Rapid detection of intracellular lipid content in oleaginous yeast

Rhodotorula glutinis

Motivation/Introduction

Acknowledgements/References

Conclusions and Outlook

a Technische Universität Berlin, Department of Biotechnology, Chair of Bioprocess Engineering,

Ackerstrasse 76 ACK24, D-13355 Berlin, http://www.bioprocess.tu-berlin.de

b Research and Teaching Institute for Brewing in Berlin, Department Bioprocess Engineering and Applied

Microbiology, Seestrasse 13, D-13353 Berlin, http://www.vlb-berlin.org/beam

Lorenz, E.a, Runge, D.a, Ngo, H. T. A.a, Marbà-Ardébol, A. M.a, Schmacht, M.b, Junne, S.a, Neubauer, P. a, Senz, M.b

The objective of the cooperative project FENA is the partial substitution of fish meal and fish oil by algae and oleaginous yeast for a sustainable and high-value fish

nutrition. Oleaginous yeasts are characterized by lipid contents of more than 20 % (Ratledge and Cohen 2005). They are able to accumulate up to 70 % of lipid of their own

mass (Ageitos et al., 2011). Typical representative yeast genera are Candida, Cryptococcus, Lipomyces, Yarrowia, Rhodosporidium and of course Rhodotorula.

Rhodotorula glutinis is an often used organism with a wide range of industrial usage reaching from biodiesel to carotenoids or as feed for breeding animals as well as

potential producer of industrial enzymes for food industry (Kot et al., 2016). For accumulation of high lipid contents, the limitation of minimum one or more essential

nutrients for cell proliferation, e.g. nitrogen, phosphor, iron, zinc or magnesium, is required. However, for biotechnology applications nitrogen limitation is preferably used to

induce lipid accumulation (Beopoulos et al., 2009; Granger et al., 1993). Therefore, cultivations of oleaginous yeasts are usually carried out in a medium with a high C to N

ratio. As long as nitrogen is present, the organism proliferates and produces (mainly lipid-free) biomass. After exhaustion of nitrogen, cell proliferation slows down and the

remaining carbon source is channeled into lipid formation.

For the development of a fermentation process, there are a lot of optimization parameters, e.g. medium components, C/N-ratio, pH, DO or temperature. Therefore, process

development takes a lot of time and experiments resulting in a large amount of samples. The upcoming samples should be analyzed as fast as possible. Traditional

methods like the gravimetric analysis described in detail by Bligh and Dyer, 1959 or the extraction of lipids by organic solvents followed by GC-FID analysis takes several

days. We developed three different methods for a rapid detection of intracellular lipid in the oleaginous yeast R. glutinis. Thereby, flow cytometry and fluorescence

spectroscopy combined with the staining dye Nile Red were applied for fast lipid detection.

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Concept and Results

Staining dye Nile Red

Figure 1. Kimura et al., 2004

characterized various oleaginous

yeast and fungi strains regarding

intracellular lipid content by means

of the fluorescence dye Nile Red.

This established staining method

was improved and simplified for a

rapid estimation of lipid content in

yeast, especially for R. glutinis.

Based on a wavelength screening,

the excitation and emission were

adjusted to 480 nm and 580 nm,

respectively.

96-well-plate (Nile Red)

Figure 3. Nile Red can also be used in 96-well-

plates (Greiner Inc., Fluotrac 200). Thereby,

OD600 should be adjusted in a range of 0.3 - 0.6.

200 µl cell suspension were stained with 4 µl of

100 µg∙ml-1 of Nile Red stock solutio, mixed by

pipetting and measured immediately in the

plate. 100 µl stained cell suspension was

discarded before measurement was executed.

Obtained RFU-values were divided by

previously determined OD600 (range of 0.3 -

0.6). Measurements were conducted at

480ex/580em (nm) with the Spectramax M2e

from Molecular Devices. Correlation with GC-

FID analysis resulted in an adjusted R-square of

0.889 and can be considered as valid method.

