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Bioresource Technology 263 (2018) 358–364

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Isolation, characterization and strain selection of a Paenibacillus species for use as a probiotic to aid in ruminal methane mitigation, nitrate/nitrite detoxification and food safety

T



Elizabeth A. Lathama,b, , William E. Pinchakb, Julian Trachselc, Heather K. Allenc, Todd R. Callawayd, David J. Nisbetd, Robin C. Andersond a

Department of Animal Science, Texas A&M University, College Station, TX 77843, USA Texas A&M AgriLife Research, Vernon, TX, USA United States Department of Agriculture, Agricultural Research Service, Food Safety and Enteric Pathogens Research Unit, 1920 Dayton Ave, Ames, IA 50010, USA d United States Department of Agriculture, Agricultural Research Service, Southern Plains Agricultural Research Center, Food and Feed Safety Research Unit, College Station, TX, USA b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Nitrate Nitrite Nitrite toxicity Paenibacillus Rumen methanogenesis

The effects of dietary nitrate and Paenibacillus 79R4 (79R4), a denitrifying bacterium, when co-administered as a probiotic, on methane emissions, nitrate and nitrite-metabolizing capacity and fermentation characteristics were studied in vitro. Mixed populations of rumen microbes inoculated with 79R4 metabolized all levels of nitrite studied after 24 h in vitro incubation. Results from in vitro simulations resulted in up to 2 log10 colony forming unit reductions in E. coli O157:H7 and Campylobacter jejuni when these were co-cultured with 79R4. Nitrogen gas was the predominant final product of nitrite reduction by 79R4. When tested with nitrate-treated incubations of rumen microbes, 79R4 inoculation (provided to achieve 106 cells/mL rumen fluid volume) complemented the ruminal methane-decreasing potential of nitrate (P < 0.05) while concurrently increasing fermentation efficiency and enhancing ruminal nitrate and nitrite-metabolizing activity (P < 0.05) compared to untreated and nitrate only-treated incubations.

1. Introduction Methane is a greenhouse gas, which according to the United States Environmental Protection Agency is the second largest contributor to global warming (US-EPA, 2016). Methane produced by ruminants is also recognized as an important loss of gross energy in feedstuffs consumed by ruminants, accounting for up to 12% loss of gross energy for animals consuming foraged-based feedstuffs and as much as 3% of gross energy for animals consuming concentrate-based feedstuffs (Johnson and Johnson, 1995). It is thought that preferred strategies for decreasing methane emissions in ruminants will divert the flow of reducing substrates away from methanogenesis and into alternative electron sinks (McAllister and Newbold, 2008). The inclusion of dietary nitrate is currently one of the few practices that fall into this strategy, with microbial reduction of nitrate to ammonia or to dinitrogen gas consuming just as many electrons as the 8 electron reduction of carbon dioxide to methane by methanogenic bacteria (Hulshof et al., 2012; Jeyanathan et al., 2014; Latham et al., 2016; Leng, 2014; Lin et al., 2013; van Zijderveld et al., 2011). However, despite harboring bacterial



populations capable of denitrifying nitrate to dinitrogen gas this process appears to be inconsequential within the rumen ecosystem as only traces of nitrous oxide (up to 0.3% of added nitrogen) are produced in the rumen as a byproduct of nitrite reduction (de Raphélis-Soissan et al., 2014; Kaspar and Tiedje, 1981). Consequently, nitrate metabolism within the rumen normally occurs mainly via dissimilatory reduction of nitrate to nitrite and then the further reduction of nitrite to ammonia which differentiates it from what often occurs in many other ecosystems. For instance, microbial populations contributing to denitrification of wastewaters can metabolize nitrate to nitrite which is then further metabolized by different bacterial populations via pathways yielding nitrous oxide, dinitrogen gas or ammonia (Meng et al., 2016). As recently discussed in several comprehensive reviews, a major limitation of current nitrate-supplementation strategies for ruminants is the risk of toxicity due to excessive accumulations of nitrite, an intermediate produced during nitrate metabolism in the rumen (Latham et al., 2016; Lee and Beauchemin, 2014; Yang et al., 2016). Nitrate-metabolizing bacteria are diverse and abundant in the rumen ecosystem; however, the reduction of nitrite to ammonia is

Corresponding author at: Department of Animal Science, Texas A&M University, College Station, TX 77843, USA. E-mail address: [email protected] (E.A. Latham).

https://doi.org/10.1016/j.biortech.2018.04.116 Received 27 January 2018; Received in revised form 25 April 2018; Accepted 28 April 2018 Available online 30 April 2018 0960-8524/ © 2018 Elsevier Ltd. All rights reserved.

