The conundrum of the occurrence of nitrate toxicities in ruminants reported in the past where nitrate is ingested by ruminants is addressed.

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1 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

2 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 Abstract R A Leng 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

3 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

4 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 ) 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 4

5 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

6 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. NO H + H 2 O+ NO Equation 1 NO H + H 2 O+ NH 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 CO 2 +NH H 2 O Equation 3 3H 2 + NO H + NH H 2 O Equation 4 6

7 3HS - + NO H + 3S 0 + NH H 2 O 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 SO 4 - reduction takes place1) in cytoplasm, where carbon metabolism takes place and 2) at the cell membrane where the energetic reactions occur SO 4 - 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). 4H 2 +SO - 4 +H + 4H 2 O+H 2 S 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) 7

8 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 S 2 -, S o, HS -, or HSO 3 - (Odom and Singleton, 1993). The pka for H 2 S 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 (H 2 S). 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

9 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 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: CH 3 CH 2 COO - + 3H 2 O CH 3 COO - + H + + HCO H 2 9

10 Butyrate Acetate: CH 3 CH 2 CH 2 COO - + 2H 2 O 2CH 3 COO - + H + + 2H 2 Ethanol Acetate: CH 3 CH 2 OH + H 2 O CH 3 COO - + H + + 2H 2 Lactate Acetate: CH 3 CHOHCOO - + 2H 2 O CH 3 COO - + HCO H + + 2H 2 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

11 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

12 Hubert et al (2007, 2009) raised the question of relative contribution of hnrb and NR- SOB 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

13 Conc. of S cmp. (mg/s/l) or N cmp. (mgn/l) 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 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 Total dissolved S Sulfate Nitrate Sulfide Sulfite Thiosulfate Poly. (Nitrate) Time (hours) 13

14 Conc. of S-cmp. ( mg-s/l) or N cmp. ( mg- N/L) Suppresion of hydrogen sulphide after 4 exposures of reactor to nitrate with concomitant production of nitrite Total dissolved S Sulfate Nitrate ThioS Nitrite Sulfide Linear (Nitrate) Linear (Sulfide) Linear (Nitrite) Microbial -2 ecology of anaerobic ecosystems may explain some of the interactions 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 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

15 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

16 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

17 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

18 Conc. in rumen fluid (mm Fermentable OM NO 3 - NRB CO 2 + VFA Fermentable OM NH 4 + SO 4 2- Competitive Exclusion of SRB by NRB SRB CO 2 + VFA H 2 S H 2 S NO 3 - ADP NR-SOB SO 4 2- NH 3 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 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

19 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 nitrate- N 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

20 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

21 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 H 2 S 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 H 2 S 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 H 2 S 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

22 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 CH 4 -production was subsequently determined in respiration chambers. CH 4 -production was significantly decreased by both supplements (nitrate: - 32%, sulphate: -16% and nitrate + sulphate: - 47% relative to the control). The decrease in CH 4 -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

23 Methane production ( L/hr) 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

24 Lowered methane production (L/hr) owing to supplementation with sulphate 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. Figure 9.The effect of sulphate supplementation in lowering methane production. (Calculated from data supplied by Zijderveld et al 2010a) 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

25 Reduction in methane production ( liters/hr) 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). 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) y = x R 2 = Time of day(00hrs) Methane inhibition by sulphate in urea diet Methane inhibition by sulphate in a nitrate diet 25

26 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

27 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

28 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

29 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 = 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 H 2 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

30 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: CH 3 CH 2 COO - + 3H 2 O CH 3 COO - + H + + HCO H 2 Butyrate Acetate: CH 3 CH 2 CH 2 COO - + 2H 2 O 2CH 3 COO - + H + + 2H 2 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 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

31 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

32 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 (MoO 4 ) 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 MoO 4 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 MoO 4 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, MoO 4 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

33 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