Development of a defined medium for the heterotrophic cultivation of Metallosphaera sedula

Abstract

The heterotrophic cultivation of extremophilic archaea still heavily relies on complex media. However, complex media are associated with unknown composition, high batch-to-batch variability, potential inhibiting and interfering components, as well as regulatory challenges, hampering advancements of extremophilic archaea in genetic engineering and bioprocessing. For Metallosphaera sedula, a widely studied organism for biomining and bioremediation and a potential production host for archaeal ether lipids, efforts to find defined cultivation conditions have still been unsuccessful. This study describes the development of a novel chemically defined growth medium for M. sedula. Initial experiments with commonly used complex casein-derived media sources deciphered Casamino Acids as the most suitable foundation for further development. The imitation of the amino acid composition of Casamino Acids in basal Brock medium delivered the first chemically defined medium. We could further simplify the medium to 5 amino acids based on the respective specific substrate uptake rates. This first defined cultivation medium for M. sedula allows advanced genetic engineering and more controlled bioprocess development approaches for this highly interesting archaeon.

Keywords: Metallosphaera sedula; Amino acids; Casamino Acids; Defined cultivation medium; Media development; Tetraether lipids.

Fig. 1

Growth kinetics of Metallosphaera sedula on Tryptone (Black square), Yeast Extract (White medium square), Casamino Acids (Black circle), N-Z-Amine (White Up-Pointing triangle) and Casein Hydrolysate (Black up-Pointin triangle) (at a concentration of 1 g/L in Brock basal medium at pH = 2.2 and 75 °C. Values for OD₆₀₀ are represented as mean ± standard deviation and derive from biological triplicates

Fig. 2

Growth kinetics of Metallosphaera sedula on Casamino Acids (Black circle) and imitation of Casamino Acids (Imitation CasA) (White Down-pointing Triangle) at a concentration of 1 g/L in Brock basal medium at pH = 2.2 and 75 °C. Values for OD₆₀₀ are represented as mean ± standard deviation and derive from biological triplicates

Fig. 3

Comparison of substrate uptake rates for the single amino acids (q_{s, amino acid}) of of Metallosphaera sedula cultivated on Brock basal supplemented with 1 g/L Casamino Acids (black) and 1 g/L imitation of Casamino Acids (white) at pH = 2.2 and 75 °C. (a) q_s for: Asp aspartate, Glu glutamate, Leu leucine, Lys lysine, Pro proline, (b) q_s for: Ser serine, His histidine, Gly glycine, Thr threonine, Arg arginine, Ala alanine, Cys cysteine, Val valine, Met methionine, Phe phenylalanine, Ile isoleucine, values are represented as mean ± standard deviation and were calculated for exponential growth phase between 24 and 44 h before substrate depletion from three replicates for every cultivation condition

Fig. 4

Proposed central metabolism in Metallosphaera sedula, including 3-Hydroxypropionate/ 4-Hydroxybutyrate cycle (3HP/4HB cycle) (red, left), incomplete tricarboxylic acid cycle (blue, center) and precursors for amino acid biosynthesis. Essential amino acids are highlighted in bold, amino acid components of the 5AA medium are in italics and amino acid components of the PS medium are underlined. Abbreviations in alphabetical order: Ac-CoA acetyl-coenzyme A, Ala alanine, Arg arginine, Asn asparagine, Asp aspartate, Cys cysteine, Gln glutamine, Glu glutamate, Gly glycine, His histidine, 3HP 3-hydroxypropionate, 4HB 4-hydroxybutyrate, Leu leucine, Lys lysine, Ile isoleucine, Met methionine, PEP phosphoenolpyruvate, Phe phenylalanine, Pro proline, Ser serine, SSA succinyl semialdehyde, Suc-CoA succinyl-coenzyme A, Thr threonine, Trp tryptophan, Tyr tyrosine, Val valine

Fig. 5

Growth kinetics of Metallosphaera sedula on Partly Simplified (Black down-Pointin triangle ) and 5AA medium (White Diamond) at a concentration of 1 g/L in Brock basal medium at pH = 2.2 and 75 °C. Growth kinetics of Casamino Acids (Black circle) and Imitation of Casamino Acids (Imitation CasA) (Black up-Pointin triangle), as seen in Fig. 2, are plotted transparently for better comparability. Values for OD₆₀₀ are represented as mean ± standard deviation and derive from biological triplicates

Fig. 6

Reflectron MALDI TOF–MS spectrum of Metallosphaera sedula lipid extract cultivated on 5AA medium in (a) positive-ion mode and (b) negative-ion mode