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for enhanced nitrite-metabolizing activity, as assessed via determination of nitrite disappearance rates. Nitrite disappearance was determined via measurement of nitrite concentrations in fluid samples collected at intervals during incubation using the colorimetric procedure of Schneider and Yeary (1973). During culture with increasing amounts of supplemental nitrite, one strain designated as nitrite-selected Paenibacillus 79R4, showed greater than average nitrite-metabolizing activity, higher growth density and a more rapid growth rate that far surpassed that observed with the other strains cultured similarly in the selective media. Fermentation characteristics of this resultant nitrite-selected Paenibacillus 79R4 were measured during 24 h pure culture at 37 °C in basal medium B with or without additions of 10 μmol nitrate or 5 μmol nitrite/mL. Spore-formation was confirmed in cells exposed for 10 min to 70% (vol/vol) ethanol and via subsequent recovery of vegetative colonies grown (39 °C) on spore-selecting egg yolk agar medium (Koransky et al., 1978). The nitrite-selected Paenibacillus 79R4 has been deposited (number B-67118) with the ARS Culture Collection (NRRL) administered within the United States Department of Agriculture’s Mycotoxin Prevention and Applied Microbiology Research Unit at the National Center for Agricultural Utilization Research in Peoria, Illinois.

slower than the reduction of nitrate to nitrite, often resulting in intoxicating accumulations of nitrite (Lee and Beauchemin, 2014), which when absorbed into the blood stream binds to hemoglobin. This bound hemoglobin is termed methemoglobin, which is unable to transport oxygen and may lead to the ruminant’s death via asphyxiation. Aside from being potentially toxic to the host, high ruminal nitrite concentrations can also be toxic to important rumen microbes that play a critical role in converting cellulosic feedstuffs unusable to the host into forms that can supply nutrition and energy to the animal (Lee and Beauchemin, 2014). It has been speculated that direct fed microbials with enhanced nitrite-metabolizing capacity may prevent nitrite toxicity in at-risk ruminants (Yang et al., 2016). Limitations to the development of such technology exist; however, dealing mainly with the culture and storage stability of strict anaerobes packaged for commercial application. The objective of the present work, therefore, was to create an aerotolerant direct fed probiotic, which is phenotypically enhanced in nitrite- and nitrate-reducing activity and thereby enhanced in its ability to detoxify nitrite. It was also expected that this probiotic, via its enhanced nitrite- and nitrate-reducing activity, would contribute to mitigation of methane production in the rumen by outcompeting methanogens for the consumption of electrons. We report here the initial enrichment, isolation, selection and characterization of a highly active nitrite- and nitrate-reducing Paenibacillus from the bovine rumen. Results from tests investigating the ability of this probiotic to suppress select foodborne pathogenic bacteria are also presented.

