1971)
Propionate → Acetate: CH3CH2COO- + 3H2O → CH3COO- + H+ + HCO3- + 3H2
Butyrate → Acetate: CH3CH2CH2COO- + 2H2O → 2CH3COO- + H+ + 2H2
Based on the potential efficiency of conversion of nitrate to ammonia and therefore
electron capture, where 100g nitrate should reduce methane production by 25.8g. If the
apparent efficiency is 60% then the additional hydrogen production that would account for
this is 10.32 g methane or 0.645M methane. Four mole hydrogen are required to produce
1 mole methane so additional hydrogen production would be 2.58 mole.
This hydrogen ( the unaccounted 2.58 mole) would be produced when 0.86mole of
propionate is converted to acetate or 1.29 mole of butyrate is directly converted to
acetate. This appears to be a tiny fraction of the potential propionate or butyrate
production rate. For example Sharpe et al (1982)found in cattle fed concentrate( corn
ground or whole ) based diets that the production per day from a diet based largely on
ground corn was, acetate 34mole/day, propionate 16 mole per day and butyrate 6.3
mole/day The cattle were fed 259g ration DM hourly and consumed 6.2 kg/day.
From 7 publications where interconversion of VFA in the rumen have been calculated
from isotope dilution studies 0.45 to 15.4% of acetate is produced directly from propionate
and 1 to 21 % of the acetate was directly produced from butyrate in the rumen,providing
evidence for syntrophic oxidation of propionate and butyrate to a limitred extent under a
variety of feeding conditions ( see Schink 1997) None of these studies however included
nitrate as a component of the diet but the direct utilisation of these VFA could have been
attributed to the activity of SRB/NRB that can utilise a diverse range of substrates(Muyzer
and Stams 2008).
Farra and Satter (1971) showed tha the ratio of VFAs was more dominated by acetate
after adaptation of dairy cows to a nitrate based diet. Following adaptation to nitrate in a
feed the proportions ( m-mole/100m-mole total VFA) of these acids changed as follows
for acetate 62.3 to 80.2(increase by 22.3%);propionate 19.6 to 14.7 ( decreased by
25%):butyrate 16.1 to 5.0(decrease of 69%). More recently Nolan et al (2010) observed a
similar trend in VFA proportions in rumen fluid from sheep fed oaten hay in equal amounts
hourly when nitrate replaced urea the acetate to propionate ratio increased markedly
from 3.22 to 4.28 and butyrate proportions were also reduced. In line with an increased
concentration of total VFA .In the research of Van Zijderveld et al ( 2010) where urea
was replaced by nitrate the reported VFA levels and proportions were not different in
sheep fed nitrate or urea but the actual VFA concentrations were measured some 24
hours after the last feed had been ingested . This last observation may be misleading
since Farra and Satter (1971)observed an increase in the acetate to propionate ratio in
rumen fluid VFA and a fairly rapid fall in butyrate proportions in the rumen fluid of cows
following consumption of feed containing nitrate, both of which returned to ‘normal’ when
the nitrate was apparently fully reduced to ammonia. Hulshof et al (2012) reported a
small increase in the proportion of acetate relative to propionate but with no effects on %
butyrate in the total VFA present in rumen fluid of cattle fed nitrate in a sugar cane /maize
silage diet where nitrate lowered methane production by 32% at an apparent efficiency of
30
87%. Clearly the diet has considerable influence on the microbial ecology of the rumen
and
therefore the response to replacing urea with nitrate as a fermentable N source. However
it does appear that this change in the microbial ecology leads to higher levels of hydrogen
production which increases the requirement for high affinity electron acceptors. In the
past a change in the proportions of VFA is attributed to the direction of carbohydrate
fermentation into either the more or less reduced VFA and the concept of VFA being
metabolized in the rumen is new. Generally metabolism of VFA is associated with slow
growth of organisms more associated with sludge fermentation. However it is emphasized
here that the nitrate reducing organisms appear to have growth rates compatible with
maintaining high population densities in the rumen and probably associated in the biofilm
mode of digestion that represents the functioning of the rumen.
The slow growth of most secondary fermentative organisms often prevents their growth in
the rumen because of the high turnover rate However, an organism termed NASF-2
which is a strictly anaerobic, non-spore forming, acetogenic, proton-reducing, butyrateoxidizing bacterium which resembles Syntrophomonas wolfei under optimum conditions
of low partial pressure of hydrogen grew exponentially and had a doubling time of
10hours ( Dwyer et al 1988) which would be compatible with substantial growth in the
rumen where the feed particulate turn over rate approximates once per day or a turnover
time of about 17hours.
The major conundrum of nitrate feeding in ruminants
From the above discussion undoubtedly ruminant can accept quite high concentrations of
nitrate in their diets and when the basal diet is low in true protein. This nitrate nitrogen is
used as a fermentable N source for microbial growth in the rumen. The conundrum is that
nitrate toxicities occur under grazing conditions and where water is contaminated which
would seem to make nitrate a too risky feed ingredient. This was well summed up by
Farra and Satter (1971) as follows;
The use of nitrate to manipulate rumen fermentation probably has little future
if for no other reason than its potential for causing nitrate toxicity. This study,
along with others point out, however, that large amounts of nitrate may be fed
to ruminants without apparent toxicity symptoms. It is difficult to reconcile
this with reported nitrate toxicity in cattle consuming water containing from
45 to200 ppm nitrate. Nitrate consumption in the present studies would be
equivalent to the nitrate intake of' a cow drinking water with 4,000 ppm
nitrate, with an average water consumption of 60 liters per day( modified
from Farra and Satter 1971).
Similar assertions have been made from time to time indicating that nitrate poisoning is
brought about when nitrate is consumed in feed by ruminants under some circumstances.
