The Effect of Nitrate, Live Yeast Culture Or Their Interaction on Methane Mitigation and Nitrate Reduction in Vitro THESIS Prese

The Effect of Nitrate, Live Yeast Culture or their interaction on Methane Mitigation and Nitrate Reduction in vitro

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Caitlyn M. Massie, BS

Graduate Program in Animal Sciences

The Ohio State University

2015

Master's Examination Committee:

Dr. Jeffrey L. Firkins, Advisor

Dr. Kelly C. Wrighton

Dr. Zhongtang Yu

Copyrighted by

Caitlyn M. Massie

2015

Abstract

Nitrates have been fed to dairy cows for years to decrease methane emissions. In the nitrate assimilatory pathway, bacteria reduce nitrate to nitrite to ammonia. The second step is rate-limiting leading to nitrite accumulation causing methemoglobinemia, which can hinder the adoption of nitrate feeding to compete with methanogens for hydrogen while assimilating the nitrogen from nitrate into microbial protein. The yeast Saccharomyces cerevisiae has the potential to anaerobically respire nitrite through its cytochrome c oxidase; it also can stimulate populations of bacteria that express nitrate and nitrite reductases. In the first trial, a 3 x 4 factorial within in vitro tubes included no yeast, an active live yeast culture (S. cerevisiae strain 1026), or autoclaved yeast dosed with nitrate, nitrite, ammonium, or a control to determine if yeast altered the nitrate reduction pathway. Differences were seen at h 0 (P < 0.01) for nitrogen treatments, that correlated with dosed metabolites. For the second project, 4 continuous cultures were used in 5 periods of 10 days. Treatments were arranged in a 2 x 2 factorial with sodium nitrate (NO3 at 1.5% of diet DM) or urea as an isonitrogenous control and without or with live yeast culture fed at a recommended 0.010 g/d (scaled from cattle to fermenter intakes). The second study showed a significant increase in NDF digestibility for the main effect of live yeast culture. Total VFA production (averaging 146 mmol/d; SEM =

15) and acetate: propionate (averaging 3.37, SEM = 0.10) did not differ (P > 0.15) among

ii treatments. The main effect of nitrate decreased (P < 0.05) methane emissions compared to the urea control (29.5 vs 21.0 mmol/d). There was no difference (P > 0.15) for hydrogen emission for nitrate or live yeast culture (averaging 0.148 ± 0.053 mmol/d), but the main effect mean of nitrate decreased (P < 0.01) aqueous hydrogen concentration compared with the main effect mean of urea (2.51 vs 3.66 ± 1.36 mM). Live yeast culture did not alter nitrite metabolism as hypothesized; however, nitrite inhibited yeast growth in batch culture. The last experiment was a yeast growth in a YPD media control combined with a 3 by 3 factorial arrangement of nitrogen sources (nitrate, nitrite, ammonium salts) replacing peptone at 25, 50 , 75% at one of three gas phases (aerobic, transition from aerobic to anaerobic, anaerobic). No interactions were detected, but the dosed nitrite inhibited yeast growth regardless of the gas phase. Overall, nitrate decreased methane production, but live yeast did not significantly influence methane production. Live yeast culture did not alter nitrite reduction, although nitrite did inhibit yeast growth.

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Dedicated to my Parents Steve and Brenda, Fiancé Jacob, Best Friend Sara and Sister

Ashley without whose support I could not have succeeded.

Acknowledgments

First, I would like to thank my advisor, Dr. Jeffrey L. Firkins, for his guidance, patience, and for sharing his wisdom with me. I am very lucky to have had the opportunity to learn from and work alongside you. Thank you for all of your assistance over my graduate experience.

I would also like to thank my other committee members, Dr. Zhongtang Yu and

Dr. Kelly C. Wrighton for all of their time and knowledge they shared with me. Without their lab managers I would have been lost; a special thank you goes out to Johanna Plank of the Ruminant Nutrition lab, Jill Stiverson recently of the Animal Science

Microbiology lab and Rebecca Daly of the Microbiology lab. My thanks also goes out to

Dr. Normand St. Pierre for his guidance and Amanda M. Gehman and Alltech, Inc. for their gracious funding of my research and graduate education.

To the Ruminant Nutrition lab, I could never say how grateful I am to all of you for your assistance, time and guidance over the years. Thank you especially to Benjamin

Wenner and Brooklyn Wagner my fellow Fermenter Fanatics, as well as the other numerous interns, farm personal and graduate students of the Ohio State University

Animal Science Department: Nicole Danszczak, Tasha Henderson, Natalia Jurcak, Logan

Morris, Sarah Waits, Jessica Pempek, Danni Ye, Bethany Keyser, Yaniris Alvarado

Luna, Thiago Bompadre and Danielle Coleman. A huge thank you goes out to Lorraine

English without whom I might have lost my sanity. Also without Rebekah Meller I could not have survived my first year of graduate school, thank you for being there in the wee morning to learn with me.

Lastly I would like to again thank my family and friends for all of your encouragement and support throughout my life and most importantly my graduate career.

Thank you Dad for opening my eyes to the possibilities in the Dairy industry, Mom for my love of learning, Jacob and Sara for your positivity and love and lastly Ashley,

Megan and Hannah for your support.

Vita

2009...... Graham High School, St. Paris, OH

2013...... BS Biology Life Science, Wilmington

College, Wilmington, OH

2013 to present ...... Graduate Research Associate, Department

of Animal Science, The Ohio State

University, Columbus, OH

Fields of Study

Major Field: Animal Sciences

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Table of Contents

Abstract ...... ii

Acknowledgments...... v

Vita ...... vii

List of Tables ...... x

List of Figures ...... xi

Chapter 1: Introduction ...... 1

Methanogenesis ...... 3

Mitigation Strategies ...... 4

Nitrate ...... 7

Thermodynamics ...... 8

Methemoglobinemia ...... 8

Nitrate Reduction Pathways ...... 9

Live Yeast ...... 12

Nitrite Reduction ...... 13

Digestibility ...... 13

Chapter 2: The Effect of Live Yeast Culture on Nitrate and Nitrite Disappearance in vitro ...... 15

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Introduction ...... 15

Methods ...... 15

Statisticsal Analysis ...... 16

Results and Discussion ...... 17

Chapter 3: The Effect of Live Yeast Culture and Nitrate on Methane Mitigation and Nitrate Reduction in a Continuous Culture System ...... 20

Introduction ...... 20

Methods ...... 20

Statisticsal Analysis ...... 23

Results and Discussion ...... 23

Chapter 4: The Effect on Live Yeast Culture Growth by Replacement of 25%, 50%, or 75% of Peptone within YPD Media with Nitrate, Nitrite, or Ammonium at aerobic, transition between aerobic and anaerobic, or anaerobic respiration phases ...... 48

Introduction ...... 48

Methods ...... 48

Statisticsal Analysis ...... 49

Results and Discussion ...... 50

Chapter 5: Conclusion...... 53

References ...... 54

List of Tables

Table 1: Nitrate, nitrite, and ammonia concentrations in mixed batch cultures at 0 and 12 h after inoculation of dosed treatments containing deionized water vehicle control without or with nitrate, nitrite, or ammonium salts and factorialized with liquid media without (NY) or with live (LY) or autoclaved yeast (AY)...... 19

Table 2: Ingredient and nutrient composition of continuous cultures treatment diets without (-) or with (+) nitrate or live yeast culture (LYC) ...... 31

Table 3: Nutrient digestibilities in continuous cultures administered diets supplemented without (-) or with (+) nitrate or live yeast culture (LYC) ...... 32

Table 4: Protozoal counts and total generation time in continuous cultures administered diets supplemented without (-) or with (+) nitrate or live yeast culture (LYC) ...... 33

Table 5: Fermentation characteristics in continuous cultures administered diets supplemented without (-) or with (+) nitrate or live yeast culture (LYC) ...... 34

List of Figures

Figure 1: Pool size of total VFA in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). Treatment by time interaction was P < 0.01. Main effect of nitrate, LYC or their interaction “at each time point” (** = P ≤ 0.01, * = P ≤ 0.05, NS = P > 0.15)...... 35

Figure 2: Pool size of acetate in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). Treatment by time interaction was P < 0.01. Main effect of nitrate, LYC or their interaction “at each time point” (** = P ≤ 0.01, * = P ≤ 0.05, NS = P > 0.15)...... 36

Figure 3: Pool size of propionate in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). Treatment by time interaction was P < 0.01. Main effect of nitrate, LYC or their interaction “at each time point” (** = P ≤ 0.01, * = P ≤ 0.05, NS = P > 0.15)...... 37

Figure 4: Acetate to propionate ratio in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). Treatment by time interaction was P < 0.01. Nitrate by LYC interaction was P > 0.15. Main effect of nitrate or LYC “at each time point” (** = P ≤ 0.01, * = P ≤ 0.05, NS = P > 0.15)...... 38

Figure 5: Pool size of butyrate in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). Treatment by time interaction was P < 0.01. Main effect of nirate, LYC or their interaction “at each time point” (** = P ≤ 0.01, * = P ≤ 0.05, NS = P > 0.15)...... 39

Figure 6: Pool size of isobutyrate in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). Treatment by time interaction was P < 0.01. Main effect of nitrate, LYC or their interaction “at each time point” (** = P ≤ 0.01, * = P ≤ 0.05, NS = P > 0.15)...... 40 xi

Figure 7: Pool size of valerate in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). Treatment by time interaction was P < 0.01. Main effect of nitrate, LYC or their interaction “at each time point” (** = P ≤ 0.01, * = P ≤ 0.05, NS = P > 0.15)...... 41

Figure 8: Pool size of isovalerate in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). Treatment by time interaction was P < 0.01. Main effect of nitrate, LYC or their interaction “at each time point” (** = P ≤ 0.01, * = P ≤ 0.05, NS = P > 0.15)...... 42

Figure 9: Incremental methane production over time in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). Treatment by time interaction was P < 0.01. Main effect of nitrate, LYC or their interaction "at each time point" (** = P ≤ 0.01, * = P ≤ 0.05, NS = P > 0.15)...... 43

Figure 10: Incremental hydrogen production over time in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). Treatment by time interaction was P < 0.01. Main effect of nitrate, LYC or their interaction "at each time point" (** = P ≤ 0.01, * = P ≤ 0.05, NS = P > 0.15)...... 44

Figure 11: Concentration of nitrate in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). Treatment by time interaction was P < 0.01. Main effect of nitrate, LYC or their interaction “at each time point” (** = P ≤ 0.01, * = P ≤ 0.05, NS = P > 0.15)...... 45