2.2. In vitro characterization Tests for the effects of the nitrite-selected Paenibacillus 79R4 during culture with mixed populations of ruminal microbes were conducted using medium B prepared under 100% carbon dioxide, 40% clarified ruminal fluid and additions of sodium nitrate and sodium nitrite as indicated. Cultures were inoculated with 10% vol/vol of freshly collected rumen fluid and with or without an overnight grown culture of the nitrite-selected Paenibacillus 79R4. Culture fluid samples were collected at 0, 2, 4, 6, 12 and 24 h intervals for colorimetric determination of nitrite as described earlier as well as for nitrate and ammonia (Cataldo et al., 1975; Chaney and Marbach, 1962). After 24 h of incubation, gas production in each tube was measured by volume displacement and gas composition and short chain fermentation acid concentrations were measured via gas chromatography (Allison et al., 1992, Lambert and Moss, 1972; Salanitro and Muirhead, 1975). Amounts produced were calculated as the difference between final and initial concentrations. Stoichiometric estimates of amounts of hexose fermented were calculated as ½ acetate + ½ propionate + butyrate + valerate and fermentation efficiency, which is based on the heats of combustion of glucose and the respective volatile fatty acids, was calculated as (0.62 acetate + 1.09 propionate + 0.78 butyrate) ÷ (acetate + propionate + butyrate) × 100 (Chalupa, 1977). To test antimicrobial capacity, replicate sets of tubes containing anaerobic medium B were separately inoculated (0.2% vol/vol) with overnight grown cultures of a novobiocin and nalidixic acid resistant Escherichia coli O157:H7 strain 933 or with Campylobacter jejuni strain CC326. Some of these cultures were then also inoculated similarly (0.2% vol/vol) with an overnight nitrite-grown Paenibacillus 79R4. The resultant pure and co-cultures, each prepared in triplicate, were incubated upright without agitation at 39 °C for 24 h. Fluids from the pure of Escherichia coli O157:H7 strain 933, Campylobacter jejuni strain CC326, nitrite-selected Paenibacillus 79R4 and co-cultures of Paenibacillus 79R4 with the respective pathogens were collected and plated at 0, 6, 24 and 48 h for viable cell count enumeration on Campy Cefex, MacConkey agar (Callaway et al., 2008; Horrocks et al., 2007), and Tryptic Soy agar, respectively.

2. Materials and methods 2.1. Strain isolation, selection and sequencing Ruminal fluid collected from a cannulated Jersey cow grazing on Bermuda grass pasture was strained through a nylon paint strainer into an insulated container. The ruminal fluid was returned to the laboratory and inoculated (1 mL/tube) within 30 min of collection into 18 × 150 mm crimp-top tubes containing 9 mL of anaerobically prepared (under 50:50 hydrogen:carbon dioxide) medium B broth (Anderson and Rasmussen, 1998) and supplemented with 36 mM sodium nitrate. This concentration was chosen to approximate ruminal nitrate concentrations that may potentially accumulate during acute nitrate toxicity. Medium B contained (mg/100 mL): K2HPO4, 22.5; KH2PO4, 22.5; (NH4)2SO4, 45.0; NaCl, 45.0; MgSO4·7H2O, 4.5; CaCl2, 2.25; thiamine, 0.2; pantothenate, 0.2; nicotinamide, 0.2; pyridoxineHCl, 0.2; riboflavin, 0.2; p-aminobenzoic acid, 0.1; biotin, 0.0005; folic acid, 0.005; lipoic acid, 0.005; vitamin B-12, 0.002; resazurin, 0.1; cysteine-HCl, 50; Na2CO3, 400; phytone peptone, 800 and clarified rumen fluid at 8% vol/vol. Cysteine-HCl and Na2CO3 were added after the pH of the medium containing the other ingredients was adjusted to 6.8. Tubes containing freshly collected ruminal fluid were incubated without agitation at 39 °C. After three consecutive 24 h batch culture series, the culture fluids from the third culture series were heated for 10 min at 100 °C to physically enrich the culture for spore-forming bacteria. Upon cooling, the culture fluids were then serially diluted to 10−5 and plated to solid, sodium nitrate-supplemented (36 µmol/mL) medium B plates prepared as described above with 2% agar and incubated in anaerobic gas jars filled with a 50:50 hydrogen:carbon dioxide gas mixture. After 24 h of incubation at 37 °C, a colorimetric nitrite detection overlay methodology was employed to pick fifteen isolates with nitrate reduction capacity according to Glaser and DeMoss (1971). These isolates were subjected to 16s rRNA gene sequence analysis to determine their phylogenetic affiliation using universal primers 27f and 1492r (Weisburg et al., 1991). The six unique strains, related to the genus Paenibacillus, were subjected to 24 h culture as described above with increasing concentrations of nitrite (0.5, 1.0, 3.0 and 6.0 μmol sodium nitrite/mL) in anaerobic medium B broth to select

2.3. Statistical analysis Tests for differences between wildtype and nitrite-selected Paenibacillus 79R4 on measured growth parameters (maximum optical densities and mean specific growth rates) as well as metabolite concentrations (headspace gas accumulations, concentrations or nitrate, 359

