Hyper Ammonia-producing Bacteria Research Articles

Optimizing media and substrate for efficient anaerobic biological ammonia synthesis: A response surface methodology approach

Ammonia ranks among the most extensively produced natural and anthropogenic chemicals due to its immense applications, especially in agriculture as fertilizer. The global demand for ammonia has thus been projected to increase. Haber process, the conventional method of ammonia production is costly, energy intensive and responsible for ∼2% of CO2 emission. The rumen Hyper Ammonia Producing Bacteria (HAB) has the potential to convert protein to ammonia. To develop an industrial feasible ammonia production alternative, this study aims to optimize soy-based media composition for biological ammonia using response surface methodology (RSM). A one-factor-at-a-time (OFAT) statistical design was used to determine the most important media components affecting biological ammonia production. Eight media components were screened, and results showed that K2HPO4, KH2PO4, Na2SO4 and soy meal protein isolate (SMPI) had the most significant impact on biological ammonia production. The four identified media components were further optimized using a central composite design (CCD). The RSM model equation for independent and response variables was computed and used to create the response surface and contour plots. Results showed a maximum biological ammonia experimental yield of ∼7,500 mg/L at 0.0492 g/100 mL K2HPO4, 0.0492 g/100 mL KH2PO4, 0.64 g/100 mL Na2SO4, and 10 % SMPI. The observed yield was ∼80 % higher than the biological ammonia production from previous studies.

93 Red clover as a functional feed: Effects on rumen microbiota of finishing ram lambs

Abstract Red clover (RC) contains bioactive isoflavones. Supplementing RC as a functional feed (as little as 7.5% of the total diet) has been shown to alter ruminal fermentation and improve feed efficiency in ram lambs fed high-concentrate finishing diets. The objectives of this study were to determine if reduced RC inclusion will produce similar rumen microbiological effects. It was hypothesized that RC supplementation below 7.5% RC will alter rumen microbial community composition during the finishing period. Sixteen individually housed Polypay ram lambs (age; 134 ± 2.5 d; initial body weight: 31 ± 0.53 kg) were used in a complete block design and fed one of four isocaloric and isonitrogenous diets (85:15 concentrate:roughage ratio; n = 4 rams diet1): a control diet in which the roughage component (15.0% wt/wt of total diet) consisted of orchardgrass hay, 2.5%-RC (roughage: 2.5% RC; 12.5% orchardgrass wt/wt), 5.0%-RC (roughage: 5.0% RC; 10% orchardgrass wt/wt), and 7.5%-RC (roughage: 7.5% RC; 7.5% orchardgrass wt/wt). Rumen fluid was collected via oral intubation at 0, 2, 4, 6, and 8 h post-feeding at the end of the adaptation period (14 d; all rams on control diet) and at 21-d intervals during the trial. Rumen functional bacterial guilds, including hyper-ammonia-producing bacteria (HAB), amylolytic, lactate-utilizing (LU), and cellulolytic bacteria were enumerated via serial dilution at 4 h post-feeding. Data were analyzed using PROC MIXED with repeated measures in SAS (v.9.4, SAS inst. Inc., Cary, NC). There were no differences between diets on d 0 for any variable (P > 0.05). At d 21, all RC-containing diets exhibited a 10- to 100-fold reduction in ruminal HAB compared with the control (P < 0.05). While suppression of HAB was no longer apparent by d 42 in 2.5%-RC, HAB had decreased 100-fold (109 to 107) in lambs fed the 5.0%-RC and 7.5%-RC diets (P < 0.01). No differences in amylolytic bacteria were detected on d 21 (P > 0.05), but were 10-fold less in all diets containing RC by d 42 in comparison with control (109 to 108; P = 0.08). In contrast, LU increased by d 21 and were 100-fold greater (106 to 108) by d 42 when compared with the control for all levels of RC supplementation (P < 0.05). Cellulolytic bacteria also increased across the study period in all lambs fed RC diets, with 100-fold greater cellulolytics (106 to 108) by d 42 (P < 0.05). Consistent with the hypothesis, supplementing RC at levels as low as 2.5% wt/wt of the total diet decreased protein-wasting HAB and amylolytic bacteria while coincidingly increasing LU and cellulolytic bacteria in the rumen. These results indicate that adding red clover to finishing diets has the potential to increase lamb growth and feed efficiency through a functional feed action.