Figure 12: Concentration of nitrite in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). Treatment by time interaction was P > 0.15. Main effect of nitrate, LYC or their interaction “at each time point” (** = P ≤ 0.01, * = P ≤ 0.05, NS = P > 0.15)...... 46

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Figure 13: Concentration of ammonia in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). Treatment by time interaction was P < 0.01. Interaction of nitrate and LYC and main effect of LYC were P > 0.15. Main effect of nitrate “at each time point” (** = P ≤ 0.01, * = P ≤ 0.05, NS = P > 0.15)...... 47

Figure 14 (one figure, 3 panels): Yeast growth by OD grown in aerobic (panel A) , transition from aerobic to anaerobic (panel B) or anaerobic (panel C) YPD media with 0%, 25%, 50% or 75% peptone replacement by nitrate, nitrite or ammonium. Means of 3 replicates (unless a value was excluded for bacterial contamination) were fit to an exponential growth model...... 52

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Chapter 1: Introduction

There is a rising concern about global warming and environmental changes among nations and the desire to decrease the predicted repercussions. The culprit is greenhouse gas emissions (GHG) such as CO2, CH4, and N2O, which enhance global surface and atmospheric temperatures through solar and thermal radiation. Each GHG has a different level of global warming potential (GWP) that determines their detriment level on the environment. GWP is based off of the gases potential in comparison to CO2; the most abundant GHG, CH4 has a GWP of 21 and N2O of 310 (EPA, 2005). In the past 100 years, the Earth’s average temperature has risen 0.8⁰C and is expected to increase by

6.4⁰C, if GHG are not controlled (The National Academy of Sciences, 2010). The agriculture industry has been estimated to make up 29% of the global GHG emissions

(EPA, 2011). In order to decrease the increasing GHG, international policies are being enacted to decrease non-CO2 emissions such as CH4 and N2O, which is cheaper than CO2 mitigation strategies (EPA, 2006; F.A.O., 2009; Shafer et al., 2011; Gerber et al., 2013).

Total CH4 has increased by 145% in the past 300 years, since the beginning of the industrial revolution (USDA, 2008). According to the USDA (2008) the agriculture industry accounts for 31% of our nation’s CH4 of which 64% comes from enteric sources.

Enteric CH4 is the result of CO2 reduction from fermentation within the rumen or hindgut;

1 this process depends upon the oxidation of reduced cofactors from volatile fatty acid

(VFA) production.

The US Dairy industry has set a goal to decrease CH4 emissions per pound of fluid milk by 25% by the year 2020 (Knapp et al., 2011). The manipulation of the ruminant biological cycle of CO2 fixation, utilization, and exhalation will help decrease

CH4 emissions (Pitesky et al., 2009).

There are other benefits besides the global impact by decreasing CH4; dairy producers can gain positive consumerism, increase production, and improve economic efficiency when CH4 is decreased. Many consumers are concerned with the carbon footprint of their food and in the future there is predicted to be more grazing land available then crop land, so the utilization of these pastures by ruminants to turn human- inedible products into high nutrient human food is key (Knapp et al., 2014). Also ruminants consume the by-products of many processes, like soy hulls that are inedible for human consumption; thereby reducing waste (Gill et al., 2010). Also through animal intensification or the increasing of production with the decreasing of animal number, the total CH4 production can be immensely reduced (Knapp et al, 2014). Through this intensification process the producers can increase their income over feed costs.

Predominately this mitigation will come through decreasing enteric CH4 by increasing ruminant production (EPA, 2005; Capper et al., 2009). Both enteric and manure CH4 contribute to 40% of GHG from milk production and they are interrelated; therefore, a decrease in enteric CH4 would decrease manure CH4 (Thoma et al., 2013; Gerber et al.,

2013; Hristov et al., 2013).

Methanogenesis

The rumen is an anaerobic environment that contains symbiotic bacteria, fungi, protozoa and archaea that function normally by maintaining a low redox potential within their anaerobic environment. Protozoa, fungi, and bacteria degrade proteins and carbohydrates (CHO) that generate reduced cofactors; if these cofactors are not reoxidized then a negative feedback could hinder normal rumen function. The degradation of CHO and sugars to VFA in order to supply nutrients to the cow is a multi- step process in which reducing equivalents (metabolic hydrogen or [2H]) are generated and reoxidized in fermentation. Each microbe needs to balance reducing equivalents, but each express different enzymatic pathways during fermentation (Hungate, 1966;

Czerkawski, 1987; Moss et al, 2000). When mixed VFA are produced, propionate production can be a sink for [2H] generated during acetate production. If microbes do not produce propionate, then they often express one or more hydrogenases capable of converting [2H] to H2. The eukaryotic fungi and protozoa typically contain hydrogenosomes in which one or more hydrogenases are membrane-bound. The [2H] can be converted by protozoan hydrogenosomes such as those in the genus Dasyticha (Yarlett et al., 1981) to H2, which can then be taken up by the methanogenic archaea through inter species H2 transfer to produce CH4 and ATP. To date, five genera have been discovered from the archaea domain amongst ruminant methanogenes, but over 33 genera are assumed to exist (McAllister et al., 2015). If methanogens were nonexistent as a H2 sink then the dissolved H2 would cause a negative feedback reducing the degradation of CHO

3 and decreasing the nutrient supply by decreased microbial growth and protein synthesis within the rumen (Wolin, 1974; McAllister and Newbold, 2008).

Mitigation Strategies

There have been many CH4 mitigation strategies proposed over the years, they can be classified into four categories. The first category is genetics, which is responsible for 50% of the milk yield increase over the past 60 years; the other half of the increase is from management practices (Knapp et al., 2014). Although no recorded increase in feed efficiency has been seen according to the Environmental Protection Agency (2005) it is the most cost effective mitigation strategy to decrease CH4 emissions. The last CH4 mitigation category is rumen modification such as inhibitors, ionophores, plant bioactive compounds, defaunation, and electron acceptors (Hristov et al., 2013).

Currently, there has been less genetic selection for decreased CH4 production in dairy cattle although there has been in other ruminants (sheep and beef cattle). However, no long term study on CH4 genetic selection and its effect on animal growth has been performed yet (Hegarty and McEwan. 2010; Clark, 2013; Pinares-Patiño et al., 2013).

For now, dairy cow genetics pertaining to CH4 mitigation is limited to improving cow efficiency. The genetic increase in milk yield is accompanied with a 64% reduction in dairy cow number and a 57% reduction of CH4 due to the increase in animal efficiency

(Capper et al., 2009). Genetic selection for improved milk yield and dry matter intake

(DMI) allow for the selection of higher producing animals with less feed intake, thereby increasing feed efficiency allotting for fewer animals needed to maintain current

4 production. Although it is not a common practice to select animals that will increase energetic efficiency due to a smaller body frame, it is theoretically possible (Knapp et al.,

2011; 2014). If body size is decreased while maintaining milk yield and components, then the energy required by the animal will be decreased; therefore, an increase in energy efficiency is possible (Knapp et al., 2011). A few cross-bred studies have seen some first generation improvements due to body size differences on energy corrected milk (ECM), but more studies are needed on future generations (Münger and Kreuzer, 2006). In general, improving the lifespan of animals by selecting against disease prevalence and other management issues will also improve herd productivity, thereby decreasing CH4 per production.

Management is important because even if animals are bred for the most efficient productivity possible and high CH4 mitigation, without proper management they will not reach their full genetic potential. According to Knapp et al. (2014) management is subdivided into three categories: reproduction, heat stress, and transition period. By improving on all of these categories milk yield will be increased improving efficiency and thereby decreasing CH4 /ECM (Johnson and Johnson, 1995; Moss et al., 2000; Boadi et al., 2004; Beauchemin et al., 2008; Pinares-Patiño et al., 2009; Clark, 2013). Overall improved feed efficiency is one of the more efficient CH4 mitigation strategies available.

Feed efficiency can be achieved through altteration of the VFA production, increase in passage rate, or increased milk production (IPCC, 1990). Feeds that can promote microbes to increase propionate production and utilize the [2H], shifting the VFA production away from acetate and butyrate can decrease CH4 (Hungate, 1966; Van Nevel

5 and Demeyer, 1996). Changing the feed via plant genetics, processing, storage and quality can also alter the VFA production, microbe population and intestinal digestibility by changing the passage rate. By increasing the diet ingredient quality, milk production can be increased and a decrease in CH4 can be seen due to raising the available energy and increased digestibility of the diet.

Modification of the rumen environment is another way to mitigate CH4. Rumen modifiers can be organized into the following categories: ionophores, enzymes, specific methanogenesis inhibitors, defaunation, probiotics, and electron acceptors (Knapp et al.,

2014). Ionophores such as monensin are capable of shifting the VFA production towards propionate thereby decreasing CH4 production (Odongo et al., 2007). The problem with ionophores is that they are not a long term effect (Guan et al., 2006; Hook et al., 2009) and often do not shift acetate: propionate in dairy cattle (Firkins and Yu, 2015).

Exogenous fibrolytic enzymes, bacteriophages, and specific methanogenesis inhibitors such as mevastatin and lovastatin have been found to reduce CH4 by up to 50% (Giraldo et al., 2007; McAllister and Newbold, 2008; Machmüller et al., 2006). The removal of all or most protozoa (known as defaunation) has decreased methane; however, because of inconsistent responses or negative effects from defaunating agents, defaunation is not recommended (Hristov et al., 2013). With time, different microbes fill the empty niche from the feed additive, thus preventing any long term effects. The ability to maintain decreased CH4 production long term is the biggest fallback of many of the mitigation strategies (Hristov et al., 2013). Electron acceptors or H2 sinks utilize the H2 from protozoa and VFA production thermodynamically outcompeteing methanogens thereby

6 decreasing CH4 production (Brown et al., 2011). Sulfate and NO3 are two electron acceptors that can suppress methanogenesis; their end products are hydrogen sulfide and

NH3, respectively. Hydrogen sulfide can have side effects with animals if eructated and breathed back into lungs (Hristov et al., 2013). Although electron acceptors can outcompete methanogens, many of them can have negative effects such as a drop in milk production, fiber digestion, or DMI (Bauman and Griinari, 2003; Beauchemin et al.,

2008; Hollmann and Beede, 2010). Nitrate is the only feed additive that over long term usage does not coincide with milk depression; NO3 reduction intermediates NO2 and NO have also been found in some instances to be toxic to methanogens (van Zijderveld et al.,

2011; Klüber and Conrad, 1998).