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nitrite and ammonia) and rates and nitrate and nitrite metabolism during pure culture were done using a general analysis of variance. Tests for effects of nitrate or nitrite additions during time of incubation with or without inoculation with nitrite-selected Paenibacillus 79R4 on nitrate- and nitrite-metabolizing activity and on concentrations of ammonia, nitrate, nitrite at each sampling time during in vitro culture with mixed populations of ruminal microbes were conducted similarly as was net production of fermentation acids and methane. Co-culture log10 reductions during in vitro co-culture experiments with E. coli O157 and C. jejuni were also tested for treatment effects by a general analysis of variance. All analyses were done using STATISTIX®8 Analytical Software (Tallahassee, FL). Means were separated using Tukey’s comparison of means. For all analysis, significance was set at P < 0.05 and trends at P > 0.05 but < 0.10.

Table 1 Results of 16s rRNA gene sequences from cultures of enriched populations of rumen microbes with nitrate and nitrite. Strain number

Closest related strain

Similarity with the closest described species in Genbank (%)

Accession number

79R1

Paenibacillus motobuensis Paenibacillus motobuensis Paenibacillus J1Ba-7 Paenibacillus Paenibacillus GM2 Paenibacillus GM2

98

MG015831

97

MG015832

sp.

97

MG015833

sp. R2 sp.

97 97

MG015699 MG015835

sp.

96

MG015836

79R2 79R3 79R4 79R5 79R6

3. Results and discussion 3.1. Strain isolation, selection and sequencing

Table 2 Biomass, max specific growth rate, and nitrite removal during pure culture of isolates incubated in medium B with 6 μmol nitrite/mL at 39 °C for 24 h.

Contemporary investigations of microorganisms involved in rumen nitrate and nitrite metabolism have focused mainly on populations selected for nitrate reduction during enrichment with added nitrate (Allison and Reddy, 1984; Asanuma et al., 2014; Lin et al., 2013; Rehberger and Hibberd, 2000). Many of these studies, however, have focused on the dissimilatory reduction of nitrate to ammonia and thus populations adapted for enhanced ability to reduce and thus detoxify nitrite, which accumulates as an intermediate during dissimilatory reduction to ammonia or denitrification to gaseous nitrogen may have been overlooked. Cheng et al. (1988) specifically focused on testing the capacity of rumen microbes to reduce nitrite. There were 51 strains tested and of those, 25 were found to grow and metabolize nitrite within seven days; this included members from the genera Bacteroides, Clostridium, Coprococcus, Lactobacillus, Megasphaera, Peptostreptococcus, Selenomonas and Veillonella. In animal studies, a Propionibacterium strain developed as a potential probiotic to prevent nitrite toxicity in ruminants, as well as a dissimilatory nitrate- and nitrite-reducing strain of E. coli W3110 investigated similarly were reported to exhibit nitrite-reducing activity but this was far exceeded their nitrate-reducing activity (Rehberger and Hibberd, 2000; Sar et al., 2005b). The enrichment procedure of the current study focused on the selection of microbes capable of complete nitrate detoxification and particularly those expressing high nitrite-metabolizing activity. Moreover, the enrichment procedure included a step to enrich for spore-forming nitrate- and nitrite-metabolizing bacteria, as it was hypothesized that spore-formers would facilitate the maintenance of culture viability during long-term storage as a commercial product. To our knowledge, endogenous Paenibacillus species have been isolated from the rumen at least twice before, as Paenibacillus woosongensis, a xylanolytic bacterium and Paenibacillus macerans, a bacterium capable of nitrogen fixation (Deng et al., 2013; Ishaq et al., 2015). The presence, diversity, and abundance of Paenibacillus in the rumen ecosystem is often revealed through DNA sequencing; although studies have found this genera at low levels (< 1% relative abundance) and without species level identification (Dai et al., 2012; Hook et al., 2011; Mayorga et al., 2016). Bioinformatics analysis revealed that the six isolated strains chosen from the present study for genotypic characterization belong in the genus Paenibacillus, formerly of the genus Bacillus (Ash et al., 1993), but likely represent previously uncultured strains (Table 1) or strains that have not specifically been found in the rumen via high-throughput or targeted 16s rRNA gene sequencing. Of these strains, Paenibacillus 79R4 (Table 1) was selected for further characterization as a potential supplemental-probiotic strain because of its extraordinary nitrite-metabolizing and spore-forming capability and successive cultivation in nitritesupplemented (6 µmol/mL) medium further enhanced its nitrite-metabolizing capability (Table 2). Spores of Paenibacillus 79R4, both before