The obvious answer is that under toxicity conditions there are interacting elements that
create the problem of nitrite spilling and methaemoglobinaemia. From the literature it
31
becomes apparent that in some way sulphur is involved and in the remainder of this paper
evidence is drawn together to attempt to explain or high light the interactions which may
lead to nitrite accumulation in the rumen.
Effects of molybdenum (Mo) on anaerobic sulphur/nitrate metabolism
If the thesis is correct, that it is the availability of hydrogen sulphide in the medium that
allows the anaerobic microbial consortium to convert nitrate to ammonia without release
of the intermediate nitrite, then any other factor that inhibits hydrogen sulphide production
should similarly affect nitrate metabolism. A series of observations indicates that
molybdenum ( Mo) has a major role to play in both sulphur and nitrate metabolism in the
rumen).Tillman et al (1965) demonstrated that Mo had marked effects on nitrate
metabolism in the rumen of sheep fed purified diets with nitrate as the sole source of
fermentable N. Two groups of sheep were used and one group had the Mo content of the
diet increased by 1ppm. 500g of each diet were each introduced into the rumen of 12 h
fasted sheep that had been acclimated to the diets. Even though the clearance of nitrate
was very similar ( Figure 13) on both diets, the additional Mo apparently increased nitrite
accumulation (peak nitrate) in rumen fluid 5-6 fold (see Figure 14). The amount of nitrite
appearing in rumen fluid is only a fraction of the amount of nitrate added to the rumen
(Figure 13) indicating that this production may be sourced from a small population of
microorganisms (possibly NR-SOB ). The minimal conversion of nitrate to nitrite is a
similar observation to that of (Lewis 1951) who injected nitrate directly into the rumen.
Mo is essential for microbial growth but also appears to be toxic or inhibitory to
some microorganisms, particularly the SRB (Bryden and Bray 1972; Huisingh et al 1975;
Gawthorne and Nader 1976; Spears et al 1977). Molybdate (MoO4) has been proposed
as an analog of sulphate that blocks the sulphate activation step (catalyzed by ATP
sulphurylase) in SRB (Oremland and Capone, 1988). Taylor and Oremland (1979)
showed that MoO4 specifically inhibits SRB in pure culture and in sediments (Oremland
and Silverman, 1979; Sorenson et al 1981)
Loneragan et al (1998) reported that supplementing sodium molybdate lowered hydrogen
sulphide concentrations in the rumen gas cap of cattle fed a high sulphur diet. Infusion of
molybdate into the rumen of sheep increased the concentration of sulphate in rumen fluid
from 2.2micro g S/ml to 7.7 and decreased the rate of reduction of sulphate to sulphide by
50%.( Gawthorne and Nader 1976). Although the rate of sulphide production was slower,
the concentration of sulphide in rumen contents was increased indicating that hydrogen
sulphide absorption may have also been decreased by Mo (Gawthorn and Nader 1976).
Bracht and Kung (1997) showed that MoO4 at concentrations greater than 10 ppm
reduced sulphide production in ruminal fermentations lowering sulphide production in
both the liquid and gas phase. Kung et al (2000) showed a 77% reduction of hydrogen
sulphide production with 25ppm in an in vitro ruminal fermentation when the sulphur
content of the diet was 2.5 times the recommended level of 0.4% of the dry matter. Under
the in vitro conditions, MoO4 appears to be a specific inhibitor of SRB because there was
no effect on methane or hydrogen production (Bracht and Kung 1997). The increased
nitrite production following the addition of Mo to a diet containing nitrate (Figure 14) is
consistent with an inhibition of nitrite reduction to ammonia in a minor population of
organisms ( i.e. NR-SOB) because of a smaller sulphide pool in the rumen when SRB
activity is limited or inhibited.
The effects of Mo on ruminal sulphide production in sheep, and its role in increasing
nitrite production from dietary nitrate( Tillman et al 1965 ) strengthens the argument that
nitrite build up when nitrate is administered to the rumen is associated with an inhibition of
32
SRB. This strengthens the research conclusions that nitrate supplementation increases
sulphur requirements of growing lambs ( Sokolowski et al 1969). However, the addition
Figure 13.. 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)
Figure 14. 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 The basal diet
was supplemented with an extra 1 mg Mo /kg dry matter (Tillman et al 1965).
33
14
Rumen fluid nitrite (mM)
12
10
8
6
4
2
0
0
2
4
6
8
10
Time after introduction of 500g feed into the rumen (h)
Rumen fluid nitrite Basal-no Mo
Rumen fluid nitrite-Basal-plus Mo
Indirect effects of nitrate and molybdenum in the feed on digestibility of forage.
Indirect effects of nitrate on the rumen ecosystem may be a critical issue if nitrate is to be
used as a major fermentable N source for inclusion in poor quality forage based diets fed
to ruminants. Marais et al (1988) have pointed out that nitrite and nitrate may more subtly
affect animal production through detrimental indirect effects on rumen organisms,
particularly toxic effects on cellulolytic organisms (Hall et al 1960; Marais et al 1988) that
may lower the apparent digestibility of forage or pure cellulose in vitro ( Spears et al
1977). Marais et al (1988) demonstrated a lowering of digestibility of Kikuyu grass in vitro
when nitrate was added to the culture medium. They suggested that the reduction in
digestibility could be the result of nitrite accumulation in the in vitro cultures inhibiting the
electron transport system of certain cellulolytic microbes and limiting ATP generation and
growth at nitrite levels as low as 0.29mM.
Spears et al (1977) showed that in an in vitro fermentation of rumen fluid from cattle fed
orchard grass 0.4 or 0.8% nitrate-nitrogen depressed cellulose ( purified wood cellulosesolka floc) digestion and increased the requirement for both sulfate and
sulfide. Depression was greater with 0.8% nitrate-nitrogen. In the presence of nitrate,
sulfide was superior to sulfate in promoting cellulose digestion suggesting inhibition of
sulphate reduction to hydrogen sulphide. When 4 or 8 ppm molybdenum were added to
the incubations, increasing concentrations of both sulfate and sulfide were required to
obtain maximum cellulose digestion. Molybdenum additions increased both the sulfate
and sulfide requirement for maximum cellulose digestion. These data focus the apparent
effect of nitrate and molybdenum on their effect on sulphur requirements and particularly
the availability of sulphide in the rumen on microbial activity.