Nitrate

Nitrates can be fed to production animals in the form of an inorganic salt as a nitrogen source or as a crude protein (CP) component in forages, especially grasses, such as perennial ryegrass and sorghum. The amount of NO3 in plants is dependent on the plant maturity and it is not distributed evenly throughout plants. Less matured grasses generally contain more NO3 as well as plants that have had access to more sunlight because NO3 reductase parallel with illumination (Pfister, 1988). Inadvertently NO3 can be increased in other forages due to a variety of circumstances, such as drought and excessive fertilization. Fed NO3 results in elevated CP levels and can be converted to NH3 to provide fermentable nitrogen for microbial growth (Lewis, 1951; Wang et al., 1961).

Nitrate poisoning has been seen as a problem when CP is between 18-38% resulting in

NH3 concentrations up to 3 times over the optimal microbial growth concentrations

(Preston and Leng, 1985).

Thermodynamics

Nitrate works as an available electron sink that can inhibit methanogenesis.

Methanogens convert CO2 to CH4 with a Gibbs free energy (∆G’0) of -16.9 kJ/mol, whereas the NO3 assimilation pathway of NO3 reduction to NO2 by NAR is -130 kJ/mol and NO2 to NH3 via nitrite reductase (NIR) is -124 kJ/mol; NO3 can thermodynamically outcompete the methanogenesis pathway (Ungerfeld and Kohn, 2006). For every 8 electrons consumed by 1 mole of NO3 to produce 1 mole of NH3, CH4 is minimized by 1 mole and NH3 is available for microbial protein synthesis (Leng and Nolan, 1984).

Unlike other mitigation strategies NO3 can also replace urea in the diet as an N source for microbes.

Methemoglobinemia

When NO3 is fed to unadapted ruminants such as through drought stricken pasture, methemoglobinemia can occur (ATSDR, 2011). Within the rumen, the flux of

NO3 reduction to NO2 is usually rapid, but followed by a slower reduction to NH3. This causes an accumulation of NO2 that can be absorbed into the bloodstream, binding to red blood cells tightly and thereby preventing their transport of oxygen, potentially causing tissue asphyxiation. This nitrite-bound molecule is known as methemoglobin, which can be found normally within ruminants at 3% (Bradley et al., 1939). Methemoglobinemia

8 can become clinical at 20 to 40% (Johnson and Schneiderm, 1983), and death can occur by asphyxiation at 67 to 90% (Asbury and Rhode, 1964). Clinical methemoglobinemia can occur when NO3 is fed above 3.0% of the diet DM (Bruning-Fann and Kaneene,

1993). Besides the possible death by asphyxiation, methemoglobinemia can also affect other aspects of ruminants such as reduced feed intake.

Clinical methemoglobinemia is an issue when NO3 is bolus dosed, but with adaptation, it ceases to develop. In sheep gradually fed NO3 the rate of metabolized NO3 increases, allowing for the adaptation of the rumen to higher NO3 dosed diets (Alaboudi and Jones, 1985). Urea is fed currently to ruminants with little issues as a nitrogen supplement, but previously like NO3 it had caused severe repercussions; however, research strategies to feed urea by acclimation without issues have arisen and can for

NO3, also. Nitrogen from NO3 could easily replace urea-N in ruminant diets (Leng,

2005). There have been reports of seeing an increase in microbial synthesis when NO3 replaced urea in diets (Guo et al. 2009).

Nitrate Reduction Pathways

Microorganisms within the rumen are capable of reducing NO3 and NO2 with

NO3 or NO2 reduction enzymes. Dissimilatory NO3 reduction to ammonia (DNRA) reduces coenzymes acting as an electron acceptor, like methanogenes would by NADH- dependent enzymes without ATP production and is dependent on NO3 concentration

(Page et al., 1990). Ammonia can also be produced by enzymatic catalyst reducing NO3 to NH3 by assimilatory NO3 reduction to NH3 (ANRA) through membrane electron

9 transport chain ATP generation (Page et al., 1990). These enzymes are triggered by the presence of NO3 and NO2 and are down regulated by NH3 concentrations (Cole and

Brown, 1980; Dunn-Coleman et al., 1984; Page et al., 1990). Nitrate reduction by DNRA can be carried out by one of three reductase catalyst: assimilatory NO3 reductase of the cytoplasm (NAS), membrane bound NO3 reductase (NAR), or periplasmic NO3 reductase

(NAP) of gram negative bacteria (Lundberg et al., 2004). Although it is uncommon for microbes to contain multiple NO3 reductase pathways, E. coli, for example expresses both NAP and NAR enzymes (Lundberg et al, 2004). When NO3 reductase takes place through the NAR (narG operon) the NO2 is reduced by NADH-dependent NO2 reductase

(NirBD) within the cytoplasm, while following the NAP NO2 reduction still occurs within the periplasm by cytochrome c NO2 reductase (Nrf) (Lunderberg et al., 2004). In

E. coli, at high NO3 concentrations the cytoplasmic pathway (narG and nirBD operons) is more active, whereas at low concentrations, the periplasmic pathway (Nap and Nrf operons) is most efficient.

Typically, bacteria that can reduce NO3 to NO2 also have the capability of reducing NO3 to NH3. Each NO3 reduction pathway utilizes 8 electrons to form NH3; the

ANRA is regulated by NH3 so that reduction occurs at a required rate with little accumulation within the culture (Hewitt, 1975; Payne, 1973). In DNRA, NO3 and NO2 are rapidly reduced to NH3 regardless of the NH3 concentration within the culture, possibly leading to a large accumulation (Hewitt, 1975; Cole and Brown, 1980).

2- 2- Organisms capable of both reducing NO3 and S are called NO3 reducing, S oxidizing bacteria (NR-SOB). One ruminant bacterium, Wolinella succinogenes, is thought to have

10 this capability and can convert NO2 to NH3 in order to generate ATP to enhance its own growth through the ANRA pathway (Simon, 2002; Bokranz et al., 1983). The bacterium

Veillonella alcalescens is a NO3 reducing bacterium (NRB) that is thought to contain both the ANRA and DNRA as demonstrated by Inderlied and Delwiche (1973) using 15N.

In this study, NO3 was dissimilated into NO2 then NH3 or assimilated into microbial protein. Yordy and Delwiche (1979) concluded that NIR was induced by the presence of

NO3 and NO2, but high NH3 concentrations inhibited this enzyme. There have been some studies showing that Selenomas ruminantium also are assimilatory NRB (Iwamoto et al.,

1999). Besides the previous discussed bacteria, Campylobacter fetus and Veillonella parvula are capable of reducing NO3 (Stewart et al., 1997; Lin et al., 2011).

Nitrate is also an efficient CH4 mitigation strategy because of its instantaneous results and its minor effects on the cellulolytic microbes. Zhou et al. (2011; 2012) detected a decrease in the bacteria Fibrobacter succinogenes, Ruminococcus albus, and

R. flavefaciens and in total methanogens when 12 mM of NO3 was added to unadapted rumen fluid cultures. In contrast, when 1.0 to 2.0% NO3 (DM %) was fed to cannulated steers, F. succinogenes, R. flavefaciens, and S. ruminantium increased; contrary to predictions, no changes were seen for R. albus and V. parvula (Zhao et al., 2015). Nitrate,

NO2, N2O, and NO all had inhibitory effects on methanogens Methanobacterium bryantii and Methanosarcina barkeri, resulting in decreased CH4 production (Klüber and Conrad,

1998).

Nitrate disappearance within the rumen has been seen to decrease within the first hour post-dose followed by a slow decrease; at higher doses (120mM), a decrease of up

11 to 80% has been seen within the first hour (Tillman et al., 1965). Ammonia concentrations gradually rise and NO2 peaks between 2 and 4 hours; therefore, a number of processes must act on the NO3 decrease (Leng, 2005). This differs from urea, which was noted to increase NH3 concentrations 7 fold 30 minutes after dosing (Kaye et al.,

15 2001). The NO3 doses are quickly decreased, indicating the entrance of unlabelled NO3 or NO2 into the NO3 pool (Wang et al., 1961). Assuming that no NO3 was being procured by the feed, there are a few possibilities as to where the NO3 was coming from: NO3 being absorbed quickly into the bloodstream and being recycled back into the rumen over time, temporary sequestration of NO3 by microorganisms then released into the rumen as

NO3 concentrations decrease, NO3 bound to a protein and released as concentrations diminish, NO3 within the fluid pool is moved to another N pool within the rumen to be regurgitated with NO2 or a rapid growth of NRB that reduce NO3 to NH3 to be stored for growth (Leng, 2005). The NO3 reduction and peak concentration depends on the amount dosed and acclimation of the ruminant; in Allison and Reddy (1984), it took rumen microbes 6 days to adapt and within 1 hour post-feeding NO3 was undetectable.

Live Yeast

The yeast Saccharomyces cerevisiae could possibly prevent NO2 toxicity.

Cytochrome c oxidase electron transport expressed in some eukaryotic cells can form NO from NO2 under anaerobic conditions, which the yeast S. cerevisiae is thought to contain

(Castello et al., 2006). S. cerevisiae has been shown to stimulate lactic utilizing bacteria, which help maintain pH, promoting cellulolytic bacteria fiber digestion (Dawson, 1995).

Besides the possible reduction of methemoglobinemia, yeast also are capable of improving digestibility (Desnoyers et al., 2009), enhancing NH3 utilization and increasing VFA production particularly propionate (Dawson, 1995).

Nitrite Reduction

Nitric oxide is respired by some yeast through their mitochondrial respiratory chain with an alternative electron acceptor (David and Poyton, 2005). Castello et al.

(2006) discovered that yeast mitochondria were capable of reducing NO2 to NO via a cytochrome c oxidase (Paitian et al., 1985). If S. cerevisiae has this ability to reduce NO2, then it might assist in preventing methemoglobinemia when NO3 salts are fed in the diet.

S. cerevisiae has decreased NH3 concentrations in cattle by 20 to 34% and 7 to 12% in rumen-simulation cultures due to inducing NH3 utilization by cellulolytic bacteria growth

(Edwards, 1991; Dawson and Newman, 1988; Newbold and Wallace, 1992; Williams and

Newbold, 1990).