ODmax (OD600 nm) μmax (h−1) Nitrite removalmax (μmol mL−1 h−1) ab

Wild-type Paenibacillus 79R4

Nitrite-selected Paenibacillus 79R4

0.279b ± 0.02 0.026b ± 0.008 2.24b ± 0.12

0.545a ± 0.03 0.075a ± 0.009 5.85a ± 1.00

Means across rows with unlike superscripts differ at P < 0.05.

and after nitrite-selection were readily regenerated in both aerobic and anaerobic media, thus indicating this strain possesses attractive preservation characteristics that should aid in the production of stable inoculum preparations with a long shelf life. Denitrification as well as the ability to produce spores, are common characteristics of this facultative bacterium known for its genetic diversity (Iida et al., 2005). Paenibacillus are thought to possess a complete denitrification pathway and therefore ought to be able to reduce nitrate completely to nitrogen gas, in an energy conserving process. This is in contrast to Propionibacterium acidipropionici which is reported to reduce nitrate to nitrite and then further reduce nitrite to nitrous oxide, possibly for detoxification purposes (Kaspar, 1982). Nitrogen gas was the predominant end-product produced by Paenibacillus 79R4 when it was incubated in medium B containing 5 μmol nitrite/mL and under 100% carbon dioxide (1.66 μmol/mL nitrogen which is the equivalent to the consumption of 3.32 μmol/mL nitrite). When Paenibacillus 79R4 was incubated with added nitrate, nearly all the nitrate was metabolized to nitrite, which was almost entirely metabolized (Table 3). However, the final quantitative recovery of all products has not yet been conclusively determined, as accumulations of ammonia observed in culture fluids after incubation potentially accounted for some but not all of the nitrate or nitrite metabolized. Hence, we cannot exclude the possibility that at least some ammonia may have been an end product of Paenibacillus 79R4 metabolism and consumed by other bacteria for their nitrogen metabolism.

3.2. In vitro characterization Results from in vitro incubations revealed a cumulative benefit of the combined supplementation of nitrate and the nitrite-selected Paenibacillus 79R4 on decreasing methane production and decreasing the potential negative effects of nitrite accumulation. For instance, main effects of nitrate treatment, nitrite-selected Paenibacillus 79R4 inoculation and their interaction were observed (P < 0.05). In the case of the interaction, amounts of methane produced by cultures not inoculated with the nitrite-selected Paenibacillus 79R4 were less than that 360

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during the metabolism of nitrate is much more reactive than nitrate and at concentrations as low as 0.5 µmol nitrite/mL can inhibit not only the growth or rumen methanogens but also the growth of important fiberdegrading bacteria (Iwamoto et al., 2002; Latham et al., 2016; Yang et al., 2016). This inhibition of fiber-degrading bacteria undoubtedly would adversely affect the conversion of ingested feedstuffs into compounds usable by the host. Measurement of nitrite accumulations in the present in vitro incubation of mixed ruminal microorganisms supplemented with nitrite revealed a beneficial effect of the nitrite-selected Paenibacillus 79R4 on nitrite clearance (Fig. 1). For instance, inoculation of the nitrite-selected Paenibacillus 79R4 to mixed cultures containing 0.5, 1.5 and 3.0 μmol/mL nitrite resulted in the elimination of nitrite to near or below the level of detection. Conversely, mixed cultures incubated with 0.5, 1.5 or 3.0 μmol/mL added nitrite but not inoculated with the nitrite-selected Paenibacillus 79R4 showed 57, 77 and 19% reduction of nitrite after 24 h, respectively. Nitrite metabolism in mixed cultures incubated with 6.0 μmol/mL added nitrite was greater with cultures inoculated with the nitrite-selected Paenibacillus 79R4 than in cultures not inoculated with this bacterium but remained above 4 μmol nitrite/ mL even after 24 h incubation. Profiles of nitrite and nitrate accumulations in the in vitro incubations of mixed ruminal microorganisms supplemented with or without nitrate, and with or without the nitrite-selected Paenibacillus 79R4 are shown in Figs. 2 and 3. Addition of the nitrite-selected Paenibacillus 79R4 into the in vitro cultures incubated with 9, 18 or 36 μmol/mL added nitrate resulted in more rapid metabolism of nitrate (Fig. 2) and less rapid and in some cases lower peak accumulations of nitrite than in cultures incubated without Paenibacillus 79R4. By comparison, peak nitrite accumulations by mixed populations of ruminal microbes incubated with additions of 10 mM added nitrate and inoculated with nitrate- and nitrite-metabolizing strains of E. coli (Sar et al., 2005a) occurred earlier than in the present incubations. Nitrite disappearance commenced rapidly after peak accumulations were achieved in both the present study and the study of Sar et al (2005a) although the clearance of nitrite after attainment of peak concentrations appeared more rapid with Paenibacillus 79R4. These observations are again consistent with earlier in vitro studies showing increased rates of nitrate metabolism and nitrite accumulation in mixed ruminal populations inoculated with nitrate-metabolizing bacteria (Anderson and Rasmussen, 1998;