34
According to the thesis presented here and the research of Spears et al (1977) a more
likely explanation of the observation of Marais et al (1998) could be that nitrate
metabolism inhibits the conversion of inorganic and organic sulphur, reducing the pools of
S intermediates required for cellulolytic microbes. In the rumen this would particularly
apply to the phycomycetous fungi that are normally present in the rumen (see Gordon and
Phillips 1998). When herbage diets with a low content of S have been fed to ruminants,
anaerobic fungi were either apparently absent from the rumen or their numbers were
greatly reduced and feed intake and digestibility were low. Supplementation of these diets
with several different sources of S allowed fungi to proliferate in the rumen and resulted in
increased voluntary feed intake. At the same time, there was little or no change in the
ruminal populations of bacteria and ciliate protozoa due to dietary supplementation (Akin
et al. 1983; Gulati ef al. 1985; Morrison et al. 1990) Diets low in S (including straw,
digiteria, spear grass and maize stover forages) have been successfully supplemented
with S which greatly increased the population density of anaerobic fungi in the rumen and
forage intake by sheep (see Gordon and Phillips 1998). Different strains of anaerobic
fungi grown in vitro required reduced forms of S (Orpin & Greenwood,1986; Phillips &
Gordon, 1991) indicating the need for reduction of supplementary sulphate to sulphide
before it would be available for these organisms. The level of molybdenum in the diet may
then dictate the severity of the inhibition of hydrogen sulphide production
Kikuyu, is a grass that under ideal conditions has about the same content of protein as
Perennial Rye Grass but the S-amino acid content is lower by 68%( methionine) and
57%(cysteine) (Reeves et al 1996) and therefore the sulphide pool in the rumen is likely
to be low in livestock on this forage and therefore more likely to be lowered below a
critical level by additional nitrate. This may explain the more recent results where nitrate
as a major proportion of the fermentable nitrogen in a diet fed to ruminants has had no
effects on dry matter digestibility as there has been additional sulphur above the
recommended levels added with the nitrate.
In support of the above conclusion, de Villiers and Ryssen (2001) working with Perennial
Rye Grass grown under increasing N fertilization ( and increasing nitrate content in its dry
matter) found that in vitro dry matter digestibility and intake of the forage by lambs and
sheep in pens were not different even though nitrate intake increased. The apparent
relationship between a low S content of pasture and a declining ruminal fungal population
did not apply to a ryegrass pasture in Scotland (Millard et al. 1987) where anaerobic
fungal populations were higher in sheep consuming an unfertilized hay (containing 0.9 g
S/kgDM)) than in those fed a hay prepared from fertilized pasture (2-4 g S/kg DM). The
additional S of the fertilized pasture was predominantly contained in the sulphate and
non-protein fractions. This was similar to the S distribution in the fertilized D. pentzii
where fertilization of the soil on which it was grown increased S content of the plant and
this in turn increased fungal population in the rumen of sheep fed the hay. In this case the
additional S was found in the soluble, non-protein fraction (Akinet al. 1983) The form and
distribution of S in herbage of low total S content appears to be as important as the total S
content in determining the size of the fungal population in the rumen. Clearly this is an
area for more research particularly where the level of molybdenum is considered.
Practical observations linking dietary Mo to nitrate poisoning.
In general nitrate poisoning in livestock is associated with grazing of high quality pasture
from highly fertile soils. High protein pastures are produced when either N fertilized or
have a high proportion of legumes. Low Mo in soils is associated with poor pasture growth
and quality (Savage 1982 ). Legumes in particular need Mo fertilization particularly on
35
acid soils to ensure root nodulation for bacterial N fixation and for growth of the plant. Mo
is often deficient in pastures as it is removed in grazed plant materials, bound in the soil
matrix or is leached. Large tracks of grazing country are now fortified with Mo, provided as
a seed coating or as molybenised super phosphate, the latter particularly in Australia and
New Zealand. In Australia particularly, in the rain fed pasture growing areas, super
phosphate is applied containing Mo. The amount applied is often calculated to supply
plant requirements for Mo for between 3 to 10 years. Under these circumstances the Mo
content of pasture can vary widely and be particularly high soon after fertilizer application,
possibly exposing grazing animals to excessive Mo in consumed forage which at times
reaches toxic levels.
Figure 15. Relationship between nitrate and crude protein content of perennial rye grass
(Lovett et al 2004)
4
y = 0.032x - 4.08
3.5
R2 = 0.94
Nitrate (g/kg DM)
3
2.5
2
1.5
1
0.5
0
100
120
140
160
180
200
220
240
260
280
300
Crude protein (g/kgDM)
The nitrate content of pasture increases when total crude protein content exceeds a
critical level( see Eckard 1990). This occurs whether manure, nitrate or urea fertilizers are
applied and whether super phosphate is used to promote legume based pastures. The
relationships between crude protein in rye grass and nitrate content is shown in Figure 15
and the effects of manure fertilization of mixed temperate pastures on the amount of
nitrate nitrogen in crude protein is shown in Figure 16. The relationship between nitrate
and crude protein in alfalfa (lucerne) re-growth produced in growth chambers is also of
interest since lucerne appears to have a much greater accumulation of nitrate at higher
crude protein contents then the grasses (see Figure 17). This may be important as
lucerne has been the basal diet where nitrate metabolism of ruminants has been studied
On the other hand, perennial rye grass appears to accumulate nitrate at lower crude
protein content (Figure 15). Overall the three Figures15,16and17 indicate that the higher
the soil fertility and the conditions for growth the greater the likelihood of nitrate
accumulation. The point being made is that because of the key role of Mo in plant growth,
these high levels of crude protein (and nitrate) must be related to, among other issues,
the Mo content of the organic matter.