Ionophore antibiotics and hop cones as regulators of digestion and metabolism in ruminants

The general characteristics of ionophore antibiotics and the mechanisms of their antimicrobial action were analyzed. Two types of ionophore antibiotics are known: those that transport ions across the membrane, and those that form a channel in the cell membrane through which ions pass. Ionophore antibiotics used in animal husbandry belong to the former group (monensin, lasalocid, salinomycin, narasin). They are synthesized by bacteria of the Streptomyces genus. Bacterial cells and rumen fluid differ in ionic composition, which is regulated by active ion transport. As a result, the cytoplasm of bacteria contains more potassium ions, while the rumen fluid, on the contrary, has more sodium ions. Ionophores transport potassium inside the cell and remove sodium outside. The bacteria try to correct this imbalance and require ATP energy to carry out this process. Eventually, energy deficit develops in the bacterial cell and it dies. Ionophore antibiotics affect only the Gram-positive bacteria in the rumen of ruminants, because they cannot penetrate through the cell wall of Gram-negative bacteria. Thus, there is a selective destruction of some types of bacteria, the most sensitive among which are the so-called hyper-ammonia-producing bacteria (HAB). Ionophore antibiotics, which change the breakdown of protein and carbohydrates, change the ratio of volatile fatty acids by increasing the proportion of propionic acid and thus inhibit methanogenesis in the rumen. Ionophore antibiotics are an important antiketotic agent because they reduce the concentration of β-hydroxybutyrate and non-esterified fatty acids in the blood of ruminants. During the transition period, cows have metabolic disorders so they are more prone to infectious diseases due to a decrease in resistance. The use of ionophore antibiotics affects the immune function and resistance to inflammatory processes, which is caused by an indirect influence associated with a lower pathological effect of ketosis and steatosis. Ionophore antibiotics reduce the negative energy balance and its pathological impact on metabolism. The influence of ionophore antibiotics on cow’s milk productivity is mostly absent or insignificant. The study presents characteristics of biologically active substances of hop cones. Hop cones contain biologically active components similar to ionophore antibiotics by action. These are prenylated flavonoids: humulone (α-acid), lupulone (β-acid) and their derivatives. These components of hop cones can be regarded as a potential substitute for ionophoric antibiotics. In particular, lupulone and some other components of hop cones inhibit the activity of Gram-positive bacteria, causing reactions similar to ionophore antibiotics. In addition, hop cones have many other biologically active compounds: phenols, essential oils, and resins, which have antimicrobial, antioxidant, sedative, phytoestrogen, insulin stimulating, immunomodulatory, and antitumor effects.

Biological Ammonia Production via Anaerobic Fermentation of Soy Meal Protein.

Conventional ammonia production methods, notably the energy-intensive Haber-Bosch process, are costly and contribute substantially to about 2% of the world's CO2 emissions. This study focuses on the biological approach to convert protein to ammonia via hyper-ammonia-producing bacteria (HAB) fermentation. A consortium of ruminal microbes was employed in this work to ferment soybean meal protein under varying processing conditions. The parameters investigated included pH (7-11), inoculum concentrations (1-10%), substrate concentrations (5-20%), and fermentation time (0-168 h). Optimal conditions for microbial growth and biological ammonia production were observed at pH 7, fermentation duration of 72 h, inoculum concentration of 10%, and substrate concentration of 10%. ~8000 mg/L biological ammonia was produced following HAB fermentation. By leveraging the capabilities of rumen HAB, this study contributes to the ongoing efforts to develop environmentally friendly processes for ammonia production that will mitigate both economic and environmental concerns associated with traditional methods.

Isolation and Characterization of Effective Bacteria That Reduce Ammonia Emission from Livestock Manure.

