Critical parameters and procedures for anaerobic cultivation of yeasts in bioreactors and anaerobic chambers

Abstract

All known facultatively fermentative yeasts require molecular oxygen for growth. Only in a small number of yeast species, these requirements can be circumvented by supplementation of known anaerobic growth factors such as nicotinate, sterols and unsaturated fatty acids. Biosynthetic oxygen requirements of yeasts are typically small and, unless extensive precautions are taken to minimize inadvertent entry of trace amounts of oxygen, easily go unnoticed in small-scale laboratory cultivation systems. This paper discusses critical points in the design of anaerobic yeast cultivation experiments in anaerobic chambers and laboratory bioreactors. Serial transfer or continuous cultivation to dilute growth factors present in anaerobically pre-grown inocula, systematic inclusion of control strains and minimizing the impact of oxygen diffusion through tubing are identified as key elements in experimental design. Basic protocols are presented for anaerobic-chamber and bioreactor experiments.

Class-II dihydroorotate dehydrogenases from three phylogenetically distant fungi support anaerobic pyrimidine biosynthesis

“…The gas mixture supplied to the anaerobic workstation contained 10% CO 2 , 5% H 2 and 85% N 2 . Measures to minimize inadvertent oxygen entry were implemented as described by [ 102 ]. When anaerobic precultures reached stationary phase, they were used to inoculate cultures on SMUD and SMUD + ura supplemented with Tween 80 and ergosterol.…”

“…[ 103 ] Optical density at 660 nm of aerobic cultures was measured using an Jenway 7200 spectrophotometer (Bibby Scientific, Staffordshire, UK) after accurate dilution to an OD 660 between 0.1 and 0.3. Anaerobic cultures were first diluted to an optical density at 600 nm (OD 600 ) between 0.15 and 0.35, followed by optical densities measurements at 600 nm on an Ultrospec 10 cell density meter (Biochrom, Harvard Biosience, Holliston, MA) that was placed in the anaerobic workstation [ 102 ].…”

A squalene–hopene cyclase inSchizosaccharomyces japonicusrepresents a eukaryotic adaptation to sterol-limited anaerobic environments

“…To reassess conclusions from an early literature report on sterol- and UFA-independent anaerobic growth of Sch. japonicus ( 21 ), we performed serial transfer experiments in an anaerobic chamber ( 36 , 41 ) using phosphate-buffered synthetic medium with glucose as carbon source (SMPD), with and without supplementation of ergosterol and/or Tween 80 as source of UFAs. Parallel experiments with the S. cerevisiae laboratory strain CEN.PK113-7D were included to assess low-level contamination with oxygen, as reflected by residual slow growth in sterol-free media ( 41 ).…”

“…Samples from these precultures were then used to inoculate experiments on regular SMPD supplemented with either Tween 80 and ergosterol, only Tween 80 or ergosterol, or neither. Cultures of S. cerevisiae CEN.PK113-7D on SMPD without Tween 80 or ergosterol were included in all anaerobic chamber experiments to assess low-level oxygen contamination ( 41 ). All growth experiments were performed by monitoring optical density at 600 nm in independent duplicate cultures.…”

Identification of fungal dihydrouracil-oxidase genes by expression in Saccharomyces cerevisiae

“…cultures were grown in a Bactron 300-2 anaerobic workstation (Sheldon Manufacturing Inc, Cornelius OR) with equipped with a Pd-catalyst and lled with a mixed gas atmosphere (85% N 2 , 10% CO 2 , 5% H 2 ). To minimise oxygen entry, the protocols described by Mooiman et al (2021) for cultivation in anaerobic chambers were followed. Flasks were placed on an IKA KS 260 basic orbital shaker (IKA-Werke, Staufen im Breisgau, Germany) at 240 rpm and temperature in the workspace was maintained at 30°C.…”