36
The sulphur in plants, high in crude protein is largely in the S-amino acids in proteins
which are mainly contained in Rubisco( 60% total). This protein is highly soluble and
rapidly hydrolyzed to amino acids which in turn are fermented. Cysteine is desulphurated
with the production of hydrogen sulphide. The extent of hydrogen sulphide production
from various S sources is indicated by its concentrations in the gas cap in the rumen
which is in equilibrium with that in rumen fluid (see Figure 18 and 19)
Nitrate-N in Herbage herbage(g/100g DM)
Figure 16. Relationship between percentage crude protein and nitrate nitrogen in herbage
(After ap Griffiths (1960).
0.35
y = 0.0007e 0.1934x
R2 = 0.8583
0.3
0.25
0.2
0.15
0.1
0.05
0
12
17
22
27
32
Crude Protein in herbage (g/100g DM)
It is also recognized that nitrate accumulates in good quality pastures when
photosynthesis is slowed by adverse environmental conditions such as restriction in soil
moisture or reduced solar energy as occurs when cloud cover increases. Under these
circumstances, nitrate poisoning often results in ruminants grazing such pastures. The
suggested effect may be the result of sudden high nitrate intake and from the above
discussion which could be aggravated if Mo content of the pasture is high. The level of Mo
in leguminous forages is related to the soil pH. Simply liming lucerne pastures can have a
large effect increasing molybdenum uptake and tissue content of molybdenum
Figure 17. Relationship between nitrate and crude protein in alfalfa grown under
increasing N fertilizer application (Cherney et al 1994)
37
8
y = 0.077x - 15
R2 = 0.86
Nitrate-N (g/kg DM)
7
6
5
4
3
2
1
0
200
210
220
230
240
250
260
270
280
290
Crude protein (g/kg DM)
Hydrogen sulphide in rumen head-space
gas(p.p.m.)
Figure 18. Effects of iso-S-supplements on the concentration of hydrogen sulphide in the
rumen head space gas of dairy cows. The cows had been without feed for 4hr prior to the
administration of the S-source ( Dewhurst et al 2007)
600
500
400
300
200
100
0
0
20
40
60
80
100
120
140
160
Time from injection of S-source(minutes)
Control
Cysteine
Methionine
Sodium sulphate
.
Linking Sulphur and Mo content of a diet to incidence of nitrate poisoning.
38
Sulphide in gas space of the rumen is in equilibrium with that in rumen fluid ( see Figure
19) and in dairy cows hydrogen sulphide levels in the gas space was zero 4 hours after
consuming a meal indicating the rapid turnover rate of the rumen fluid sulphide pools.
Figure 19. The concentrations of hydrogen sulphide in rumen fluid and gas head space
(after Gould et al (1997)
Concentration of sulphide in rumen
-3
head-gas space( ppm x 10 )
10
9
8
y = 0.022x + 0.52
R2 = 0.55
7
6
5
4
3
2
1
0
0
50
100
150
200
250
300
350
Concentration sulphide in rumen fluid (micro-mole /L)
The research of Dewhurst et al (2007) produced the un expected finding that the
hydrogen sulphide in the rumen gas pool increased following a meal of high quality
perennial rye grass but was considerably less then that in cattle of similar intake of dry
matter from a white clover pasture (Figure 20). The clover was much higher in crude
protein than perennial rye grass (25.8% v 16% CP) and therefore contained much more
readily fermentable protein. This suggests that either there is much less hydrogen
sulphide produced from clover or more of the hydrogen sulphide was metabolized in the
rumen from clover relative to perennial rye grass. Dewhurst et al (2007) suggested that
the lower level of hydrogen sulphide might be a result of diversion of sulphur to the
production of thiocyanate in the detoxification of the cyanide generated in the rumen from
cyanogenic glucosides present in white clover. Whilst this is a possibility it is also possible
that the high quality white clover would have contained considerably higher levels of Mo
and nitrate with the potential to inhibit sulphur reducing organisms. It seems that the likely
effect was a result of a reduction in hydrogen sulphide production owing to a high intake
of nitrate or Mo or both The nitrate content of the white clover fed to the cows is not given
but the CP was 25.8% CP . Extrapolating from Cherney et al (1994) the nitrate content
could have been as high as 0.5% N. but no values are available for the Mo content.
However, such a high crude protein concentration in a legume would indicate a high
availability and uptake of Mo essential for bacterial nitrogen fixation in clover root nodules.
39
Hydrogen sulphide in rumen heas space
gas(p.p.m.)
Figure 20. The effects of a meal ( 9.5kgwet weight) of white clover or perennial rye grass on the
concentration of hydrogen sulphide in the rumen head space gas of a dairy cows ( Dewhurst et al
2007)
800
700
600
500
400
300
200
100
0
0
30
60
90
120
150
180
210
Time from 4hour post fasting
white clover
Ryegrass
Is there evidence for nitrate poisoning being increased by dietary Mo?
Cheng et al (1985) confirmed the ability of rumen microbes to reduce nitrite and nitrate
was induced by slowly increasing the amounts given to cattle over time. In their studies a
K-nitrate drench was increased every three days from 0 to 0.16 to 0.32 to 0.48 to 0.54 g
K-nitrate/kg body weight of cows (800kg live weight) fed lucerne cubes. The ability to
reduce nitrate and nitrite by the cow’s rumen fluid was about 0.1 of that in the sheep fed
non legume diets. The ability to reduce nitrate in cows increased only slowly as each
drench level was stepped up at 3 day intervals. At a drench rate of 0.54 g K-nitrate/kg
live-weight, all cows died with methaemoglobinaemia on the day following drench being
given. This was a major set back for research on nitrate utilization by ruminants but in
this study the toxicity may have stemmed from the lucerne hay. Lucerne can vary in Mo
and nitrate contents as discussed above depending on the soil Ph and the fertility of the
soil on which the lucerne was produced. A possible explanation for the mortality rate from
methaemoglobinaemia could have been related to a high Mo intake on the basal diet,
which from the hypothesis proposed here would inhibit hydrogen sulphide production and
establish a spilling of nitrite by NR-SOB. The level of NR-SOB may have been enhanced
by the presence of nitrate in the lucerne fed prior to the start of the drenching program.