Ammonia from livestock manure reacts with chemical components discharged from various emission sources to produce airborne particulate matter. This study aimed to investigate a novel effective microbial agent to suppress ammonia gas emitted from manure. Both isolated L12I and 12III strains, identified as Pediococcus acidilactici (PA), were selected for their superior activity in assays performed with the evaluation criteria such as acid production, ammonia decomposition, and urease inhibition, which are key factors influencing ammonia excretion. The survivability of PA strains was confirmed by an increase in DNA abundance in the manure. PA strains lowered the pH of manure and suppressed the growth of hyper-ammonia-producing bacteria (HAB) possessing urease activity. The L12I and 12III treatment groups showed 23.58% and 38.00% emission reductions, respectively. Especially, the 12III strain was proven to be the more effective strain for reducing ammonia gas emission, with the best ability to reduce pH and inhibit HAB. The strains could have an additive effect in improving the manure quality as a nitrogen fertilizer by preserving the total nitrogen and urea content. These results suggest that PA strains can be used as unprecedented microbial agents to improve manure-derived environmental pollution and improve fertilizer quality.

Ruminal tryptophan-utilizing bacteria degrade ergovaline from tall fescue seed extract.

The objectives of this study were to evaluate degradation of ergovaline in a tall fescue [ (Schreb.) Darbysh.] seed extract by rumen microbiota ex vivo and to identify specific bacteria capable of ergovaline degradation in vitro. Rumen cell suspensions were prepared by harvesting rumen fluid from fistulated wether goats ( = 3), straining, and differential centrifugation. Suspensions were dispensed into anaerobic tubes with added Trypticase with or without extract (∼10 μg kg ergovaline). Suspensions were incubated for 48 h at 39°C. Samples were collected at 0, 24, and 48 h for ergovaline analysis and enumeration of hyper-ammonia producing (HAB) and tryptophan-utilizing bacteria. Ergovaline values were analyzed by repeated measures using the mixed procedure of SAS. Enumeration data were log transformed for statistical analysis. When suspensions were incubated with extract, 11 to 15% of ergovaline disappearance was observed over 48 h ( = 0.02). After 24 h, suspensions with added extract had 10-fold less HAB than controls ( = 0.04), but treatments were similar by 48 h ( = 1.00). However, after 24 h and 48 h, suspensions with extract had 10-fold more tryptophan-utilizing bacteria ( < 0.01) that were later isolated and identified by their 16S RNA gene sequence as . The isolates and other known rumen pure cultures ( JB1, B159, HD4, B, F, MD1, SR) were evaluated for the ability to degrade ergovaline in vitro. Pure culture cell suspensions were incubated as described above and samples were taken at 0 and 48 h for ergovaline analysis. Data were analyzed using the ANOVA procedure of SAS. All HAB, including the isolates, tested degraded ergovaline (54 to 75%; < 0.05). B14 was also able to degrade ergovaline but to a lesser capacity (12%; < 0.05), but all other bacteria tested did not degrade ergovaline. The results of this study indicate which rumen bacteria may play an important role in ergovaline degradation and that microbiological strategies for controlling their activity could have ramifications for fescue toxicosis and other forms of ergotism in ruminants.

Biochanin A (an Isoflavone Produced by Red Clover) Promotes Weight Gain of Steers Grazed in Mixed Grass Pastures and Fed Dried‐Distillers’ Grains

Biochanin A (BCA) is an isoflavone produced by red clover (Trifloium pratense L.) that can inhibit hyper‐ammonia‐producing bacteria (HAB) to reduce deamination in the rumen and increase the feed amino acids available for gastric digestion. An ex vivo experiment was conducted to evaluate the effect of dried distiller's grains (DDG) and BCA (30 μg mL−1) on HAB in bovine rumen fluid. Following a 24‐h incubation, HAB and ammonia concentrations in the media increased with addition of DDG. However, DDG fermentations with BCA had 100‐fold fewer HAB and 37% less ammonia than DDG‐only fermentations. A grazing experiment was conducted with crossbred steers grazing a mixture of predominately endophyte‐free tall fescue [Lolium arundinaceum (Schreb.) Darbysh], Kentucky bluegrass (Poa pratense L.), and orchardgrass (Dactylis glomerata L.) in the early and late growing season. Average daily gains (ADG) were compared among treatments of pasture‐only control, daily feeding of 1.4 kg DDG steer−1, and daily feeding of 1.4 kg DDG plus BCA (6.3 g steer−1, representing ∼30% red clover diet). Averaged over the early and late growing seasons, feeding DDG (0.83 ± 0.05 kg d−1) increased ADG by 15% over the pasture‐only treatment (0.72 ± 0.03 kg d−1), but addition of BCA to the DDG (0.93 ± 0.04 kg d−1) provided an additive increase in ADG of 29% over the control treatment. Results of the ex vivo and grazing experiments provide evidence that BCA inhibits HAB in the rumen and that the reduced deamination in the rumen can enhance weight gain performance of steers.