Digestibility

Yeast are a rich source of vitamins, enzymes, and nutrients for microbes; they are capable of maintaining metabolic activities under anaerobic activities and withstanding large amounts of physical stress (Dawson, 1995). S. cerevisiae stimulate rumen microorganisms especially the lactic acid utilizing and cellulolytic bacteria (Desnoyers et al., 2009; Wiedmeier et al., 1987; Dawson et al., 1990; Callaway and Martin, 1997). By stimulating bacteria such as S. ruminantium, S. cerevisiae can stabilize rumen pH by

13 stimulating the conversion of lactate to propionate (Callaway and Martin, 1997; Stewart et al, 1977). Cellulolytic bacteria can be stimulated to increase VFA (Gray and Ryan,

1988; Martin et al., 1989) resulting in a decreased acetate: propionate (Harrison et al.,

1988; Martin et al., 1989; Williams and Newbold, 1990; Dawson et al., 1990). Salvati et al. (2014) discovered that S. cerevisiae have the capability to reduce lactate and butyrate in vivo. Treatment of live yeast has stimulated cellulolytic bacteria in dairy cattle to increase fiber digestion resulting in an increased DMI and milk production (Williams et al., 1991; Wohlt et al., 1991). S. cerevisiae stimulate cellulolytic bacteria including F. succinogenes, R. flavefaciens, and R. albus; lactic acid utilizing bacteria including S. ruminantium and Megasphaera elsdenii; and carbohydrate-utilizer Prevotella ruminicola

(Dawson, 2005).

S. cerevisiae can alter protein synthesis increasing the microbial protein flow and amino acid concentration (Erasmus et al., 1992). This bacteria stimulation seems to shift specific bacteria concluding that S. cerevisiae can selectively stimulate bacteria (Girard and Dawson, 1994). S. cerevisiae increased daily intestinal absorption of non-ammonia nitrogen (Williams and Newbold, 1990) and increased digestibility of NDF (Shaver and

Garrett, 1997; Miller et al., 2002; Harrison et al., 1988; Callaway and Martin 1997).

Chapter 2: The Effect of Live Yeast Culture on Nitrate and Nitrite Disappearance in vitro

Introduction

When NO3 salts are fed to ruminants the reduction to NO2 is quick, but a slower reduction to NH3 leads to a buildup of NO2, that can be absorbed into the bloodstream leading to methemoglobinemia (ATSDR, 2011). The yeast S. cerevisiae is thought to be able to respire NO2 through its cyctochrome c oxidase (Castello et al, 2006). Besides the possible reduction of NO2 by yeast, S. cerevisiae is capable of stimulating cellulolytic bacteria that utilize NH3 for growth, thereby reducing NH3 concentrations (Edwards,

1991; Dawson and Newman, 1988; Newbold and Wallace, 1992; Williams and Newbold,

1990). We hypothesized that live yeast would help reduce NO2 over autoclaved or no yeast culture in vitro and also examined the effect of live yeast on other NO3 reduction intermediates, such as a possible decrease of NH3.

Methods

S. cerevisiae was grown anaerobically in liquid YPD media and dosed as live or autoclaved cells at approximately constant dosages (9 x 106 cells/mL at an OD of 600 nm reading at 0.30) with comparison to a control without yeast. These three yeast treatments were factorialized with the four nitrogen treatments: sodium nitrate (NO3 at 1.5% of diet

DM, 9.4 mM), or isonitrogenous sodium nitrite, or ammonium sulfate compared with a

15 control without N supplementation (deionized water). The in vitro tubes contained 0.5 g of 60: 40 alfalfa and concentrate pellets (ground through a 1-mm screen)/ Tubes were inoculated with 30 mL of 50: 50 mineral solution (Simplex Buffer without L-cysteine hydrochloride) and rumen fluid that was collected from two multiparous Jerseys from

Waterman Dairy and strained through 2 layers of cheesecloth (The Ohio State University,

Columbus, OH). Cows were housed according to approved animal usage protocols. After live yeast culture and nitrogen treatments were dosed, the in vitro tubes were then incubated in a 39⁰C shaking water bath. Tubes were removed from the water bath at h 0 and 12, when 10 mL were sampled for NO3, NO2, and NH3 analysis. Samples were centrifuged at 15,000 x g for 15 minutes at 4⁰C, then filtered through a 0.2-μm sterile filter unit (Fisher Scientific) into four 2-mL microcentrifuge vials and frozen at -80⁰C until analyzed. NO3 and NO2 samples were measured by ion chromatography in the

Wrighton lab (Dionex ICS-2100 Ion Chromatography System, courtesy of K. C.

Wrighton, The Ohio State University, Columbus, OH). The baseline buffer was subtracted out of NO2 values due to IC peak contamination. For NH3, a colorimetric assay was run according to Chaney and Marbach (1962).

Statistical Analysis

The following model was used to analyze samples by SAS MIXED procedure with repeated measures (SAS Institute Inc., Cary, NC v9.4): Yijm = μ + Ni + Sj + Hm +

NSij +NSHijm+ eijm. In the model Yijm is the dependent, continuous variable, μ is the

th overall population mean, Ni is the fixed effect of the i N treatment (i = 1, 2, 3, 4), Sj is

th th the fixed effect of the j yeast treatment (j = 1, 2, 3), Hm is the fixed effect of the m hour

th th (m = 1, 2), NSij is the fixed effect of the interaction of the i nitrate treatment, and j

th th yeast treatment, NSHijm is the fixed effect of the interaction of the i nitrate treatment, j

th yeast treatment, the m hour, and eijm is the residual error, assumed independent and ~N

2 (0, σ e). The N treatments were compared by least significant difference protected by a significant F-test (N by yeast or main effect of N when there was no interaction with yeast). Trends were declared between 0.05 < P < 0.15 and significant at P ≤ 0.05.

Results and Discussion

Nitrogen source (NO3, NO2, and NH4 salts plus control) and yeast treatments

(none, autoclaved, or live yeast) interacted with time (P < 0.01) for concentrations of

NO3, NO2, and NH3 (Table 1). There was no significant effect of the interaction of N source and yeast treatments or main effect of yeast on NO3, NO2, or NH3 concentrations.

The main effect of N source (P < 0.01) resulted from NO3 concentration at 0 h being higher for the dosed NO3 treatments than other treatments, as expected. Over time, a post-dose decrease of NO3 was seen in many studies and is attributed typically to NO3 reduction to NO2 (Lewis, 1951; Takahashi and Young, 1991; Sar et al., 2004; Tillman et al., 1965). In batch culture, there is no absorption of NO3; therefore, the lack of difference at 12 h in NO3 concentration for the N treatments versus control documents extensive reduction. The main effect of N source at 0 h (P < 0.01) was explained by the higher NO2 concentration for the NO2 treatment, as planned, but this effect was lost at 12 h ( P >

0.15). The significance of time was seen for NH3 concentration due to the main effect of

NH4 (P <0.0) at 0 h, which is expected due to NH4 salts being dosed.

The main effect of N source at 0 h can be explained by the metabolite dosed reflective to experimental error with main effect means of NO3, NO2, and NH4 salts of approximately 9.4, 8.6, and 6.1 mM, respectively. Regardless of dosed N salt or yeast treatment the end product of metabolism, NH3, was consistent for all treatments at 12 h averaging 13.5 mM. This can be expected because of two things 1) the N salt dosed was isonitrogenous and 2) yeast can stimulate bacteria. The isonitrogenous doses should bring in the same amount of N in the doses; therefore, we discovered the same end N amount should be present. Since there were no 12 h significant differences the end N concentrations were consistent across treatments. Secondly the yeast whether autoclaved or live could have promoted bacteria growth to utilize NH3, although without yeast the same effect occurred, so there was no statistical effect of dosing yeast alive or autoclaved on NH3 utilization. Even though yeast did not significantly impact NO2 reduction or NH3 utilization, at higher live yeast doses or over a prolonged period of time, there might be an interaction with live yeast and NO3 reduction.

Interaction

Yeast

-value of effects of -value

<0.01

N source

2.1

0.73

0.64

1.50

0.34

0.82

SEM

Pooled Pooled

16.6

6.06

1.56

2.89

0.55

0.52

17.5

5.91

3.33

0.44

0.52

<0.01

Ammonium

1,2

14.9

6.25

3.82

0.06

0.46

<0.01

14.0

2.16

1.65

7.54

0.40

0.60

> 0.15). >

11.6

2.08

0.66

0.40

0.17

10.36

Nitrite

12.0

2.26

7.78

0.55

<0.01

cells/mL) at log phase or autoclaved in log phase. log in autoclaved or phase log at cells/mL)

14.4

2.22

1.92

2.09

0.55

8.31

< 0.01). At 0 h, the N salt dosed increased (P < 0.05) its concentration relative to that of all other treatments, which were were which treatments, other all of that to relative concentration its 0.05) < (P increased dosed N salt the 0 h, At 0.01). <

11.7

2.30

0.06

4.14

0.58

9.12

Treatments Dosed Into Batch Cultures Batch Into Dosed Treatments

Nitrate

13.3

2.19

3.00

<0.01

10.85

14.4

2.20

2.92

0.59

0.46

<0.01

9.6

grown anaerobically in liquid YPD media (9 x 10 x (9 media YPD liquid in anaerobically grown

2.13

0.44

3.42

0.53

0.49

Control

11.7

2.73

4.24

0.39

0.37

<0.01

4,5

Nitrate added at 1.5% diet DM; nitrite and ammonium salts were added to the diet isonitrogenously with the nitrate treatment. nitrate the with isonitrogenously diet the to added were salts ammonium and nitrite DM; diet 1.5% at added Nitrate

Saccharomyces cerevisiae Saccharomyces

Nitrite quantified by ion exchange chromatography minus baseline buffer without any extra salts added. salts extra any without buffer baseline minus chromatography exchange ion by quantified Nitrite

Interaction of yeast and N source dosage treatments with time ( time with treatments dosage N source and yeast of Interaction

Main effects of yeast or N source dosage treatments or their interaction. NS = not significant ( significant NS not = interaction. their or treatments dosage N or source yeast of effects Main

Item, m Item,

not different (P > 0.15) from each other, but this effect was negated at 12 h. 12 at negated was effect this but other, each from 0.15) > (P different not

nitrate, nitrite, or ammonium salts and factorialized with liquid media without (NY) or with live (LY) or autoclaved yeast (AY). yeast autoclaved or (LY) live with or (NY) without media liquid with factorialized and salts ammonium or nitrite, nitrate, Table 1: Nitrate, nitrite, and ammonia concentrations in mixed batch cultures at 0 and 12 h after inoculation of dosed treatments containing deionized water vehicle control without or with with or without control vehicle water deionized containing treatments dosed of inoculation after h 12 0 and at cultures batch mixed in concentrations ammonia and nitrite, Nitrate, 1: Table 19

Chapter 3: The Effect of Live Yeast Culture and Nitrate on Methane Mitigation and Nitrate Reduction in Continuous Culture

Introduction

Nitrates have been fed to ruminants to decrease CH4 emissions, which has been decreased by up to 50% in some studies (Giraldo et al., 2007; McAllister and Newbold,

2008; Machmüller et al., 2006). Methanogenes are thermodynamically outcompeted by

NO3 salts, preventing the archaea from producing CH4. The problem when feeding NO3 salts is the slower assimilation of the intermediate NO2 to NH3 than that of NO3 to NO2.