Unfortunately the composition of the lucerne was not recorded.
40
Is the major group of sulphur reducing bacteria in the rumen( without nitrate in the diet)
also facultative nitrate reducing bacteria?
It is possible in Cheng and colleagues( Cheng et al 1985) research that the NRB activity
in the rumen as the nitrate load was increased, belonged to the SRB. In general SRB
have the ability to reduce a number of different terminal electron acceptors and use a
wide range of organic and inorganic compounds ( see Moura et al 2007) and the
population density of these organisms could have been suppressed by Mo should it have
been present in high amounts in the animals fed only lucerne. This seems feasible since
as already stated, the nitrate reducing activity of the cow’s rumen fluid was only 0.1 of that
recorded in sheep. The suggested scenario based on this hypothesis is that the hydrogen
sulphide concentration in the cow’s rumen fluid would have been low so inhibition of SRB
by nitrate again leads to sulphide deficiency and the NR-SOB produce nitrite as the end
product. If the lucerne had been fed for sometime to the cows, prior to administration of
nitrate, then the populations of SRB that preferentially take up nitrate when that is
available (see Moura et al 2007) could have been much reduced in agreement with the
very low capacity of the cows rumen to metabolize nitrate (10% of that recorded in sheep)
In contrast to this in studies with sheep fed purified diets with nitrate as a sole fermentable
N source an additional 1 ppm of Mo in the feed increased both ammonia and nitrite
production rate in sheep force fed a portion of their diet (Tillman et al 1965). However the
sheep had been on the lower Mo diet continuously and obviously the NRB and SRB that
evolved under the feeding conditions may be quite different as against feeding systems
where nitrate is suddenly introduced. These results are suggestive of higher true NRB
and NR-SOB populations since the animals were well acclimated to nitrate and SRB
activity would have been low and had an increased sulphur requirements as shown by
Sokolowski et al (1969) This observations also suggests that when nitrate is a high
proportion of the crude protein in a diet of ruminants, that had been slowly adjusted to the
diet, there is a permanent change to bacteria that are obligate nitrate reducers therefore
it may not have been possible to maintain SRB and hydrogen sulphide production in order
to prevent nitrite accumulation ( according to the hypothesis posed here).
Some evidence of Mo accumulation by legumes grown under high fertility soil conditions
Further observations on the variability of Mo in forages consumed by ruminants
Acidic soils, are low in available phosphorus and Mo. Large areas of grazing country,
particularly in Australia and New Zealand, are fertilized with super phosphate containing
Mo at irregular intervals that extend from once every few years to up to once every
10years. Pasture growth, and particularly its protein content, is consistently increased
depending on rain fall. In leguminous pastures Mo is required in greater amounts for
nodulation and nitrogen fixation. Liming (2000mg/kg soil) and addition of both phosphorus
and Mo can affect the content of Mo in leaf materials. The concentration of molybdenum
in clover in Victoria,Australia, ranges from less than 0.1 to more than 4 mg/kg DM, with
60% of samples analyzed containing 0.5 mg/kg DM or less (Brown 1982) The
recommended requirements for Mo for ruminants is about 0.1mg/kg forage DM. The
effects of added P and Mo to soil on the Mo content of red clover is shown in Figure 21
In a study with soil in pots, Ribera et al (2010) found the concentration of Mo in red clover
leaf tissue increased as Mo application rates were increased both, in non limed and limed
soil( Figure 21). Similar results have been reported previously by Adams (1997), Vistoso
(2005) and Lopez et al. (2007). These results indicate that Mo concentration in leaf
shoots was significantly correlated (r = 0.579; P < 0.05) with Mo availability in the soil
41
(Figure 21).Shoot Mo concentration in red clover plants untreated with Mo and P was 0.29
mg /kg DM in non limed soil and 0.39mg/kg DM in limed soil. In limed soil the effect of P
application to the soil on shoot Mo concentration was higher than those detected in non
limed soil. With P supply of 200 and 400 mg P/ kg in limed soil, shoot Mo concentrations
increased to 4.9mg and 7.8 mg/ kg DM, respectively. “Normal” levels of Mo in an animals’
diet are below 5.0 mg/kg DM and the requirement is 0.1 mg/kg DM
Lucerne also responds to liming of acid soils and application of Mo- fertilizers. John et al
(1972) examined the changes in the Mo composition of lucerne which resulted from the
application of 4 lime treatments to 7 acid soils. The trials were run in a growth chamber.
Concentration of molybdenum in lucerne foliage increased progressively with increased
lime application to most soils and the concentration increased, on average, from 5.8
mg/kg to 16.3 mg/kg dry matter at the optimal lime application rates. The highest levels
found in lucerne aerial parts were 40 mg/kg DM (Table 1).