Degradation of spent craft brewer's yeast by caprine rumen hyper ammonia-producing bacteria.

Spent yeast from craft beers often includes more hops (Humulus lupulus L.) secondary metabolites than traditional recipes. These compounds include α-and β- acids, which are antimicrobial to the rumen hyper ammonia-producing bacteria (HAB) that are major contributors to amino acid degradation. The objective was to determine if the hops acids in spent craft brewer's yeast (CY; ~ 3·5mgg(-1) hops acids) would protect it from degradation by caprine rumen bacteria and HAB when compared to a baker's yeast (BY; no hops acids). Cell suspensions were prepared by harvesting rumen fluid from fistulated goats, straining and differential centrifugation. The cells were re-suspended in media with BY or CY. After 24h (39°C), HAB were enumerated and ammonia was measured. Fewer HAB and less ammonia was produced from CY than from BY. Pure culture experiments were conducted with Peptostreptococcus anaerobiusBG1 (caprine HAB). Ammonia production by BG1 from BY was greater than from CY. Ammonia production was greater when exogenous amino acids were included, but similar inhibition was observed in CY treatments. These results indicate that rumen micro-organisms deaminated the amino acids in CY to a lesser degree than BY. Spent brewer's yeast has long been included in ruminant diets as a protein supplement. However, modern craft beers often include more hops (Humulus lupulus L.) than traditional recipes. These compounds include α- and β- acids, which are antimicrobial to the rumen hyper ammonia-producing bacteria (HAB) that are major contributors to amino acid degradation. This study demonstrated that hops acids in spent craft brewer's yeast protected protein from destruction by HABinvitro. These results suggest that the spent yeast from craft breweries, a source of beneficial hops secondary metabolites, could have value as rumen-protected protein.

Effects of a specific blend of essential oils on apparent nutrient digestion, rumen fermentation and rumen microbial populations in sheep fed a 50:50 alfalfa hay:concentrate diet.

ObjectiveAn experiment was conducted to investigate the effects of a specific mixture of essential oils (MEO), containing thyme, clove and cinnamon EO, on rumen microbial fermentation, nutrient apparent digestibility and blood metabolites in fistulated sheep.MethodsSix sheep fitted with ruminal fistulas were used in a repeated measurement design with two 24-d periods to investigate the effect of adding MEO at 0 (control), 0.8, and 1.6 mL/d on apparent nutrient digestibility, rumen fermentation characteristics, rumen microbial population and blood chemical metabolites. Animals were fed with a 50:50 alfalfa hay:concentrate diet.ResultsRuminal pH, total volatile fatty acids (VFA) concentration, molar proportion of individual VFA, acetate: propionate ratio and methane production were not affected with MEO. Relative to the control, Small peptides plus amino acid nitrogen and large peptides nitrogen concentration in rumen fluid were not affected with MEO supplementation; while, rumen fluid ammonia nitrogen concentration at 0 and 6 h after morning feeding in sheep fed with 1.6 mL/d of MEO was lower (p<0.05) compared to the control and 0.8 mL/d of MEO. At 0 h after morning feeding, ammonia nitrogen concentration was higher (p<0.05) in sheep fed 0.8 mL/d of MEO relative to 1.6 mL/d and control diet. Ruminal protozoa and hyper ammonia producing (HAP) bacteria counts were not affected by addition of MEO in the diet. Relative to the control, no changes were observed in the red and white blood cells, hemoglobin, hematocrit, glucose, beta-hydroxybutyric acid, cholesterol, total protein, albumin, blood urea nitrogen and aspartate aminotransferase and alanine aminotransferase concentration. Apparent total tract digestibility of dry matter, crude proten, organic matter, and neutral detergent fiber were not influenced by MEO supplementation.ConclusionThe results of the present study suggested that supplementation of MEO may have limited effects on apparent nutrient digestibility, ruminal fermentation and protozoa and HAP bacteria count, blood cells and metabolites.