The slower reduction of NO2 leads to a buildup and eventual absorption of NO2 into the bloodstream, potentially causing tissue asphyxiation and methemoglobinemia (ATSDR,

2011). Theoretically, the yeast S. cerevisiae should be able to reduce NO2 within the rumen via its cytochrome c oxidase (Castello et al., 2006). Besides a possible reduction of NO2, live yeast are also capable of improving digestibilities and increasing VFA production (Dawson, 1995; Desnoyers et al., 2009). We hypothesize that the addition of

NO3 will decrease CH4 production and that the interaction of NO3 and live yeast culture will decrease the NO2 concentration in continuous cultures from which fermentation production can be measured for quantitative stoichiometric analyses.

Methods

Four dual flow continuous cultures designed to retain protozoa (Karnati et al.,

2009) were used in a 5 x 4 Youden square design with 5 periods of 10 d; 7 d of 20 adaptation followed by 3 d of collection. All four fermenters were fed 40 g DM daily of a

50: 50 forage and concentrate pellets (32.4% starch and 37.35% NDF in the total diet;

Table 2). The treatments were either NO3 (1.5% of diet DM, 1.85 mM dosed concentration) or urea on an isonitrogenous basis and without or with live yeast culture

(Yea-Sacc®1026; Alltech Inc., Nicholasville, KY) fed at a recommended 0.010 g/d

(approximately 10 x 106 CFU, scaled from cow to fermenter) administered in a 2 x 2 factorial arrangement of treatments. Four liters of rumen fluid were collected as previously stated in the prior experiment. The rumen fluid was pooled and added to 39⁰C anaerobic buffer at half fermenter volume, as per the protocol of Hoover et al. (1976).

Fermenter volumes ranged from 1600 to 1850 mL. The vessels were maintained anaerobically at a pH range between 6.0 and 7.0 by a 7.0% influx of buffer, temperature at 39°C, and agitation at 50 rpm.

Daily protozoa counts, pH, and flow rate were recorded during the duration of the adaptation period. The continuous culture vessels were monitored constantly for CH4 and

H2 via a Micro-Oxymax Respirometer (Columbus Instruments, Inc., Columbus, OH). On d 6 at h 0, 4, 8, and 12, a 10-mL sample was pulled from fermenters with a 60 mL pipette and combined with 10 mL of formalin for protozoa counts. Isotrichia, Dasytrichia,

Epidinium, Ophryoscolex, total Entodinium, large Entodinium (> 100 μm), and the subfamily Diplodiniinae were characterized per Dehority (1993).

On d 7, 8, and 9, 40 mL of effluent were withdrawn from the liquid effluent line of each fermenter at h 0, 0.5, 1, 1.5, 2, 4, 8, and 24. From these samples, 10-mL was used for NO3, NO2, and NH3; to the second 10-mL aliquot was added 1 mL of 6N HCl to stop

21 fermentation and frozen at -20⁰C for subsequent VFA analysis; and the last 20-mL of sample was injected into a culture bottle with 5 mL 15N H3PO4 flushed with N2 for aqueous H2 analysis (Wenner et al., 2014). The samples were prepped and analyzed for

NO3, NO2, and NH3 as in the previous experiment. Nitrite IC results were contaminanted by carbonate from within the buffer, so an average carbonate value was subtracted from each IC NO2 value. VFA samples were thawed, centrifuged at 15,000 x g for 15 min then filtered through a Whatman #1 filter paper. A 1.6-mL aliquot was then placed into a 2- mL microcentrifuge vial with 0.4 mL of 25% m-HPO3 and micro-centrifuged at 12,500 rpm for 15 min; a 1.0-mL aliquot was combined with 0.1 mL of internal standard (2-ethyl butyric acid), pipetted into a glass GC vial, and frozen until analysis by GC according to

Firkins et al. (2009). VFA pool size was calculated by concentration multiplied by fermenter volume.

Daily solid and liquid effluents were pooled from each fermenter at h 24 on d 7, 8, and 9 after which samples were aspirated for sub-sampling. A 10 mL sample was combined with 10 mL of formalin for protozoal generic counts as described previously.

Based on fermenter pool sizes and effluent flow, total protozoa generation time was calculated (Karnati et al, 2009). From the effluent, a 50-mL aliquot was saved for VFA as described previously. VFA production was calculated by multiplying VFA concentration by fermenter’s average effluent flow Approximately 20% of effluent volume was sampled, stored frozen, and subsequently dried in a 55⁰C oven prior to grinding. A 2-g aliquot was used for DM and OM analysis (AOAC, 1990), 0.6-g aliquot was used for CP

22 analysis via a Kjeldahl (AOAC, 1990), and 0.5-g for NDF analysis (Van Soest et al.,

1991),

Statistical Analysis

The daily samples were analyzed by MIXED procedure of SAS (SAS Institute

Inc., Cary, NC v9.4) using the following model Yijkl = μ + Ni + Sj + Pk + fl + NSij + eijkl.

In the model Yijkl, μ, Ni (i = 1, 2), Sj (j = 1, 2), NSij, and eijkl are the same variables as the

th previous study with Pk as the fixed effect of the k period (k = 1, 2, 3, 4, 5), and fl as the random effect of the lth fermenter (l = 1, 2, 3, 4). The data from hourly and continuous samples were ran with repeated measures using the model of Yijkl = μ + Ni + Sj + Pk + fl +

Hm + NSij + NSHijm + eijkl, where the variables are as stated prior. One fermenter’s gas production was omitted for all periods (unrelated to treatment). Treatments were analyzed by ANOVA, trends proclaimed between 0.05 < P < 0.15 and significant at P ≤ 0.05.

Results and Discussion

Neither NO3 nor live yeast culture affected apparent OM or apparent N digestibilities, but NDF digestibility increased significantly when live yeast culture was added relative to the control treatment (Table 3). van Zijderveld et al. (2011) found no differences between urea or NO3 diets for NDF digestibility. Although not studied in our trial, van Zijderveld et al. (2011) noted no differences in starch or fat digestibilities in vivo between urea and NO3 diets. As long as the methemoglobinemia can be avoided from a digestibility standpoint, NO3 appears to be an acceptable replacement for urea in

23 ruminant diets at these concentrations. However, palatability needs further testing. Live yeast culture has increased NDF digestibility and other fermentation characteristics like

VFA production (Harrison et al., 1988; Callaway and Martin, 1997; Shaver and Garrett,

1997; Miller et al., 2002).

There were no significant interactions of protozoa counts between NO3 and live yeast culture treatments (Table 4). The main effect of NO3 had a tendency to be significant for total Entodinium, Isotricha, and Ophryoscolex (P = 0.11, 0.15, and 0.07, respectively). Total Entodinium increased when NO3 was added to the diet over the control (691 versus 552 cells/mL). Because of the negligible number of Isotricha and

Ophryoscolex within the continuous culture vessels, these trends may not be entirely accurate. Live yeast culture did not affect protozoa populations, as seen in Mathieu et al.

(1996), but they noted a trend for an increase in Entodinium for live yeast culture.

Arakaki et al. (2000) reported that, over a prolonged period of time (124 d), yeast culture increased protozoal concentration. There was no difference of generation time between the main effects or interaction of NO3 and live yeast culture in our study, longer term experiments should be repeated.

The interaction and main effects of NO3 and live yeast culture were not affected by treatment (P > 0.15) for total or individual daily VFA production (Table 5). The total daily VFA production averaged 146 ± 15 mmol/d. Acetate: propionate averaged 3.37 ±

0.10. Nitrate has been seen in other studies to alter VFA by increasing acetate and decreasing butyrate concentrations (Bryant, 1965; Farra and Satter, 1971; Alaboudi and

Jones, 1985; Guo et al., 2009). Guo et al. (2009) reported an increase in the molar

24 percentage of acetate between urea and NO3 diets (61.7 and 67.5) and a decrease in butyrate (9.82 and 5.4); in our study, there were no significant changes for acetate and butyrate. Similarly to our study, van Zijderveld et al. (2010) noted no VFA differences between nitrate and control (urea) treatments. Desnoyers et al. (2009) reported an increase in VFA production when yeast was added.

Fermenter pool size VFA (concentration scaled to fermenter volume for comparison to production rates) had treatment by time interactions for all individual and total VFA, so data were analyzed per each time point. The NO3 by live yeast culture interaction (P < 0.05) was detected at 24 h for total VFA (Figure 1). Live yeast culture increased the pool size without NO3 but decreased it with NO3. Total VFA pool size had a tendency for significance for NO3 at 0, 0.5, and 2 h. Live yeast culture was higher (P

<0.05) at 2 h relative to the main effect of control. Total VFA concentration was increased as seen in Desnoyers et al. (2009) and Arcos-García et al. (2000); the improvement in NDF digestibility by yeast might not have been enough to increase the cumulative VFA production.

Acetate pool size had a significant NO3 by yeast culture interaction for 24 h, where yeast alone increased acetate pool size and decreased it with the interaction NO3

(Figure 2). The main effect mean of NO3 was greater than the main effect of control for acetate pool size at h 0.5, 1.5, and 2 and tended to be higher at h 0 and 8. The main effect of live yeast culture was only lower (P < 0.05) at 2 h for acetate pool size, with a trend at

1 h. Acetate molar proportion is often increased by yeast culture (Bryant, 1965; Farra and

Satter, 1971; Alaboudi and Jones, 1985; Guo et al., 2009; Zhao et al., 2015). The

25 interaction of NO3 and live yeast culture was P < 0.05 at 24 h, with a trend at 2 h for propionate pool size (Figure 3). The live yeast culture increased the pool size without

NO3 and decreased it with NO3. The main effect of NO3 had a trend (P = 0.08) for lower pool size of propionate at 24 h and was higher for live yeast culture at 2 h (P < 0.05).

Unlike in Bryant (1965) and Farra and Satter (1971) in which propionate concentration decreased by the addition of NO3, our study aligned with Guo et al. (2009) and Zhao et al. (2015), who reported no significant differences between NO3 treatments and control on propionate production. The interaction of NO3 and live yeast culture was not significant at any time point for the acetate to propionate ratio (Figure 4). However, the acetate to propionate ratio increased (P < 0.05) at all times for the main effect of NO3.

Live yeast also had a tendency for a higher acetate to propionate ratio at 0 h (P = 0.07), and it increased significantly at h 2 and 4. Nolan et al. (2010) reported a similar increase in the acetate to propionate ratio when ruminants were fed NO3 treatments. The live yeast

S. cerevisiae decreased the acetate to propionate ratio in the study of Arcos-García et al.

(2000), but Nisbet and Martin (1991) noted a slight increase in this ratio.