Molybdenum in red clover shoots (mg/kg DM)
Figure 21 . The effects of adding P and Mo to an acid Andisol (that had received a
dressing of lime) on the concentration of Mo in red clover shoots ( Ribera et al 2010 )
9
8
7
6
5
4
3
2
1
0
0
200
400
Amount of Phosphorus added to soil (mg/P/kg soil)
Zero Mo added to soil
0.58 mg Mo/kg soil
0.96 mg Mo/kg soil
From the above and depending on the fertilizer practices, Mo levels in legume foliages
can vary from 0 .29 mg/kg DM up to 8 mg/kg/DM with even higher levels in lucerne A high
Mo-clover may explain the apparent very low H2S content of the rumen gas cap in cattle
fed clover as against perennial rye grass (Dewhurst et al 2007) and it also indicates that
lucerne fed animals could develop similar conditions where Mo concentrations are high in
the foliage The concept being that the higher content of Mo in the clover reduces the
activity of SRB in the rumen and when the H2S pool is lowered in the presence of nitrate,
nitrite is spilled by the NR-SOB that oxidize hydrogen sulphide and reduce nitrite to
ammonia. This is possible where high quality lucerne chaff is fed to ruminants and could
explain the results of Cheng’s research in which a cattle were killed by a relatively small
42
dose of nitrate given orally (see Cheng et al 1985). If the lucerne came from a soil either
naturally high in Mo or where recent liming had occurred or both, then nitrite production
could have been enhanced. In studies of nitrate metabolism in the rumen Tillman et al
(1965) showed that increasing the Mo content by a mere 1mg/kg of feed dry weight
increased the peak nitrite concentration in the rumen from 2.5 to about 13 mg nitrite
N/100ml rumen fluid and peak nitrite in blood from about 2 mcg/100 ml to about 30
mcg/100m blood.
Table 1. The effects of increased lime application to soil growing lucerne on the
concentration of Mo (mg/kg DM) in lucerne foliage grown on a variety of soils(John et al
1972)
Soil series
No Lime
40% base
70% base
100%
saturation
saturation
base saturation
Ladner
7.1
7.5
24.7
25.4
Hazelwood
2.8
3.6
8.7
11.3
Monroe
6.0
5.2
4.4
10.6
Fairfield
4.4
4.8
9.1
12.1
Wheatcom
5.6
10.0
8,3
9.5
Marble Hill
12.5
13,5
29.7
40.0
Annis
2.4
1.2
5.2
5.2
Average
5.8
6.6
12.9
16.3
Do any rumen organisms act in the same way as NR-SOB from terrestrial anaerobic
ecosystems?
Evidence for the presence of NR-SOB in the rumen
Wolinella succinogenese was first isolated from a bovine rumen by Wolin et al (1961). It
has been extensively studied because of biochemical similarities with pathogenic
organisms belonging to the Helicobacter and Campylobacter species. It has been shown
to metabolize nitrate to nitrite by the dissimilatory pathway and nitrite to ammonia by
respiratory ammonification coupled to the generation of an electro-chemical proton
potential across the membrane and ADP phosphorylation to ATP (Bokranz et al 1983;
Simon 2002). The proposed pathway of nitrite ammonification in NR-SOB is shown below
(see Simon et al 2002)
3HS- +NO2- +8H+  3S0 +NH4++2H2O -----------------------------------------Equation 7
43
W succinogenese has physiological similarities to the NR-SOB, Sulphurospirillium
deleyianum isolated from water from oil wells treated with nitrate to prevent hydrogen
sulphide release. These similarities include almost identical three- dimensional structures,
location of heme groups, similar substrate and product channels and similar architecture
of the catalytic site (see Simon 2002). These similarities suggest that they have similar
metabolic roles; S deleyianum having the capacity to reduce nitrate and oxidize sulphide
(that is it belongs to the NR-SOB family) suggests that W succinogenese can also be
considered to belong in the same group. Growth of W. succinogenese has been
demonstrated with sulphide as an electron donor and fumarate as an electron acceptor
(Macy et al 1986). Intact cells of W. succinogenese grown with formate and nitrate
catalyze electron transport from formate to nitrate at extremely rapid rates (Simon et al
2002). As discussed earlier, W. succinogenese and Desulfovibrio species are rumen
organisms that can be readily isolated from the rumen of a wide range of animals on
different feeds and diverse rumen ecosystems albeit their population densities ( often
(around 10-2 / ml )are small relative to the total bacterial population in the rumen( around
10-12./ml).However, this maybe misleading as only fluid phase microbes were counted
and it is highly probable that the majority of these organisms would be sessile in a mixed
biofilm matrix in the rumen. The available evidence appears to support the concept that
NR-SOB are present in the rumen and the diversity of substrates they are able to use
allow them to be permanent members of most rumen ecosystems. It is also necessary to
hypothesize that these organisms are capable of rapidly adjusting to nitrate when it is first
introduced into the rumen.
It seems likely that the population of NR-SOB in the rumen would be controlled by the low
concentration of sulphide (approx. 0.1mM) but it is more logical to assume that higher
populations would exisit closely associated sulphide producing SRB in the biofilm matrix
that forms on feed materials in the rumen. The presence of NR-SOB would be
advantageous as they would maintain pools of various oxidized and reduced forms of
sulphur for the use of the mixed microbes in the rumen ecosystem which could be
depleted rapidly by the rate at which sulphide is protonated and lost to the gas phase in
the rumen. As discussed earlier, accumulation of nitrite in the rumen, when a quantity of
nitrate is ingested or administered, would then be dependent on pool size of the NR-SOB.
This in turn would be associated with the availability of protein (S-amino acids) and other
sources of sulphur. The nitrate to sulphur ratios in the rumen during experiments where a
nitrate load is placed on the rumen are extremely high. The initial nitrate levels vary from
20-120mM (see Table 4) and the hydrogen sulphide levels may be 0.1-0.3mM (cf. up to
2.5mM hydrogen sulphide in oil field’s water). The concentration of nitrate in the rumen in
the clearance studies are much greater than the levels used to control sulphide
production in oil field waters (10mM) but the oil fields medium would be much lower in
organic matter and thus in supply of electron donors.