Board-invited review: Rumen microbiology: Leading the way in microbial ecology1,2

Robert Hungate, considered the father of rumen microbiology, was the first to initiate a systematic exploration of the microbial ecosystem of the rumen, but he was not alone. The techniques he developed to isolate and identify cellulose-digesting bacteria from the rumen have had a major impact not only in delineating the complex ecosystem of the rumen but also in clinical microbiology and in the exploration of a number of other anaerobic ecosystems, including the human hindgut. Rumen microbiology has pioneered our understanding of much of microbial ecology and has broadened our knowledge of ecology in general, as well as improved the ability to feed ruminants more efficiently. The discovery of anaerobic fungi as a component of the ruminal flora disproved the central dogma in microbiology that all fungi are aerobic organisms. Further novel interactions between bacterial species such as nutrient cross feeding and interspecies H2 transfer were first described in ruminal microorganisms. The complexity and diversity present in the rumen make it an ideal testing ground for microbial theories (e.g., the effects of nutrient limitation and excess) and techniques (such as 16S rRNA), which have rewarded the investigators that have used this easily accessed ecosystem to understand larger truths. Our understanding of characteristics of the ruminal microbial population has opened new avenues of microbial ecology, such as the existence of hyperammonia-producing bacteria and how they can be used to improve N efficiency in ruminants. In this review, we examine some of the contributions to science that were first made in the rumen, which have not been recognized in a broader sense.

Reconsidering rumen microbial consortia to enhance feed efficiency and reduce environmental impact of ruminant livestock production systems

Because cultivation-based approaches to assess rumen microbiology assess only the minority of microbial groups and can miss opportunities to assess important functions, molecular-based approaches have flourished in microbial ecology and have been adapted to the rumen. Current approaches are described herein, specifically for a robust adaptation to nutrition studies or future opportunities. These included automated profiling techniques, density gradient gel electrophoresis, and future adaption of microarray or high-throughput sequencing technologies. Based on current knowledge, a more holistic approach is needed to describe various functional groups of microbes within the context of how they influence, and are influenced by, the whole consortium (combination of microbial groups). Such a perspective is applied to issues related to increasing fiber digestibility when feeding concentrate or unsaturated fats to high producing beef and dairy cattle. These same microbial populations should help to provide growth factors for fibrolytic bacteria while competing against the hyperammonia-producing bacteria such that there would be less need for excessive rumen-degraded protein as a safety factor. Moreover, these same dietary conditions influence the processes of biohydrogenation and methanogenesis. After accounting for population structures of bacteria, protozoa, methanogenic archaea, and even fungi, efforts to integrate molecular-based rumen microbial ecology with dietary conditions should allow us to better explain and therefore predict conditions that will improve feed efficiency and reduce environmental impact of ruminant production systems.

Antimicrobial Effect of Red Clover (Trifolium pratense) Phenolic Extract on the Ruminal Hyper Ammonia-Producing Bacterium, Clostridium sticklandii

Ruminal proteolysis and subsequent amino acid degradation represent considerable economic loss in ruminant production. The hyper ammonia-producing bacteria (HAB) are largely responsible for amino acid deamination in the rumen. HAB can be controlled with ionophores, but they are also susceptible to antimicrobial plant secondary metabolites. Red clover (Trifolium pratense) is rich in soluble phenolics, and it is also more resistant to proteolysis than other legumes. The goal of this study was to identify phenolic compounds from Trifolium pratense cultivar Kenland, and determine if any of the compounds possessed antimicrobial activity against the bovine HAB, Clostridium sticklandii SR. HPLC analysis revealed that clover tissues were rich in the isoflavonoids formononetin and biochanin A, particularly in plants left to wilt for 24 h. Biochanin A inhibited C. sticklandii in bioassays that employed thin-layer chromatography (TLC). Both clover extracts and biochanin A inhibited the growth of C. sticklandii in broth culture, but formononetin had no effect. These results indicate that clover phenolic compounds may have a role in preventing amino acid fermentation.

