Anaerobic (N2-sparged) batch fermentations were performed in triplicate using 10% fecal slurry (1% fecal inoculum) under controlled conditions [water-jacket vessels (Soham Scientific, Soham, United Kingdom), pH 6.8, temperature 37°C]. Basal medium contained per liter: 2 g peptone (Oxoid, Basingstoke, United Kingdom), 2 g yeast extract (Oxoid), 0.1 g NaCl (Fisher Scientific, Fair Lawn, NJ, United States), 0.04 g K2HPO4 (BDH, Toronto, ON, Canada), 0.04 g KH2PO4 (BDH), 0.01 g MgSO47H2O (BDH), 0.01 g CaCl26H2O (Honeywell, Morris Plains, NY, United States), 2 g NaHCO3 (Oxoid), 2 mL Tween 80 (Sigma–Aldrich, Oakville, ON, Canada), 0.05 g Hemin (Sigma–Aldrich) dissolved in 1 mL of 4 M NaOH (Fisher Scientific), 10 μL Vitamin K (Sigma–Aldrich), 0.5 g l-Cysteine HCL (Sigma–Aldrich), 0.5 g Bile Salts (Oxoid), and 4 mL of Resazurin (Sigma–Aldrich) (0.025 g/100 mL). Vessels were dosed with the substrates (1% wt/vol) after simulated in vitro upper gastrointestinal digestion and dialysis (Mandalari et al., 2008 (link)) and inoculated with 10% fecal slurry. The final volume of each culture was 200 mL. Samples were harvested at time points 0 (immediately after incubation) and 24 h.
K2hpo4
Manufactured by BD
Sourced in United States, United Kingdom
K2HPO4 is a chemical compound that is commonly used in various laboratory applications. It is a white, crystalline solid that is highly soluble in water. K2HPO4 serves as a buffering agent, maintaining a specific pH level in solutions.
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Lab products found in correlation
Kh2po4, by BD (4 mentions)
Yeast extract, by Thermo Fisher Scientific (3 mentions)
Mgso4, by BD (3 mentions)
Sodium chloride (nacl), by BD (3 mentions)
Bile salt, by Thermo Fisher Scientific (2 mentions)
Vitamin k, by Merck Group (2 mentions)
L cysteine hcl, by Merck Group (2 mentions)
Mgso4 7h2o, by BD (2 mentions)
Tween 80, by Merck Group (2 mentions)
Cacl2, by BD (2 mentions)
Nahco3, by Thermo Fisher Scientific (2 mentions)
Peptone water, by BD (2 mentions)
Bacto casamino acids, by BD (2 mentions)
Peptone, by Thermo Fisher Scientific (1 mentions)
Resazurin, by Merck Group (1 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (1 mentions)
Hemin, by Merck Group (1 mentions)
4 m naoh, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by BD (1 mentions)
Mgcl2, by Merck Group (1 mentions)
12 protocols using k2hpo4
1
Anaerobic Batch Fermentation of Fecal Slurry
2
Bacterial Strain Storage and Cultivation
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Bacterial strains used in this study are listed in Table 1 , and in Petrovski et al. [29 (link)], and methods for their storage and cultivation were those described by Petrovski et al. [29 (link)]. These were all grown on R2A medium (0.5 g/L Yeast extract (Oxoid, Adelaide, Australia), 0.5 g/L Proteose peptone (Difco, North Ryde, Australia), 0.5 g/L Casamino acid (Difco, North Ryde, Australia), 0.5 g/L Glucose, 0.5 g/L soluble starch (Difco, North Ryde, Australia), 0.3 g/L K2HPO4, 0.005 g/L MgSO4.7H2O, 0.3 g/L sodium pyruvate (BDH, Murarrie, Australia)) broth and agar R2A + 14 g/L agar (Oxoid, Adelaide, Australia) at 25 C. All remaining chemicals were obtained from Sigma (Sydney, Australia), unless otherwise noted.
