K amiar

The tryptic soy broth (TSB, CASO Buillon, X938.2) was from Carl Roth (Karlsruhe, Germany). The kanamycin sulfate (≥750 I.U./mg, T832.1), ampicillin sodium salt (≥97%, K029.5), chloramphenicol (≥98.5%, 3886.1), geneticin disulfate (Bio-Science grade, 2039.2), and erythromycin (≥98.0%, 4166.1) were from Carl Roth. The tetracycline (≥98.0%, 87128) was from Fluka (Vienna, Austria) and poly-(R)-3-hydroxybuttersäure (quality level 200, 363502) from Sigma-Aldrich (Vienna, Austria). Other chemicals were from Sigma-Aldrich/Fluka or Carl Roth and were of the highest purity available. An enzymatic assay for ammonium (K-AMIAR) was obtained from Megazyme International (Wicklow, Ireland). The strain C. necator H16 DSM 428 (aka ATCC 17699, NCIB 10442) was from DSMZ, Deutsche Sammlung für Mikroorganismen und Zellkulturen.

Ammonia was determined using an ammonia rapid assay (Megazyme, Bray, Ireland, Cat. no.: K-AMIAR). It is a highly specific assay involving the enzymatic conversion of 2-oxoglutarate, NADPH, and dissolved ammonia into L-glutamic acid, NADP+, and water under the action of glutamate dehydrogenase. Originally, the assay was not developed for NH₃ determination of In vitro fermentation, and, therefore, protocol optimization was executed.
Final optimized experimental protocol: 100 µL fermentation samples were centrifuged at 14,000 rpm (Spectrafuge 24D, Labnet, Woodbridge, New Jersey, USA) for 5 min, then 2 µL of supernatants (larger volume might result in assay saturation), ammonia standard (0.04 mg/mL) and water as blank were added with 30 µL buffer and 20 µL NADPH with 208 µL PBS (pH 9.8). Absorbance (A1) was taken at 340 nm with a microplate reader (Molecular Devices SpectraMax Plus, San Jose, California, USA). Then, 2 µL glutamate dehydrogenase was added to each well and allowed to react for 5 min, followed by second absorbance (A2) at 340 nm. The final ammonia concentration (mg/mL) was calculated as

Fermentations were carried out in a Sauvignon Blanc grape must harvested in 2016 (SB16) in the Bordeaux Area, provided by Vignobles Ducourt (Ladaux, France). Before fermentation, grape must was sterilized by membrane filtration (cellulose acetate 0.45 μm Sartorius Stedim Biotech, Aubagne, France). Fermenting sugars (205 g/l) were estimated by measuring glucose and fructose following the enzymatic method described by Stitt et al. (1989) (link). The Yeast Assimilable Nitrogen (YAN) content of SB16 was estimated enzymatically using enzymatic kits K-PANOPA and K-AMIAR (Megazyme) following the manufacturer’s instructions. SB16 contains 99 mg N/l of YAN (80 mg/N from primary amino acids and 19 mg N from ammonium), which represented the low nitrogen condition. A normal nitrogen modality containing 250 mg N/l was obtained by spiking SB16 with 170 mg/l of a mixture of amino acid and 80 mg/l of ammonium chloride. The composition of amino acid mixture is described elsewhere (Marullo et al., 2006 (link)).