Table 3 Fermentation characteristics of nitrite-selected Paenibacillus 79R4 (79R4) during 24 h pure culture (at 37 °C) in basal medium B with or without additions of 10 μmol nitrate or 5 μmol nitrite/mL. 79R4 alone Total gas (mL) Hydrogen (μmol/mL)1 Nitrogen (μmol/mL)1 Nitrite metabolized (μmol/mL)1 Nitrate metabolized (μmol/mL)1 Ammonia formed (μmol/ mL)1

−3.0 0.51b 0.00c 0.10c

b

79R4 and nitrate a

79R4 and nitrite

± 0.0 ± 0.1 ± 0.1 ± 0.12

4.5 ± 0.5 0.40b ± 0.1 0.23b ± 0.1 7.56a ± 1.03

4.5a ± 0.5 0.60a ± 0.1 1.66a ± 0.1 4.90b ± 0.12

0.49b ± 0.12

8.58a ± 0.12

0.14b ± 0.22

−0.03c ± 0.1

2.37a ± 0.1

2.22b ± 0.1

abc

Means across rows with unlike superscripts differ at P < 0.05. per mL of incubation fluid. 2 Represents net amount metabolized and was calculated as the endogenous or added amounts at 0 h minus residual amounts measured after 24 h incubation. 3 Represents the difference between the amount of nitrate metabolized to nitrite and residual amount of nitrite measured after 24 h incubation. 1

of the non-nitrate treated controls only in cultures treated with 18 or 36 µmol nitrate/mL. Conversely, methane production was decreased at all nitrate addition levels for cultures that had been co-treated with the nitrite-selected Paenibacillus 79R4 (Table 4). When mixed populations of ruminal microbes were incubated with additions of 0.5 or 1.5 μmol nitrite/mL, amounts of methane production were decreased (P < 0.05) more than 98% compared to that of untreated controls regardless of whether or not the cultures were inoculated with Paenibacillus 79R4 (not shown). These findings are consistent with earlier in vitro studies reporting that additions of 5 µmol nitrate/mL or 0.5 to 3 µmol nitrite/ mL to mixed populations of ruminal microbes cultured in vitro resulted in approximately 50% to near complete inhibition of methane production, with nitrite being more potent than nitrate (Iwamoto et al., 2002). As comprehensively reviewed in Latham et al. (2016), nitrate and nitrite can inhibit methane production within rumen ecosystems by serving as thermodynamically favorable alternative electron sinks for the disposal of electrons that accumulate during anaerobic digestion of feedstuffs. Nitrite, however, which is produced as an intermediate

Table 4 Fermentation characteristics of mixed populations of ruminal microbes incubated without or with added nitrate and inoculated without or with nitrite-selected 79R4 (79R4). Products produced (µmol/mL) Head space hydrogen Head space methane Acetate Propionate Butyrate Isobutyrate Valerate Isovalerate Lactate Total acids Hexose fermented (µmol/ mL) Fermentation efficiency (%)