The antimicrobial effects of hops (Humulus lupulusL.) on ruminal hyper ammonia-producing bacteria

To assess the antimicrobial effects of hops (Humulus lupulus L.) on hyper ammonia producing-bacteria (HAB), which catabolize amino acids and peptides in the bovine rumen. When media were amended with dried hops or hops extract (30.7% lupulone), ammonia production by mixed rumen bacteria was inhibited. The growth and ammonia production of pure cultures (Peptostreptococcus anaerobius, Clostridium aminophilum, or Clostridium sticklandii) was inhibited by 30 ppm lupulone at pH 6.7, and bactericidal activity was observed at pH 5.6. When hops extract was added to energized cell suspensions, the intracellular pH rapidly decreased and intracellular potassium was lost. The three HAB species were sensitive to the antimicrobial components in hops, and the inhibition of ammonia production by mixed rumen bacteria indicates that similar effects could be expected in the rumen. As much as half of the amino acids consumed by ruminants can be lost due to microbial degradation in the rumen. This study supports the idea that biologically active plant metabolites can be used to mitigate this wasteful process.

The effects of monensin on amino acid catabolizing bacteria isolated from the Boer goat rumen

When ruminants consume feed, as much as half of the amino acid nitrogen can be lost due to microbial degradation in the rumen. Hyper ammonia-producing bacteria (HAB) are primarily responsible for amino acid loss in sheep and cattle, and ionophores (e.g. monensin) are used to inhibit the HAB. Ionophores are sometimes included in the diets of meat goats, but the effects on caprine HAB have not been investigated. Five amino acid catabolizing bacteria were isolated from the rumina of Boer goats, which were consuming a supplement that contained monensin. Two of the isolates were most closely related to the bovine HAB, Peptostreptococcus anaerobius, but the other three were more closely related to Peptoniphilus indolicus. When the isolates were exposed to monensin, intracellular potassium was lost, and ammonia production was inhibited. However, the P. indolicus-like isolates demonstrated a greater capacity to overcome this inhibition in growth experiments. These results indicate that the monensin sensitivity of these bacteria can be variable, even when previous exposure to monensin occurred in the rumen environment.

Bovicin HC5 inhibits wasteful amino acid degradation by mixed ruminal bacteriain vitro

Streptococcus bovis HC5 produces a broad spectrum lantibiotic (bovicin HC5) that inhibits pure cultures of hyper ammonia-producing bacteria (HAB). Experiments were preformed to see if: (1) S. bovis HC5 cells could inhibit the deamination of amino acids by mixed ruminal bacteria taken directly from a cow, (2) semi-purified bovicin was as effective as S. bovis HC5 cells, and 3) semi-purified and the feed additive monensin were affecting the same types of ammonia-producing ruminal bacteria. Because purified and semi-purified bovicin HC5 was as effective as S. bovis HC5 cells, it appeared that bovicin HC5 was penetrating the cell membranes of HAB before it could be degraded by peptidases and proteinases. Mixed ruminal bacteria that were successively transferred and enriched nine times with trypticase did not become significantly more resistant to either bovicin HC5 (50 AU mL(-1)) or monensin (5 microM), and amplified rDNA restriction analysis indicated that bovicin HC5 and monensin appeared to be selecting against the same types of bacteria.