3
Struvite Production and Characterization
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Natural and synthetic struvite were used as the struvite source in solid and liquid media. Natural struvite (fragmented form of approximate diameters 1-4 mm and powdered forms of diameter 335 μm) was kindly obtained from Veolia Water Outsourcing (London, UK) from their operating site in Exeter, UK. Synthetic struvite was prepared by adding 3.038 g MgCl 2 (Sigma-Aldrich), 5.408 g NH 4 Cl (Sigma-Aldrich) and 7.728 g K 2 HPO 4 (BDH) to 1 L of Milli-Q water to obtain an equimolar Mg: N: P ratio. Then, 5 M NaOH was used to adjust the pH to 8.5 and stirred continuously using a magnetic stirrer for 5 min. Precipitated struvite was collected by filtration through Whatman filter paper and dried in a desiccator to constant weight at ambient temperature for at least 2 days. XRD analysis confirmed that the precipitate produced was the monohydrate form, dittmarite (MgNH 4 PO 4 ÁH 2 O).
4
Preparation and Characterization of Zearalenone Transformation Products
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ZEA was obtained from Sigma-Aldrich (St. Louis, MO, USA). ZEA-14-phosphate and ZEA-16-phosphate were chemically synthesized by TripleBond (Guelph, ON, Canada) according to the proposed ZEA transformation products in this study. NBM contained 8 g nutrient broth (BD, Mississauga, Canada), 2.5 g K2HPO4, 2.5 g KH2PO4, 1.0 g (NH4)2HPO4, 0.2 g MgSO4·7H2O, 0.01 g FeSO4, and 0.007 g MnSO4 per liter. Nutrient agar (NA) plates were prepared using 8 g of nutrient broth and 15 g agar per liter. CSE or ASE was prepared by blending 50 g corn or alfalfa silage with 200 mL of deionized water for two minutes and was filtered through a Whatman No. 1 filter paper (Whatman, Maidstone, Kent, UK). CMB was prepared by soaking 40 g of corn meal in 1 L of deionized water at 58 °C for 4 h. After standing for 2 h, the broth was filtered through a Whatman No. 1 filter paper and 3 g (NH4)2SO4, 1 g K2HPO4, 0.5 g MgSO4·7H2O, 0.5 g K2SO4, 0.01 g FeSO4, and 0.007 g MnSO4 were added. All the media were autoclaved at 121 °C for 15 min before using.
5
Preparation of Basal Media for Anaerobic Cultivation
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The basal media was made by mixing yeast extract (Oxoid, Hampshire, UK), Peptone water (BDH, Poole, UK), KH2PO4 (BDH), MgSO4 × 7H2O NaCl, K2HPO4 (BDH), (Fisher Scientific, Loughborough, UK), CaCl2 × 6H2O (0.01 g/L), NaHCO3 (Fisher Scientific), tween-80, hemin (0.05 g/L), vitamin K (10 µL/L), L-cysteine HCl (0.5 g/L), bile salts (0.5 g/L) (Oxoid, Basingstoke, UK) and resazurin solution 0.025 g/100 mL (4 mL/L, pH 7). The obtained solution was heated, cooled, poured into Duran bottles, autoclaved and stored until use.
6
Carbohydrate Quantification Protocol
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Sucrose, D-(−)-fructose, D-(+)-glucose, D-(+)-xylose, D-(+)-galactose, D-(+)-mannose, D-(+)-cellobiose, Folin–Ciocalteu′s phenol reagent, albumin from bovine serum (BSA), KNaC4H4·4H2O, NaOH, Na2CO3, CuSO4, and 3,5-Dinitrosalicylic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). The electrophoresis reagents were from Bio-Rad (Hercules, CA, USA). K2HPO4, MgSO4, HCl, CaCl2, NaCl, MnSO4, and FeSO4 were from J.T. Baker (S.A. de C.V., Toluca, Estado de México, México) Bacto Yeast Extract from BD Biosciences (San Jose, CA, USA).
7
Nematode and Bacterium Culture Protocol
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All chemicals used for nematode culture and bacterium culture were from Sigma-Aldrich (NaCl [7647–14–5]; cholesterol [57–88–5]; MgSO4 [10034–99–8]; CaCl2 [10035–04–8]; KH2PO4 [7778–77–0]; K2HPO4 [7758–11–4]) or BD (peptone [211820]; tryptone [211699]; yeast extract [212750]). Spermidine was from Sigma-Aldrich (124–20–9), LysoTracker Blue DND-22 was from Life Technologies (L-7525), and Recombinant SLO protein was from Sigma-Aldrich (401470–29–9).