The interaction between NO3 and live yeast culture was P < 0.05 for butyrate pool size at 24 h and trend at 2 h, with an increased pool size by the addition of live yeast and a decrease with NO3 and yeast (Figure 5). The main effect of NO3 was significantly lower at h 1, 1.5, and 2 for butyrate pool size, with a trend at 4 h. The butyrate decrease as a result of NO3 treatments over the control is consistent with previous studies (Bryant,

1965; Farra and Satter, 1971; Alaboudi and Jones, 1985; Guo et al., 2009). Butyrate pool size also had a trend at 2 h for the main effect of yeast. Interactions (P < 0.05) of NO3 and

26 live yeast culture at 24 h were noted for isobutyrate and valerate and a trend (P = 0.10) for isovalerate resulted from live yeast culture increasing pool sizes without NO3 but decreasing pool sizes when combined with NO3 (Figures 6, 7, and 8). Because this effect or trend was consistent across all VFA, it is related to total VFA pool size (Figure 1). The main effect of NO3 was higher (P < 0.05) at 0 h for isobutyrate and at h 0.5 and 1 for isovalerate. There were also NO3 trends at h 0.5 for isobutyrate, h 2 and 24 for valerate, and h 1.5, 2, and 8 for isovalerate. Live yeast culture’s main effect had a trend to increase pool size at 2 h for isobutyrate (P = 0.08) and isovalerate (P = 0.06) and was significant for valerate. Zhao et al. (2015) noted no differences for isobutyrate, valerate, or isovalerate concentration between 1.0% and 2.0% (DM basis) addition of NO3 when compared to a control, which agrees with our study from a cumulative measurement. All

VFA production over time followed a similar pattern to that of Stewart et al. (1958) or a normal VFA production over time after feeding.

Methane was decreased by the NO3 treatment (P < 0.05), although the interaction of NO3 and live yeast culture was not significant (P > 0.15). Nitrate thermodynamically outcompetes methanogens and its reduction pathway intermediates NO2 and NO are known inhibitors of methanogenesis, as was seen in continuous culture (Sar et al., 2004; van Zijderveld et al., 2010; 2011). On average the CH4 was decreased by 29.0% (29.5 vs

21.0 ± 7.7 mmol/d) when NO3 was added to the diet (Table 5). According to Knapp et al.

(2014) electron acceptors NO3 and SO3 can reduce CH4 by 16 to 57%. van Zijderveld et al. (2010) showed a decrease in CH4 of 16 to 47% with NO3 and SO3 treatments. There was a significance of treatment by time for incremental rate of CH4, but there was no

27 significant interaction of time between NO3 and live yeast culture (Figure 9). The main effect of NO3 decreased (P<0.05) at hours 2, 2.5, 3, 4, 5, and 6 (Figure 9). The CH4 production followed a pattern similar to that of Nolan et al. (2010), where post feeding the CH4 concentration fell in the first hour and increased in the second; our study had a more obvious decline for the NO3 treatment over the control. The overall shape of our

CH4 production curve follows that of van Zijderveld et al. (2010) with peak CH4 production occurring 6 h post feeding and significant differences between the control and

NO3 from h 2 to 6. Although there was no significance of time for live yeast culture, it closely follows the control pattern until h 8, then it resembles the NO3 pattern. The interaction of NO3 and live yeast culture might have been seen as significant with a higher NO3 or live yeast culture dose.

Daily production of gaseous H2 was not different among treatments, averaging

0.148 ± 0.053 mmol/d (Table 5). In contrast, aqueous H2 concentration was about 46% higher for the main effect means of NO3 (3.66 mM) compared with diets without NO3,

(2.51 mM; Table 5). There was a significant interaction by time for incremental rate of H2 emission (Figure 10). There were no significant interactions, but the main effect of NO3 and live yeast culture both tended to be lower at several time points < 8 h. Zhou et al.

(2012) reported an inhibition of H2 production when NO3 was fed to a culture due to inhibition of H2-producing bacteria; although we cannot determine yet if this is the case in our study, it seems an unlikely because most of the difference peaked at 3 h (Figure

10). With prolonged feeding, abundance of archaea decreased when fed NO3 salts (van

Zijderveld et al., 2010). Archaea probably have a higher affinity for aqueous H2

28 compared with NO3 reducing bacteria; therefore, a decreased abundance of archaea would allow a greater aqueous H2 concentration while still effectively using it to reduce

NO3 or NO2 (Hristov et al., 2013).

Both concentrations of NO3 (Figure 11) and NH3 (Figure 13) interacted with time

(P < 0.01), but NO2 (Figure 12) did not (P > 0.15). Interactions of NO3 and live yeast culture had a trend for increased NO3 concentration at 4 h and an increase in NO2 concentration at 2 h. Nitrate concentration increased (P < 0.01) at h 0.5 and 1 post feeding of NO3 and had tendencies for increased concentrations at h 2 and 4. This response to NO3 feeding had minimal effect on NH3 concentration, which only decreased for the NO3 treatments at 1 h. Nitrate rapidly increased in the first 0.5 hour after dosing and then declined until 2 h (Lewis, 1951; Takahashi and Young, 1991; Tillman et al.,

1965; Sar et al., 2004). In our study, the NO2 concentration coincided with NO3 dosing until 2 h and then numerically persisted at 4 h (trends for NO3 and live yeast culture treatment main effects, explaining the lack of treatment by time). Although these values must be qualified because of indirect calculation (subtracting of carbonate anion that coeluted with NO2). McAllister et al. (1996) found that NO2 peaked 4 to 6 hours post

NO3 dose, but our study had two peaks for NO2 production, at 0.5 h for the main effect of

NO3 and with the addition of live yeast culture at 4 h. The addition of live yeast culture hypothesized to decrease NO2 production actually had the reverse effect. Although not significant, live yeast culture combined with NO3 had a higher concentration of NO3 and

NO2 than just a solo NO3 dose. In part based on studies with aqueous bacteria (Sayama,

2001), Leng (2005) postulated that nitrate sequestered in ruminal bacteria. Although NO3

29 is not assimilatory in S. cerevisiae, they should have the ability to efflux NO3 or NO2 from stores produced in NO signaling pathways (Cabrera et al., 2014), so the live yeast also could be sequestering NO3 and/or NO2 for subsequent efflux over time. Hinze and

Holzer (1985) noted that when NO2 was added to yeast culture a decrease in ATP levels could be noted, which can be explained by an inhibition of glyceraldehydes-3-phosphate dehydrogenase by NO2 (Hinze and Holzer 1986). This inhibition of yeast by NO2 could explain why there was no significant interaction between NO3 and live yeast culture.

For NH3 the only significant main effect was at 1 h at which time NO3 was lower

(P < 0.01) when compared to the control at (Figure13). The overall shape of the NH3 curve follows that demonstrated by Lewis et al. (1951) until 8 h, where NH3 concentration gradually declined followed by a shift upward in concentration to 24 h, returning to beginning NH3 concentrations. Unlike in Guo et al. (2009), in which the urea treatment produced more NH3 than the NO3 treatment, our study shows no difference We cannot conclude whether or not NO3 with the addition of live yeast has a beneficial enough impact on ruminants in order to replace urea in the diet and prevent methemoglobinemia, but there is a likely case.

Table 2: Ingredient and nutrient composition of continuous cultures treatment diets without (-) or with (+) nitrate or live yeast culture (LYC).1,2,3

NO3 - - + + LYC Item - + - + Ingredients, % of DM Alfalfa meal 44.4 44.9 44.4 44.9 Soybean hulls 16.4 16.6 16.4 16.6 Corn, ground 17.9 18.1 17.9 18.2 Distillers grains with solubles 4.4 4.5 4.4 4.5 Soybean meal, 48% CP 4.0 4.1 4.0 4.1 Corn oil 0.80 0.81 0.80 0.81 TM Salt 0.40 0.40 0.40 0.40 Dicalcium phosphate 0.34 0.35 0.34 0.35 Magnesium oxide 0.09 0.09 0.09 0.09 Nitrate 1.9 0 1.9 0 Urea 0 0.66 0 0.66 Yea-Sacc®4 0 0.02 0.02 0

Nutrients DM (% As Fed) 90.6 90.5 90.6 90.5 OM (% of DM) 89.7 91.5 89.7 91.5 NDF (% of DM) 40.9 41.5 40.9 41.5 CP (% of DM) 15.0 17.2 15.0 17.2

1 Diets were fed 40 g/d of 50:50 alfalfa to concentrate pellets. 2 Nitrate was added to the diet at 1.5% of diet DM. 3 Live yeast culture (LYC) was added to the diet at 0.010 g/d. 4 Strain 1026; Alltech Inc., Nicholasville, KY.

Interaction

LYC

<0.01

-value of effects of -value

> 0.15). >

1.6

3.0

3.9

SEM

Pooled Pooled

40.5

33.9

55.0

+ LYC +

+ NO +

43.8

38.7

50.6

-LYC

41.2

32.3

56.6

+ LYC +

-NO

41.0

34.1

45.8

-LYC

Treatment Dosed into Continuous Cultures Continuous into Dosed Treatment

1,2

LYC added at 0.010 g/d (scaled from cow to fermenter). to cow from (scaled g/d 0.010 at added LYC

Nitrate added at 1.5% diet DM; urea was added to the diet isonitrogenously with the nitrate treatment. nitrate the with isonitrogenously diet the to added was urea DM; diet 1.5% at added Nitrate

Main effects of nitrate or LYC treatments or their interaction. NS = not significant ( significant NS not = interaction. their or treatments LYC or nitrate of effects Main

2 2

1 1

OM,Apparent

N,Apparent

NDF

Item, % Item,

live yeast culture (LYC) culture yeast live Table 3: Nutrient digestibilities in continuous cultures administered diets supplemented without (-) or with (+) nitrate or or nitrate (+) with or (-) without supplemented diets administered cultures continuous in digestibilities Nutrient 3: Table

Interaction

0.07

LYC

-value of effects of -value

0.07

0.15

0.11

> 0.15). >

4.8

2.9

3.4

3.2

2.8

8.2

4.9

SEM

Pooled Pooled

6.7

6.4

732

29.8

12.7

12.8

<0.01

+ LYC +

+ NO+

0.2

3.2

3.9

650

35.1

<0.01

- LYC

2.4

3.2

503

36.6

12.7

19.1

18.8

<0.01

+ LYC +

1,2

- NO

0.2

9.6

3.2

6.4

5.8

541

32.7

<0.01

- LYC

Treatment Dosed into Continuous Culture Continuous into Dosed Treatment

cell length >100 >100 length cell

(cells/mL)