A review of plant-derived essential oils in ruminant nutrition and production

Public concern over use of antibiotics in livestock production has increased in recent years because of their possible contribution to emergence of antibiotic resistant bacteria, and their transmission from livestock to humans. Accordingly, ruminant microbiologists and nutritionists have been exploring alternative methods of favorably altering ruminal metabolism to improve feed efficiency and animal productivity. Plant extracts contain secondary metabolites, such as essential oils (EO), that have antimicrobial properties that make them potential alternatives to antibiotics to manipulate microbial activity in the rumen. Essential oils are naturally occurring volatile components responsible for giving plants and spices their characteristic essence and color. Over the last few years, a number of studies have examined effects of EO, and their active components, on rumen microbial fermentation. However, many of these studies are laboratory based ( i.e., in vitro) and of a short-term nature. Nevertheless, results from in vitro batch culture studies provide evidence that EO and their components have the potential to improve N and/or energy utilization in ruminants. Effects of EO on ruminal N metabolism is more likely mediated by their impact on hyper-ammonia producing (HAP) bacteria resulting in reduced deamination of amino acids (AA) and production of ammonia N. However, these responses are only observed with high doses of EO, which also can inhibit the process of ruminal fermentation as reflected by a decline in total volatile fatty acid production. Effects on methane production are inconsistent, but evidence to date indicates that there is potential to select EO, or active components, that selectively inhibit ruminal methanogenesis. Results from in vitro continuous culture studies suggest that rumen microbial populations may adapt to EO, which may explain the lack of an effect of EO on ruminal metabolism and animal performance in long-term in vivo studies. Several studies have examined the activity of a number of EO against a wide variety of food-borne pathogens. Data available show a strong bactericidal activity against pathogenic bacteria such as Escherichia coli O157:H7 and Salmonella spp. Essential oils hold promise as feed additives in ruminant nutrition to improve feed efficiency and control the spread of pathogens in livestock. However identification of EO, or their active components, that favorably alter fermentation without resulting in broad overall inhibition of rumen fermentation, continues to be a major challenge for researchers.

Isolation and identification of hyper-ammonia producing bacteria from swine manure storage pits.

Storage of swine manure is associated with the microbiological production of a variety of odorous compounds, including ammonia, organic acids, and alcohols, phenolics, and sulfides. Until recently, little was known about the microorganisms responsible for their production. Results from our laboratory have demonstrated that the predominant microbial populations of stored swine manure are anaerobic, low (G + C), Gram-positive bacteria. However, studies on pure cultures isolated from manure have found few microorganisms that produce appreciable ammonia concentrations. Therefore, selective and enrichment techniques were employed to isolate ammonia-producing bacteria from stored swine manure by using media containing peptone and amino acids as carbon and energy sources. We now report on the isolation of 40 bacterial cultures, a number of which are capable of producing at least 40 mM ammonia in peptone-amino acid medium, concentrations similar to those produced by hyper-ammonia producing (HAP) bacteria isolated from the rumen of cattle. The manure HAP isolates are phylogenetically distinct from the ruminal isolates and may prove to be intimately involved in the production of ammonia during storage of swine manure.

Mathematical estimations of hyper-ammonia producing ruminal bacteria and evidence for bacterial antagonism that decreases ruminal ammonia production(1).

Mixed ruminal bacteria (MRB) from cattle fed hay produced ammonia from protein hydrolysate twice as fast as MRB from cattle fed mostly grain, and a mathematical model indicated that cattle fed hay had approximately four-fold more hyper ammonia-producing ruminal bacteria (HAB). HAB had a high maximum velocity of ammonia production (V(max)) and low substrate affinity (high K(m)), but simulations indicated that only large changes in V(max) or K(m) would cause a large deviation in HAB numbers. Some carbohydrate-fermenting ruminal bacteria produced ammonia at a slow rate (CB-LA), but many of the isolates had almost no activity (CB-NA). The model indicated that the ratio of CB-LA to CB-NA had little impact on HAB numbers. Validations based on predicted ratios of HAB, CB-LA and CB-NA over-predicted the specific activity of ammonia production by MRB, but co-culture incubations indicated that washed MRB from cattle fed grain could inhibit HAB. Because autoclaved MRB had virtually no effect on HAB and the incubations were always carried out at pH 7.0, the inhibition was not simply a chemical effect (e.g. low pH).