8
Salbutamol Sulfate Quantification via Spectrophotometry
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4-amino-3-hydroxylnaphthalene sulfonic acid (AHNSA) (Sigma-Aldrich); KH2PO4, and K2HPO4 (98–101%) (BDH, England); salbutamol sulfate (99.92%) (Emmellen Biotech Pharmaceuticals Limited); H2SO4 (98%), and HNO3 (69.0–70.0%) (Loba Cheime Pvt. Ltd, India); NaOH and HCl (Riede-De Haen, Germany) were used. All reagents were of analytical grade and were used directly without further purification. Distilled water was used for the preparation of all solutions throughout this work.
Phosphate buffer solutions (PBS) prepared by mixing equi-molar (0.1 M) of K2HPO4 and KH2PO4 were used to prepare salbutamol sulfate solutions. 1.0 M of NaOH and HCl solutions were used to adjust the desired pH of the buffer solutions.
Phosphate buffer solutions (PBS) prepared by mixing equi-molar (0.1 M) of K2HPO4 and KH2PO4 were used to prepare salbutamol sulfate solutions. 1.0 M of NaOH and HCl solutions were used to adjust the desired pH of the buffer solutions.
9
Gonococcal Growth Media Formulation
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Gonococcal (GC) base agar was made from 10 g/l dehydrated agar (BD Biosciences), 5 g/l NaCl (Roth), 4 g/l K2HPO4 (Roth), 1 g/l KH2PO4 (Roth), 15 g/l Proteose Peptone No. 3 (BD Biosciences), 0.5 g/l soluble starch (Sigma-Aldrich) and supplemented with 1% IsoVitaleX (IVX). IVX was made from 1 g/l D-glucose (Roth), 0.1 g/l L-glutamine (Roth), 0.289 g/l L-cysteine-HCL x H2O (Roth), 1 mg/l thiamine pyrophosphate (Sigma-Aldrich), 0.2 mg/l Fe(NO3)3 (Sigma-Aldrich), 0.03 mg/l thiamine HCL (Roth), 0.13 mg/l 4-aminobenzoic acid (Sigma-Aldrich), 2.5 mg/l β-nicotinamide adenine dinucleotide (Roth), and 0.1 mg/l vitamin B12 (Sigma-Aldrich). GC liquid medium is identical to the base agar composition but lacks both agar and starch.
10
Fecal Slurry Model for Gut Microbiome Dynamics
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Cell-free culture supernatants from the proximal and distal colon, both in resting conditions (basal) and after immune challenges (LPS and IL-15), were subsequently used to explore their effect on a stable fecal population via the fecal slurry model. For this purpose, we used a basal media composed of 2 g/l peptone water [Becton, Dickinson and Company (BD), Franklin Lakes, NJ, USA] 2 g/l yeast extract (BD), 0.1 g/l NaCl, 0.04 g/l K2HPO4, 0.04 g/l KH2PO4, 0.01 g/l MgSO4, 0.01 g/l CaCl2.⋅2H2O, 2 g/l NaHCO3, 2.5 g/l l -Cysteine-HCl, 0.5 g/l bile salts, 2 ml/l Tween-80, 1 g/l arabinogalactan, 2 g/l pectin, 1 g/l xylan, 4 g/l starch, 0.4 g/l glucose, and 0.4 g/l mucin type III (all purchased to Sigma-Aldrich). The mixture was homogenized and autoclaved for 15 min at 121°C, and the following components were added to the cooled media after sterilization by filtration (0.20 μm): 0.05 g/l bovine hemin (Sigma-Aldrich) and 10 μg/l vitamin K (Sigma-Aldrich). Before use, the basal media was maintained overnight at 37°C in anaerobiosis (10% v/v H2, 10%CO2, and 80% N2) in an anaerobic chamber Mac 500 (Don Whitley Scientific, West Yorkshire, UK).
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