Entodinium =

Entodinium

LYC added at 0.010 g/d (scaled from cow to fermenter). to cow from (scaled g/d 0.010 at added LYC

Nitrate added at 1.5% diet DM; urea was added to the diet isonitrogenously with the nitrate treatment. nitrate the with isonitrogenously diet the to added was urea DM; diet 1.5% at added Nitrate

Large Large

Main effects of nitrate or LYC treatments or their interaction. NS = not significant ( significant NS not = interaction. their or treatments LYC or nitrate of effects Main

Generation Time (h) Time Generation

Dasytrichida

Epidinium Epidinium

Ophryoscolex Ophryoscolex

Isotrichida Isotrichida

Diplodiniinae (cells/mL) Diplodiniinae

Large Large

Total Total

Item

with (+) nitrate or live yeast culture (LYC) culture yeast live or nitrate (+) with Table 4: Protozoal counts and total generation time in continuous cultures administered diets supplemented without (-) or or (-) without supplemented diets administered cultures continuous in time generation total and counts Protozoal 4: Table

Table 5: Fermentation characteristics in continuous cultures administered diets supplemented without (-) or with (+) nitrate or live yeast culture (LYC)1,2 Treatment Dosed into Continuous Culture 3 - NO3 + NO3 Pooled P -value of effects

Item - LYC + LYC - LYC + LYC SEM NO3 LYC Interaction Total VFA (mmol/d) 157 136 147 143 15 NS NS NS

Acetate (mmol/d) 101 88.2 96.8 94.6 10.2 NS NS NS

Propionate (mmol/d) 30.7 26.8 28.5 27.2 3.0 NS NS NS

Acetate: Propionate 3.28 3.35 3.38 3.49 0.10 NS NS NS

Isobutyrate (mmol/d) 1.30 1.16 1.16 1.15 0.15 NS NS NS

Butyrate (mmol/d) 17.6 15.1 15.6 15.1 1.1 NS NS NS

Isovalerate (mmol/d) 3.39 3.08 3.24 3.15 0.29 NS NS NS

Valerate (mmol/d) 2.41 2.03 2.12 2.14 0.20 NS NS NS

CH4 (mmol/d) 29.8 29.3 22.5 19.5 7.7 <0.05 NS NS

H2 (mmol/d) 0.196 0.153 0.153 0.092 0.053 NS NS NS

4 aqH2 (mM) 2.64 2.38 3.42 3.90 1.36 <0.01 NS NS 1 Nitrate added at 1.5% diet DM; urea was added to the diet isonitrogenously with the nitrate treatment. 2 LYC added at 0.010 g/d (scaled from cow to fermenter). 3 Main effects of nitrate or LYC treatments or their interaction. NS = not significant (P > 0.15). 4 aqH2 = aqueous hydrogen.

0.05, 0.05,

≤

0.01, * = = * 0.01,

≤

Nitrate*LYC

LYC

Time (h) Time

Nitrate

Control

< 0.01. Main effect of nitrate, LYC or their interaction at each time point (** = = (** point time each at interaction their or LYC nitrate, of effect Main 0.01. <

P P

NS NS NS NS NS NS NS NS NS NS NS NSNS NS

0.15 0.13 NS NS 0.06 NS NS NS NS 0.06 NS 0.13 0.15 NS

> 0.15). >

100

110

120

130 140

Total VFA (mmol) VFA Total

LYC NS NS NS NS * NS NS NS NS NS * NS NS LYC

Interact Interact

NS =

Treatment by time interaction was was interaction time by Treatment Figure 1: Pool size of total VFA in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). (LYC). culture yeast live or nitrate with or without supplemented diets administerd cultures continuous in VFA total of size Pool 1: Figure

P < < 0.05 P

Nitrate*LYC

LYC

Time (h) Time

Nitrate

Control

< 0.01. Main effect of nitrate, LYC or their interaction at each time point (** = P ≤ = P = ≤ * 0.01, NS 0.05, (** P = > point time each at interaction their or LYC nitrate, of effect Main 0.01. <

P P

0.07 * NS * ** NS 0.13 0.13 NS NS * ** 0.07 *

3 100

Acetate (mmol) Acetate

LYC NS NS 0.14 NS * NS NS NSNS * NS NS 0.14 NS LYC

Interact NS NS NS NS NS Interact NS NS

0.15).

by time interaction was was interaction time by Figure 2: Pool size of acetate in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). Treatment Treatment (LYC). culture yeast live or nitrate with or without supplemented diets administerd cultures continuous in acetate of size Pool 2: Figure

0.08

P < < 0.05 P

Nitrate*LYC

LYC

Time (h) Time

Nitrate

Control

< 0.01. Main effect of nitrate, LYC or their interaction at each time point (** = P ≤ = P = ≤ * 0.01, 0.05, (** point time each at interaction their or LYC nitrate, of effect Main 0.01. <

NS NS NS NS NS NS NS NS NS NS NS NS NS

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5 30.0

Propionate (mmol) Propionate

LYC NS NS NS NS * NS NS NS NS NS * NS NSLYC

Interat Interat NS NS NS NS 0.11 NS NS

NS P 0.15). = >

Treatment by time interaction was was interaction time by Treatment Figure 3: Pool size of propionate in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). (LYC). culture yeast live or nitrate with or without supplemented diets administerd cultures continuous in propionate of size Pool 3: Figure

P < < 0.05 P

Nitrate*LYC

> 0.15. Main effect of nitrate or LYC at each time point (** = P = (** point time each at LYC or nitrate of effect Main 0.15. >

LYC

Time (h) Time

Nitrate

Control

< 0.01. Nitrate by LYC interaction was was interaction LYC by Nitrate 0.01. <

** ** ** ** ** ** * * ** ** ** ** ** **

3.0

3.1

3.2

3.3

3.4

3.5

3.6

3.7

3.8

3.9

4.0 3

Acetate: Propionate Acetate:

LYC 0.07 NS NS NS * * * * NS NS NS 0.07 LYC

≤ P = ≤ * 0.01, NS 0.05, P 0.15). = >

Treatment by time interaction was was interaction time by Treatment Figure 4: Acetate to propionate ratio in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). (LYC). culture yeast live or nitrate with or without supplemented diets administerd cultures continuous in ratio propionate to Acetate 4: Figure

* *

P < < 0.05 P

Nitrate*LYC

LYC

Time (h) Time

Nitrate

Control

< 0.01. Main effect of nirate, LYC or their interaction at each time point (** = P ≤ = P = ≤ * 0.01, NS 0.05, (** P = > point time each at interaction their or LYC nirate, of effect Main 0.01. <

NS NS ** * * 0.15 NS 0.15 * * ** NSNS

17 19

Butyrate (mmol) Butyrate

LYC NS NS NS NS 0.11 NS NS NS NS NS 0.11 NS NSLYC

Interact N NS NS NS 0.12 NSInteract NS

0.15).

by time interaction was was interaction time by Figure 5: Pool size of butyrate in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). Treatment Treatment (LYC). culture yeast live or nitrate with or without supplemented diets administerd cultures continuous in butyrate of size Pool 5: Figure

P < < 0.05 P

Nitrate*LYC

LYC

Time (h) Time

Nitrate

Control

< 0.01. Main effect of nitrate, LYC or their interaction at each time point (** = P ≤ = P = ≤ * 0.01, NS 0.05, (** point time each at interaction their or LYC nitrate, of effect Main 0.01. <

* 0.13 NS NS NS NS NS

0.5

0.6

0.7

0.8

0.9

1.0 1.1

Isobutyrate (mmol) Isobutyrate

LYC NS NS NS NS 0.08 NS NS NS NS NS 0.08 NS NSLYC

Interat Interat NS NS NS NS NSNS N

= P 0.15). = >

Treatment by time interaction was was interaction time by Treatment Figure 6: Pool size of isobutyrate in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). (LYC). culture yeast live or nitrate with or without supplemented diets administerd cultures continuous in isobutyrate of size Pool 6: Figure

0.13

P < < 0.05 P

Nitrate*LYC

LYC

Time (h) Time

Nitrate

Control

NS NS NS NS 0.14 NS NS NS NS 0.14 NS NS NSNS

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50 2.75

Valerate (mmol) Valerate

LYC NS NS NS NS ** NS NS NS NS NS ** NS NSLYC

Interact NSNS NS NS Interact NS NS NS

0.15).

by time interaction was was interaction time by Figure 7: Pool size of valerate in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). Treatment Treatment (LYC). culture yeast live or nitrate with or without supplemented diets administerd cultures continuous in valerate of size Pool 7: Figure

0.10

†

* * * * * *

P < < < < < < < 0.05 0.05 0.05 0.05 0.05 0.05 PPPPPP

P < < 0.05 P

S S

Nitrate*LYC

LYC

Time (h) Time

Nitrate

Control

< 0.01. Main effect of nitrate, LYC or their interaction at each time point (** = P ≤ = P = ≤ * 0.01, NS 0.05, (** point time each at interaction their or LYC nitrate, of effect Main 0.01. <

P P

* * NS 0.09 0.06 NS 0.09 0.09 NS NS * * 0.06 0.09

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00 3.25

NO (mmol) Isovalerate

LYC NS NS NS NS 0.06 NS NS NS NS NS 0.06 NS NS LYC

Interact N NS NS NS NS NS Interact NS

= P0.15). = >

Treatment by time interaction was was interaction time by Treatment Figure 8: Pool size of isovalerate in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). (LYC). culture yeast live or nitrate with or without supplemented diets administerd cultures continuous in isovalerate of size Pool 8: Figure

†

ns ns

NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS

NS 0.14 NS 0.09 NS 0.06 NS * NS * NS 0.09 NSNS 0.06 NS 0.14

NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS

P < < 0.05 P

Nitrate*LYC

LYC

Time (h) Time

Nitrate*Yea-Sacc

Nitrate

Time Time (h)

Control

Yea-Sacc

Control

NS NS * * * 0.10 0.08 * NS 0.12 NS 0.10 NS NS NS NS 0.10 NS * 0.08 0.10 * * * NS NS NS 0.12 NS

† * ns * * ns ns ns ns **ns * †

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

100

YS ns ns ns ns * ns ns ns ns * ns ns nsns YS NO Acetate (mmol) Acetate

NO (mmol/h) Methane

LYC NS LYC NS NS NS NS 0.07 NS NS NS NS NS NS NS NS NS

Interact 0.10 NS NS NS NS NS NS NS NS NS NS NS NS NS NS Interact

interaction was P < 0.01. Main effect of nitrate, LYC or their interaction at each time point (** = P ≤ = P = ≤ * 0.01, NS 0.05, (** P 0.15). = > point time each at interaction their or LYC nitrate, of effect Main P 0.01. < was interaction Figure 9: Incremental methane production over time in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). Treatment by time time by Treatment (LYC). culture yeast live or nitrate with or without supplemented diets administerd cultures continuous in time over production methane Incremental 9: Figure