2.5 µmol mL−1

None

9 µmol mL−1

36 µmol mL−1

Without 79R4

With 79R4

Without 79R4

With 79R4

Without 79R4

With 79R4

Without 79R4

With 79R4

Without 79R4

With 79R4

1.2 ± 1.0

0.7 ± 0.6

0.1 ± 0.1

0.9 ± 1.4

3.8 ± 2.0

2.9 ± 0.1

3.5 ± 1.8

3.0 ± 1.0

3.3 ± 1.7

3.1 ± 0.9

18.6ab ± 1.9

18.4ab ± 4.5

26.6a ± 8.4

5.5c ± 4.8

8.6bc ± 4.0

0.1c ± 0.1

5.6c ± 3.6

0.1c ± 0.1

4.3c ± 1.9

0.9c ± 1.4

14.7 ± 3.0 1.7 cd ± 1.5 2.2 fg ± 1.0 0.1bc ± 0.1 0.6b ± 0.1 0.4 ± 0.1 0.1 ± 0.2 19.8 ± 5.8 12.8ef ± 1.8

29.3 ± 8.8 16.3a ± 1.3 5.3ef ± 1.6 0.2abc ± 0.1 0.5bc ± 0.1 0.5 ± 0.1 1.0 ± 0.1 53.1 ± 11.8 28.6ef ± 6.6

27.0 ± 1.8 2.0 cd ± 3.4 1.5 fg ± 2.6 0.1bc ± 0.2 1.2a ± 0.1 0.7 ± 0.1 0.1 ± 0.2 32.6 ± 6.7 17.2ef ± 4.4

30.3 ± 0.4 11.1abc ± 0.9 5.6ef ± 0.4 0.4ab ± 0.1 1.6a ± 0.1 0.9 ± 0.1 0.2 ± 0.3 50.0 ± 2.0 27.9ef ± 1.0

13.2 + 2.9 0.6d ± 1.1 0.7 g ± 0.9 0.2abc ± 0.1 0.4bcd ± 0.2 0.4 ± 0.1 0.3 ± 0.1 15.7 ± 5.1 8.0f ± 3.0

37.4 + 22.6 19.6a ± 6.2 8.1e ± 1.8 0.5a ± 0.2 1.3a ± 0.1 1.0 ± 0.2 1.2 ± 0.1 69.0 ± 31.0 37.8e ± 16.0

16.9 ± 8.5 1.9 cd ± 1.9 1.1 fg ± 1.1 0.2abc ± 0.1 0.4bcd ± 0.2 0.5 ± 0.2 0.3 ± 0.1 21.2 ± 11.9 10.9ef ± 6.5

38.1 ± 29.4 12.7ab ± 6.7 3.8efg ± 2.1 0.1bc ± 0.1 0.1 cd ± 0.2 0.8 ± 0.6 1.9 ± 1.3 57.6 ± 39.7 29.3ef ± 19.7

8.0 ± 1.9 0.1d ± 0.4 0.1 g ± 0.1 0.1c ± 0.1 0.1d ± 0.1 0.1 ± 0.1 0.6 ± 0.1 8.8 ± 2.0 4.1f ± 1.1

17.1 ± 10.0 5.4bcd ± 2.2 2.0 fg ± 0.8 0.1c ± 0.1 0.1d ± 0.1 0.1 ± 0.1 2.0 ± 0.2 26.5 ± 12.6 13.2ef ± 6.7

69.6 + 1.7

79.1 + 2.5

65.1 + 5.4

75.0 + 0.6

64.1 + 3.2

78.8 + 3.0

66.0 + 3.6

75.0 + 3.4

62.1 + 0.2

74.4 + 4.1

abcd efg

18 µmol mL−1

Interaction effect with means across rows with unlike superscripts differing at P < 0.05. Error values are standard deviations from n = 3 incubations. Interaction effect with means across rows differing at P > 0.05 but < 0.10. Error values are standard deviations from n = 3 incubations. 361

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Fig. 1. Effects of supplementing only nitrite (circles) or nitrite with nitrite-selected Paenibacillus-79R4 (79R4, squares) at (a) 0.5, (b) 1.5, (c) 3.0 and (d) 6.0 μmol/mL on nitrite concentrations during anaerobic incubation with mixed populations of ruminal microbes. Nitrite effect (P < 0.05), 79R4-effect (P < 0.05) and time effect (P < 0.05) in (a), (b), (c) and (d). Error bars represent standard deviations from n = 3 cultures/treatment.