The numbers of NR-SOB in the rumen may be limited by both S and nitrate content of a
diet.The numbers of W succinogenese in the rumen is effected by nitrate inclusion in a
diet. Cell numbers of known species of nitrate- and nitrite-reducing bacteria,
Selenomonas ruminantium, Veillonella parvula and Wollinella succinogenes, in the rumen
of goats (25–30 kg) were estimated by competitive polymerase chain reaction (PCR)
(Asunama et al 2002). The number of S. ruminantium ,V. parvula W. succinogenes
5.6 × 107,6.7 × 103 and 1.6 × 103 cells/ml respectively in rumen fluid in goats fed a highroughage diet. . The number of W succinogenese was below the detectable level when a
high-concentrate diet was fed, but was significantly increased by feeding a high-roughage
diet. However when considering that rumen microbe population density may be as high
as 1012 cells /ml then the concentration of W succinogenese is extremely low. An NR-SOB
44
in the rumen would be controlled by both N and S content of the feed. The population
density is probably at their highest when high protein feed is given. The point being made
is that the pool size of the suspected NR-SOB( W succinogenese) is small and not
increased in size by very much by nitrate alone. However, the counts reported are difficult
to put into perspective where the biofilm resident population has not been monitored.
It also appears that the amounts of organic electron donors probably affect the
interactions of the SRB, NRB and NR-SOB. The concentration of nitrate at which the SRB
re-establish sulphate metabolism to sulphide - and in NR-SOB the extent of nitrate
reduction to nitrite is then limited by the re establishment of nitrite ammonification and is
probably dependent on the fermentable substrate present.
In the oil fields studies the ratio of nitrite produced to ammonia produced from nitrate was
apparently controlled largely by the availability of electron donors which was dependent
on the concentration of lactate in the medium (Hubert and Voordouw 2007) .
Hubert and Voordouw (2007) showed that complete removal of hydrogen sulphide from a
bioreactor medium inoculated with water from an oil well, required increasing the nitrate
concentrations to 10mM at concentrations of sulphate varying from 0.75, 2 and 6 mM. In
the rumen, the clearance of a nitrate load is extremely rapid initially, but at 30-60 min
following administration into the rumen of sheep, when nitrate concentrations have been
lowered to 20-30 mM, its clearance appears to slow down( see Lewis 1951 and for a
more complete coverage Leng 2008a). This may be interpreted as the point when SRB
activity returns, producing sulphide and NR-SOB cease nitrite production and recommence nitrite ammonification. The strongest support for this concept is that the
transition from a fast to a slow rate of nitrate clearance appeared to occur at the same
nitrate concentrations in the rumen in sheep administered intra-ruminally with widely
different amounts of nitrate (see Figure 5)( see also Leng 2008a).The effects of sulphate
on the pattern of methane production when nitrate or urea were fermentable N sources
(Zijderveld et al see Figures 7,8and 9) is also supportive of the proposed mechanism of
nitrite production in the rumen. It is concluded that a minor group of NRB with NR-SOB
capabilities is responsible for the release of nitrite in the rumen and that this is controlled
by the availability of hydrogen sulphide, the production of which is restricted by the NRB
that out compete SRB when nitrate is introduced unless more sulphur, both organic and
sulphate, levels are also elevated. Defining the interaction of nitrate and sulphate in the
diet is imperative but the genome of organisms such as W succinogenese are fully
described and should allow biotechnology to develop methods to prevent their
occurrence in the rumen and if the thesis is correct eliminate nitrite poisoning. Supporting
this argument is the fact that from calculated stoichiometry the amount of nitrite required
to produce significant amounts of methaemoglobin is very small relative to the amount of
nitrate that is often consumed. A 40kg sheep having a blood volume of 2 liters would have
approximately 240 g haemoglobin or .037 moles. To convert this to methaemoglobin
would require approximately O.67g nitrite which is a fraction of the nitrate that has been
injected into the rumen in experiments such as those conducted by Takahashi et al 98or
Lewis( 1951 ) . To this author this suggests that nitrite is produced by a very small
population of one species of microbes.
Overview
Global warming is becoming a primary economic consideration for most countries in the
world with governments developing legislation to limit out put of green house gases.
Within the scientific community there is recognition that it is essential that all greenhouse
gas emissions must be brought under control. Methane is an attractive target for
mitigation since it has a shorter half life then carbon dioxide and has 21 times the
irradiative forcing of a similar amount of carbon dioxide. The radiative forcing effect of
45
methane is probably going to be revised upwards with new information on the interactions
of tropospheric methane and carbon black with oxygen and a reappraisal of the half life
of methane( UNEP 2011). It appears that the value may be raised from 21 to 72 and there
appears to be a case to increase it even further. Animal agriculture produces a variable
proportion of a country’s green house gas. In general it is acknowledged that enteric
methane production from herbivorous animals is potentially manipulated. This has led to
large investment in nutritional research aimed at reducing methane by manipulation of the
diet.
Feeding nitrate to ruminants undoubtedly lowers the carbon foot print of the livestock
industries. However to be able to apply this technology there needs to be developed a
method of administration of the nitrate to the animal and minimization of a poisoning
syndrome that is associated with such systems. This will only be achieved if the
fundamental metabolism and utilization in the rumen is understood
Nitrate poisoning is due to the absorption of nitrite from the rumen of animals ingesting
nitrate in their feed. The amount of nitrite absorbed is critical as it binds haemoglobin
preventing the transport of oxygen to critical organs and tissues.
Although nitrate poisoning has been researched for a considerable time, little is known of
the microbiological changes that occur in the rumen biofilm ecosystem when nitrate is
introduced into a diet.
The microbiological changes in biofilms that that occur in sediments, oil wells and sewage
when nitrate is introduced have been subject of detailed microbiological and biochemical
studies reflecting the relative economic impact of these, particularly the problem of smell
from sewage and the souring of oil fields. However the relatively low economic effect of
nitrate poisoning in ruminants is now of much more significance because of the potential
of adding nitrate to ruminant feeds to mitigate enteric methane production. Globally
ruminants produce around 80 million tonnes of methane annually, which accounts for
about 28% of anthropogenic emissions. Recent research has demonstrated that dietary
nitrate may lower methane production from ruminants by as much as 50%. The economic
implication now become very much greater, but the uptake of the technology will be
limited unless livestock keepers can be assured that their livestock health is not
endangered. This will particularly apply to the huge number of multi-purpose ruminants
managed by small farmers in developing countries. These livestock are generally fed
agricultural by-products or forage from waste land and nitrate would have a dual role in
providing essential rumen nutrient, ammonia and acting as a high affinity electron
acceptor. Research is in progress in SE Asia to study nitrate as a source of nitrogen in
local feeds with some success already recorded (see Leng and Preston 2010).