Flow cytometry (side scatter)

Figure 4. Measurements via flow cytometry

showed that side scatter (granularity) can be

used for adequate detection of intracellular

lipids. An adjusted R-square of 0.875 indicated

a strong correlation between granularity and

lipid content. In figure 2 (right) lipid drops

inside yeast cells can be direct visualized

without previous staining.

• Rapid lipid measurement is possible via photometric or flow cytometric methods

• Online lipid detection is even possible without staining via granularity (side scatter)

• Methods were proven by real HCD production process of R. glutinis with 106 g∙L-1

biomass and 65% intracellular lipid content.

Flow cytometry (Nile Red)

Figure 5. 1 ml washed cells with a concentration of

2-6∙106 cells∙ml-1 were stained with 20 µl of 100 µg∙ml-1 of Nile Red stock

solution, mixed for 5 s and measured immediately by flow cytometry.

Fluorescence signal was captured by channel FL2 (488 nm excitation and

bandpass filter 590/50). For statistically relevant evaluation, fluorescence values

of ≥1∙105 cells were recorded. This method showed among others the highest

correlation to GC-FID exhibiting an adjusted R-square of 0.959. Measurements

were executed by CyFlow® Cube 8 (Sysmex Deutschland GmbH). Exemplarily,

the progress of lipid content (histogram) measured by flow cytometry is plotted

(right).

lipid content

time

8h

10 µm

10 µm

10 µm

10 µm

10 µm

24h

32h

72h

80h

Figure 2. The final production process ensured high biomass concentrations of more 100 g∙L-1 and

60 % lipid content in a short period of time with R. glutinis. Microscopy pictures (right) visualized the

lipid accumulation during the process. Pictures were captured by using an Axio Imager M2

microscope equipped with an Axio Cam MR3 camera and a N-Achroplan 100x/1.25 oil m27 objective

(total magnification 1000x) applying the filter set 43 HE (Carl Zeiss Microscopy GmbH).

Propagation Lipid accumulation

The course of process development is often dependent on the speed of providing the analytical data. Therefore, we investigated different methods to speed up the process

development for maximizing lipid production with R. glutinis. In comparison to traditional protocols, the established methods based on flow cytometry and fluorescence

spectroscopy could significantly enhance the time of analysis and thus the time of process development from days to minutes. These detection procedures are reliable,

valid and easy to handle, indicated by high correlation quotients of up to 0.959. Further, the 96-well-plate spectroscopy assay can also be applied for extended strain

screening. As an additional rapid detection method for lipid accumulation, granularity can be measured by side scatter via flow cytometry. Therefore, it is conceivable that

an on-line integration of flow cytometry in a production facility for monitoring the lipid formation immediately without staining is of value. However, it has to be considered if

the developed methods are suitable for lipid detection in other strains as well.

Technische Universität Berlin

Single Cell Oils: microbial and algal oils / editors Z. Cohen and C. Radledge 2nd Edition, Publisher: AOCS Press Urbana, Illinois (2005)

Ageitos J M, Vallejo J A, Veiga-Crespo P, Villa T G: Oily yeasts as oleaginous cell factories. Applied Microbiology and Biotechnology 90, 1219 (2011)

Kot A M, Błazejak S, Kurcz A, Gientka I, Kieliszek M: Rhodotorula glutinis - potential source of lipids, carotenoids, and enzymes for use in industries. Applied Microbiology and Biotechnology (2016)

Beopoulos A, Chardot T, Nicaud J M: Yarrowia lipolytica: A model and a tool to understand the mechanisms implicated in lipid accumulation. Biochimie 91, 692 (2009)

Granger L-M, Perlot P, Goma G, Pareilleux A: Efficiency of fatty acid synthesis by oleaginous yeasts: Prediction of yield and fatty acid content from consumed C/N ration by a simple method. Biotechnology and Bioengineering 42, (1993)

Kimura K, Yamaoka M, Kamisaka Y: Rapid estimation of lipids in oleaginous fungi and yeasts using Nile red fluorescence. Journal of Microbiological Methods 56, (2004)