†

ns ns

NS NS NS NS NS 0.09 NS NS NS NS NS 0.09 NS NS NS

P < < 0.05 P

NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS

NS NS NS NS NS NS NS NS NS NS NS NS NS NSNS

Nitrate*LYC

LYC

Time (h) Time

Nitrate*Yea-Sacc

Nitrate

Time Time (h)

Control

Yea-Sacc

Control

0.10 0.09 NS NS 0.09 NS 0.11 NS NS NS 0.11 NS 0.10 NS NS

† * ns * * ns ns ns ns **ns * †

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

100

YS ns ns ns ns * ns ns ns ns * ns ns nsns YS (mmol/h) Hydrogen NO

Acetate (mmol) Acetate

Interact NS NS NS NS NS NS NS NS NSNS NS NS NS NS Interact 0.11

LYC NS NS 0.09 0.10 0.12 * NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS * 0.12 0.10 0.09 NS LYC

interaction was P < 0.01. Main effect of nitrate, LYC or their interaction at each time point (** = P ≤ = P = ≤ * 0.01, NS 0.05, (** P 0.15). = > point time each at interaction their or LYC nitrate, of effect Main P 0.01. < was interaction Figure 10: Incremental hydrogen production over time in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). Treatment by time time by Treatment (LYC). culture yeast live or nitrate with or without supplemented diets administerd cultures continuous in time over production hydrogen Incremental 10: Figure

† †

* * * * * * *

* *

* * * * * * *

P < < < < < < < < 0.05 0.05 0.05 0.05 0.05 0.05 0.05 PPPPPPP

P < < 0.05 P

Nitrate*LYC

LYC

Time (h) Time

Nitrate

Control

< 0.01. Main effect of nitrate, LYC or their interaction at each time point (** = P ≤ = P = ≤ * 0.01, NS 0.05, (** point time each at interaction their or LYC nitrate, of effect Main 0.01. <

P P

NS ** ** NS 0.07 0.12 NS 0.12 0.07 NSNS ** **

0.0

0.5

1.0

1.5

2.0

2.5

3.0 3.5 Nitrate (m Nitrate M

)

LYC NS NS NS NS NS 0.15 0.15 0.15 0.15 NS NS NS NS NS LYC

Interact NSInteract NS NS NS 0.14 NS NS

= P0.15). = >

Treatment by time interaction was was interaction time by Treatment Figure 11: Concentration of nitrate in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). (LYC). culture yeast live or nitrate with or without supplemented diets administerd cultures continuous in nitrate of Concentration 11: Figure

†

P < < 0.05 P

Nitrate*LYC

LYC

Time (h) Time

Nitrate

Control

0.15. Main effect of nitrate, LYC or their interaction at each time point (** = P = ≤ P 0.01, * = ≤ (** 0.05, NS = P > 0.15). point time each at interaction their or LYC nitrate, of effect 0.15. Main

P >

NS ** * * NS 0.14 NS 0.14 NS NS * * **

-1 Nitrite (m Nitrite M

)

LYC NS NS NS NS NS 0.08 NS 0.08 NS NS NS NS NSLYC

Interact NSInteract NS NS NS 0.12 NS NS

interaction was was interaction

quantified by ion exchange chromatography minus baseline buffer without any extra salts added (including coeluting CO3). Treatment by time time by CO3). Treatment coeluting (including added salts extra any without buffer baseline minus chromatography exchange ion by quantified Figure 12: Concentration of nitrite in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). NO2 (LYC). culture yeast live or nitrate with or without supplemented diets administerd cultures continuous in nitrite of Concentration 12: Figure

†

* *

P < < 0.05 P

> 0.15. Main effect of nitrate at each each at nitrate of effect Main 0.15. >

Nitrate*LYC

LYC

Time (h) Time

Nitrate

Control

< 0.01. Interaction of nitrate and LYC and main effect of LYC were were LYC of effect main and LYC and nitrate of Interaction 0.01. <

NS NS * NS NS NS NS NS NS NS * NSNS NS

10 moi (m Ammonia M

)

time point (** = P ≤ = P = ≤ * 0.01, NS 0.05, (** P 0.15). = > point time

Treatment by time interaction was was interaction time by Treatment Figure 13: Concentration of ammonia in continuous cultures administerd diets supplemented without or with nitrate or live yeast culture (LYC). (LYC). culture yeast live or nitrate with or without supplemented diets administerd cultures continuous in ammonia of Concentration 13: Figure

Chapter 4: The Effect on Live Yeast Culture Growth by Replacement of 25%, 50% or 75% of Peptone within YPD Media with Nitrate, Nitrite , Ammonium at aerobic, transition between aerobic and anaerobic, or anaerobic respiration phases.

Introduction

Nitrates are fed to decrease CH4 emissions, but they could lead to NO2 accumulation which can cause methemoglobinemia if the rumen microbes do not fully reduce NO3 to NH3 (ATSDR, 2011). The yeast S. cerevisiae have a cytochrome c oxidase that is predicted to reduce NO2 thereby preventing the buildup of NO2 and its toxicity effects (Castello et al., 2006). We hypothesized that yeast would grow equally on N sources, but that at higher peptone replacement levels there would be a metabolite buildup. Yeast at aerobic phases were predicted to function more efficiently than at the anaerobic phase, with the transition phase being in between.

Methods

A single control and a 3 by 3 factorial arrangement of peptone % replaced (25, 50, and 75%) by N sources (NO3, NO2, and NH3) factorialized with 3 gas phases (aerobic, transition, and anaerobic) and included as treatments for live yeast cultures. S. cerevisiae was grown aerobically and anaerobically in liquid YPD media grown in a 39⁰C shaking incubator. Yeasts cultures were removed during log phase at an average OD of 0.20 for inoculation. YPD media was made per practices in the Yu lab with 0% of peptone, 25% of peptone, 50% of peptone, or 75% of peptone replaced isonitrogenously by sodium

48 nitrate, sodium nitrite, or ammonium sulfate. One third of each media was covered in foil and left aerobic, while the other two thirds were gassed with CO2 and stoppered to be maintained anaerobically. Five in vitro tubes were filled with 12 mL of each aerobic media and 10 tubes were filled with each anaerobic media. 1.2 mL of aerobic yeasts were dosed into each of the aerobic media tubes and capped with foil for the aerobic gas phase.

Aerobic yeasts was dosed into anaerobic tubes, flushed with CO2, and capped with a one- way valve stopper for the transition gas phase. 1.2 mL of the anaerobic yeast was dosed into the anaerobic tubes, flushed, and capped with a one-way valve stopper for the anaerobic gas phase. Tubes were incubated in a 39⁰C shaking water bath until sampled; one tube was removed from each treatment at h 0, 3, 6, 12, and 24. 5 mL was removed at each sampling time and placed in a glass test tube for immediate measurement of OD at

600 nm on the spectrophotometer and screened microscopically for bacterial contamination. If contamination was noted, samples were excluded from analysis, although the OD appeared unaffected. The experiment was run in 3 replicates in a completely randomized design. The 0-h OD was subtracted from the OD of each subsequent incubation time to account for minor differences among cultures.

Statistical Analysis

Repeated measures was not used because each hour was a separate incubation.

The model included treatment (N replacement source by peptone percentage), gas phase

(aerobic, transition, or anaerobic), hour, and all possible interactions. Yeast growth was analyzed by NLIN (SAS Institute Inc., Cary, NC v9.4) using an exponential growth

49 curve: OD = a + b(1-e-kt) to fit the mean values (averaged across replicates). In the model, the a term is the initial OD (standardized to approximately 0.20), b is the change from the initial OD until its predicted asymptote, k is the first-order fractional growth rate, and t is time in hours. The stated function was deemed more appropriate over other functions that fit a log phase because yeast were growing in the log phase at inoculation. All models converged except for NO2 treatments from which OD either decreased over time or growth phase lagged such that a longer experimental time would have been needed for better fitting. In a subsequent analysis, the YPD media control was omitted, and N replacement source by percentage for N and percentage was evaluated plus all possible interactions. Trends were declared between 0.05 < P < 0.15 and statistically significant at

P ≤ 0.05.

Results and Discussion

Nitrogen source by peptone replacement percentage treatments, gas phase, hour, and all interactions were P < 0.01. Results were inconsistent for gas phase over time, and most of the main effects of treatment was from the NO2 treatment being significantly less than the other treatments. Over time, there were no differences between YPD media control, NO3, and NH4 treatments. When the control was excluded to compare the N sources percentage substitution of N from YPD medium, there was no gas phase by N source by N percentage by hour interaction (P > 0.15). When analyzed by N source, the

NO2 treatment had a N percentage by hour interaction (P < 0.01), but this interaction was P > 0.15 for other N sources. Thus, the results are best illustrated in Figure 14,

50 documenting that gas phase had no consistent effect and that 50 and 75% substitution of

NO2-N from YPD stunted yeast growth almost completely, whereas 25% substitution moderately inhibited growth. The inhibition of yeast growth by the addition of NO2 to the culture is contradictory to our hypothesis that yeast would respire NO2; this still might be true in smaller doses, but more studies are needed to determine. The yeast growth inhibition by NO2 has been noted by Hinz and Holzer (1985, 1986) to be the result of the inhibition of glyceraldehyde-3-phosphate dehydrogenase, leading to a decrease in intracellular ATP content.

Panel A: Aerobic phase

Panel B: Transition phase

Panel C: Anaerobic phase

Chapter 5: Conclusion

As we hypothesized, nitrate thermodynamically outcompeted methanogens resulting in a decrease in methane production. The production of H2 gas did not increase in conjunction with decreased methanogenesis, as seen in some studies feeding NO3.

Assuming that NO3 reducing bacteria replaced archaea, the increased aqueous H2 concentration supports a higher affinity constant for aqueous H2 for NO3 reducers compared with methanogens in our continuous culures. There were no differences in digestibilities, generic protozoa, protozoan generation time, and total and all individual

VFA between NO3 and urea diets, so NO3 could replace urea as a N source in ruminant diets. The addition of live yeast culture to a diet can improve feed digestibility as expected. Although S. cerevisiae did not decrease NO2 concentrations, they might have the ability to sequester NO3 or its intermediates, such as NO2, to prevent methemoglobinemia. More studies are needed to know the exact effect of live yeast on

NO3 reduction.

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