The potential economic and ecological benefits of reducing methane production
indicates a major need for research funding .In general it has been demonstrated that
nitrate amendment of oil field, sewage and sediment ecosystems has a profound effect on
sulphur metabolism. In particular hydrogen sulphide production can be eliminated along
with reduction in methane through adding nitrate.
Extrapolating from research carried out in natural and contrived anaerobic ecosystems
could be misleading. The microbial ecosystems that exist in the rumen of animals on a
wide variety of feed and therefore substrate for anaerobic growth need comprehensive
studies. Never the less from a search of the literature some remarkable similarities have
emerged. These observations have been drawn together no matter how tenuous the
46
relationships are and a hypothesis is developed that the production of nitrite from dietary
nitrate only occurs when hydrogen sulphide in the system restricts the growth of NR-SOB.
The list of associations relevant to this hypothesis follows
1. The rumen contain SRB and NRB which increase in numbers in response to
increasing nitrate and sulphur in a diet
2. In diets apparently sufficient in S to meet recommended requirements of
ruminants , introducing nitrate caused the animal to move into a negative sulphur
balance
3. Some of the NRB and SRB appear to be able to use either nitrate/nitrite or
sulphur/sulphate as high affinity electron acceptors
4. If SRB in the rumen can use nitrate then increasing the sulphur content of a diet
will increase the potential for nitrate dissimilation to ammonia. Conversely high
dietary nitrate allows more sulphur to be incorporated in the diet which apparently
can replace the nitrate as a high affinity electron acceptor as the nitrate in solution
is depleted
5. hNRB have been demonstrated to competitively inhibit SRB
6. NR-SOB can both oxidize hydrogen sulphide and reduce sulphur
7. Wollinella succinogenese initially isolated from a bovine rumen, appears to be
able to use the same substrate as NR-SOB isolated from oil well water containing
with nitrate
8. Sulfurspirillium delayianum (the most studied NRSOB) and Wollinella
succinogenese have a range of structural and chemical similarities and the ability
to use nitrate, nitrite, sulphide and sulphate to generate ATP
9. Nitrate addition to oil well anaerobic ecosystem to control hydrogen sulphide
release appears to stimulate hNRB which inhibit SRB which lowers the sulphide
content of the media.The NR-SOB which use hydrogen sulphide appear to have a
minor role in the lowering of hydrogen sulphide
10. Nitrate added to reactors simulating sewage fermentation with adapted biofilms,,
produced hydrogen sulphide for some time after addition of nitrate but after 2 10hr
exposures hydrogen sulphide production ceased and the reactors started to
produce nitrite.
11. Nitrate clearance from the rumen appears to be initially very rapid but the rate of
disappearance slows some what abruptly perhaps indicating a depletion of a
critical nutrient possibly hydrogen sulphide for NR-SOB
12. Small additional inputs of Mo into a sheep’s diet increased nitrite production 5 fold.
Mo is a potent inhibitor of SRB and in many research papers it has been
demonstrated to reduce hydrogen sulphide production
13. Mo in high quality pasture plants, also high in crude protein is elevated by
application of molybdenised fertilizers to the soil. These are typically the pastures
where nitrate poisoning of livestock arises.
14. Leguminous plants high in crude protein and growing on soils of high Ph can
accumulate molybdenum. Lucerne in particular has been a diet where nitrate
toxicity could be induced by drenching with increasing amounts of nitrate.
15. When pasture plants such as perennial rye grass, exceed 15-20% crude protein
much of the extra crude protein N is from nitrate. This together with a high Mo
level may reduce hydrogen sulphide pool in the rumen
16. The hydrogen sulphide pool in the rumen of cattle was found to be much lower on
a high crude protein white clover forage( 25% CP) as compared to a perennial rye
grass ( 18%CP) which could be interpreted to be due to high nitrate intake or high
Mo intake or both from the white clover
47
17. In artificial rumen with low fermentable organic matter content, nitrate conversion
to nitrite was favored when glucose was added but no nitrite was produced when a
complete source of nutrient was added( dried grass) indicating that a deficient
nutrient was the cause of NRB converting nitrate to nitrite .The link possibly being
the availability of sulphate from amino acid degradation
18. Feeding a high level of cysteine to cows counteracted the build up of nitrite
following a nitrate load in cows.
19. Oil field microbiological changes when amended with nitrate suggest that the
effect of nitrate in lowering sulphide production is mediated via the competitive
inhibition of NRB activity on SRB and that NR-SOB uptake of sulphide plays only a
minor role in lowering sulphide production . However the reverse was observed
with biofilm reactors with sewage as the bulk fluid and treated with nitrate
20. It is hypothesized that In the rumen the inhibition of hydrogen sulphide production
by nitrate has the effect of inhibiting a small population of NR-SOB that survive the
crisis of low substrate( H2S) availability by generating ATP from reduction of
nitrate to nitrite.
21. Four processes may lower rumen fluid hydrogen sulphide 1)loss to the gas space,
2)competitive inhibition of SRB by hNRB, 3)inhibition of SRB by Mo and
4)oxidation to sulphate by NR-SOB
22. The interactions between methanogens,NRB,SRB and NR-SOB that are
postulated when different terminal acceptors are included in the diet of ruminants
would be supported where the organisms using the various terminal electron
acceptors are situated beneficially attached to particulate matter in biofilms which
is the predominant mechanism allowing efficient solubilization of organic matter in
the rumen.
48
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