respectively, during anaerobic co-culture with the nitrite-selected Paenibacillus 79R4 when compared to viable cell counts achieved during growth of the pathogens by themselves (9.04 ± 0.11 and 8.77 ± 0.56 log10 CFU/mL culture fluid, respectively). Paenibacillus has known antibacterial properties via the class of antimicrobial agents, polymyxins (DeCrescenzo Henriksen et al., 2007). This antimicrobial peptide has been reported to be effective against Gram-negative staining bacteria such as E. coli, Pseudomonas, Salmonella, and Shigella (DeCrescenzo Henriksen et al., 2007). In addition, related species within the Paenibacillus genus are also known to create a bacteriocin, which has been shown to be effective against Campylobacter (Svetoch et al., 2005).

Iwamoto et al., 2002). It is important to note, however, that in the absence of enhanced nitrite metabolism the resulting increased accumulations of nitrite may contribute to higher absorption rates and higher risks of toxicity due to methemoglobin formation and, as alluded to earlier, may adversely affect rumen fermentation. Analysis of fermentation acid production in the present in vitro incubations also revealed a beneficial effect of adding the nitrite-selected Paenibacillus 79R4 to the mixed microbial populations resulting from enhanced clearance of nitrite from the nitrate-treated incubations. For instance, accumulations of each of the fermentation acids as well as their cumulative totals were higher (P < 0.05) in cultures inoculated with Paenibacillus 79R4 than in cultures not inoculated with this bacterium (Table 4). Stoichiometric estimates of amounts of hexose fermented and fermentation efficiency were likewise increased (P < 0.05) in the cultures inoculated with the nitrite-selected Paenibacillus 79R4 than in cultures not inoculated with this bacterium which further indicates that nitrite-detoxification by this bacterium helped prevent accumulations of inhibitory concentrations of nitrite. Main effects of nitrate supplementation were observed on production on all of the measured acids with the exception of acetate and interactions between nitrate supplementation and Paenibacillus 79R4 inoculation were observed for some, but not all of the measured acids, due mainly to a negative effect of the 36 μmol/mL nitrate treatment (Table 4). Results from in vitro simulations revealed 2.04 ± 0.11 and 2.02 ± 0.45 log10 colony forming unit (CFU)/mL reductions in the foodborne pathogens E. coli O157:H7 and Campylobacter jejuni,

4. Conclusion Feeding nitrate can reduce enteric methane production in ruminants although risks of nitrite poisoning remains a barrier to commercial acceptance. A denitrifying bacterium designated Paenibacillus 79R4 isolated from the bovine rumen and selected for enhanced nitrite-reducing ability enhanced the methane mitigation potential of administered nitrate, increased fermentation efficiency and enhanced ruminal nitrite/nitrate detoxification thereby having potential to reduce adverse effects of nitrate supplementation to ruminants. Paenibacillus 79R4 is a facultative, spore-forming, easily cultured bacterium that yields a shelf stable product and exhibits antimicrobial activity against Gram-negative pathogens in vitro which may enhance its value as a potential Fig. 2. Effects of supplementing only nitrate (circles) or nitrate with nitrite-selected Paenibacillus-79R4 (79R4, squares) at (a) 2.5, (b) 9.0, (c) 18 and (d) 36 μmol/mL on nitrate concentrations during anaerobic incubation with mixed populations of ruminal microbes. Nitrate effect (P < 0.05), 79R4 effect (P < 0.05) and time effect (P < 0.05) in (a), (b), (c) and (d). Error bars represent standard deviations from n = 3 cultures/treatment.

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Fig. 3. Effects of supplementing only nitrate (circles) or nitrate with nitrite-selected Paenibacillus-79R4 (79R4, squares) at (a) 2.5, (b) 9.0, (c) 18 and (d) 36 μmol/mL on nitrite concentrations during anaerobic incubation with mixed populations of ruminal microbes. Nitrate effect (P < 0.05), 79R4 effect (P < 0.05) and time effect (P < 0.05) in (a), (b), (c) and (d). Error bars represent standard deviations from n = 3 cultures/treatment.

probiotic for ruminants.

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