Achieving a high‐density oleaginous yeast culture: Comparison of four processing strategies using Metschnikowia pulcherrima

Abstract

Microbial lipids have the potential to displace terrestrial oils for fuel, value chemical, and food production, curbing the growth in tropical oil plantations and helping to reduce deforestation. However, commercialization remains elusive partly due to the lack of suitably robust organisms and their low lipid productivity. Extremely high cell densities in oleaginous cultures are needed to increase reaction rates, reduce reactor volume, and facilitate downstream processing. In this investigation, the oleaginous yeast Metschnikowia pulcherrima, a known antimicrobial producer, was cultured using four different processing strategies to achieve high cell densities and gain suitable lipid productivity. In batch mode, the yeast demonstrated lipid contents more than 40% (w/w) under high osmotic pressure. In fed-batch mode, however, high-lipid titers were prevented through inhibition above 70.0 g L −1 yeast biomass. Highly promising were a semi-continuous and continuous mode with cell recycle where cell densities of up to 122.6 g L −1 and maximum lipid production rates of 0.37 g L −1 h −1 (daily average), a nearly two-fold increase from the batch, were achieved. The findings demonstrate the importance of considering multiple fermentation modes to achieve high-density oleaginous yeast cultures generally and indicate the limitations of processing these organisms under the extreme conditions necessary for economic lipid production. K E Y W O R D Scontinuous, fed-batch, high cell density, microbial lipids, oleaginous yeast, semi-continuous

The role of temperature, pH and nutrition in process development of the unique oleaginous yeast Metschnikowia pulcherrima

“…The strain of M. pulcherrima (NCYC 4331, National Collection of Yeast Cultures, Norfolk, UK) was previously evolved towards higher inhibitor tolerance and kept as 20% glycerol stock at −80 °C . One full loop from the stock was plated out on malt extract agar (MEA: agar 15 g L −1 ; malt extract 30 g L −1 ; mycological peptone 5 g L −1 ) plates, incubated at 20 °C for a minimum of 3 days and maximum of 2 weeks.…”

“…For the preculture, one colony from the agar plate was placed in 20 mL soy–malt broth (SMB: soy peptone 30 g L −1 ; malt extract 25 g L −1 ; pH 5) in 100 mL Erlenmeyer flasks and incubated at 20 °C for 24 h, unless specified otherwise . Standard medium for main cultivation was nitrogen‐limited broth with 80 g L −1 glucose (NLB: KH 2 PO 4 7 g L −1 ; (NH 4 ) 2 SO 4 2 g L −1 ; NaHPO 4 1 g L −1 ; MgSO 4 7·H 2 O 1.5 g L −1 ; yeast extract 1 g L −1 ; glucose 80 g L −1 ; pH 5; C:N ratio 60.1 g g −1 ), which was inoculated with 2.5% ( v / v ) preculture. For investigating the influence of ammonium nitrogen (NH 4 ‐N) and YE on lipid production parameters, the ammonium sulphate (NH 4 ) 2 SO 4 and YE concentration in the medium were separately adjusted to 4 (C:N ratio 33.5 g g −1 ) and 8 g L −1 (17.8 g g −1 ) or 0.5 (66.9 g g −1 ), 2 (50.0 g g −1 ) and 4 g L −1 (37.6 g g −1 ), respectively, with the carbon and nitrogen content of YE assumed as 12 and 11% ( w / w ).…”

“…In synthetic media, limiting nitrogen is often used with a carbon–nitrogen (C:N) ratio typically adjusted between 30 and 80 g g −1 . A negative effect of ammonia salts on M. pulcherrima growth has been ascertained, though it does not appear to have a detrimental effect on the lipid content . The addition of complex nutrients such as yeast extract (YE) also influences the C:N ratio, and a negative effect on the lipid content is common among oleaginous yeasts .…”

“…We therefore investigated the effects of temperature, pH, C:N ratio and YE on yeast lipid production in controlled STRs [here the DO was controlled through aeration and agitation (i.e. shear stress variation) and measured in relative (%) and not absolute units (mg L −1 ) 22 ], using a recently reported evolved strain of M. pulcherrima . The biotechnological potential of this yeast has been showcased previously, however the effect of the aforementioned process parameters has only been addressed marginally .…”

“…shear stress variation) and measured in relative (%) and not absolute units (mg L −1 ) 22 ], using a recently reported evolved strain of M. pulcherrima . The biotechnological potential of this yeast has been showcased previously, however the effect of the aforementioned process parameters has only been addressed marginally . Focus is placed on the kinetics of growth and lipid production, the major drawback of M. pulcherrima as a potential host for commercial lipid production …”