High-throughput screening of Mucoromycota fungi for production of low- and high-value lipids

Abstract

Background: Mucoromycota fungi are important producers of low- and high-value lipids. Mortierella alpina is used for arachidonic acid production at industrial scale. In addition, oleaginous Mucoromycota fungi are promising candidates for biodiesel production. A critical step in the development of such biotechnological applications is the selection of suitable strains for lipid production. The aim of the present study was to use the Duetz-microtiter plate system combined with Fourier transform infrared (FTIR) spectroscopy for high-throughput screening of the potential of 100 Mucoromycota strains to produce low- and high-value lipids.

Results: With this reproducible, high-throughput method, we found several promising strains for high-value omega-6 polyunsaturated fatty acid (PUFA) and biodiesel production purposes. Gamma-linolenic acid content was the highest in Mucor fragilis UBOCC-A-109196 (24.5% of total fatty acids), and Cunninghamella echinulata VKM F-470 (24.0%). For the first time, we observed concomitant gamma-linolenic acid and alpha-linolenic acid (up to 13.0%) production in psychrophilic Mucor flavus strains. Arachidonic acid was present the highest amount in M. alpina ATCC 32222 (41.1% of total fatty acids). Low cultivation temperature (15 °C) activated the temperature sensitive ∆17 desaturase enzyme in Mortierella spp., resulting in eicosapentaenoic acid production with up to 11.0% of total fatty acids in M. humilis VKM F-1494. Cunninghamella blakesleeana CCM-705, Umbelopsis vinacea CCM F-539 and UBOCC-A-101347 showed very good growth (23-26 g/L) and lipid production (7.0-8.3 g/L) with high palmitic and oleic acid, and low PUFA content, which makes them attractive candidates for biodiesel production. Absidia glauca CCM 451 had the highest total lipid content (47.2% of biomass) of all tested strains. We also demonstrated the potential of FTIR spectroscopy for high-throughput screening of total lipid content of oleaginous fungi.

Conclusions: The use of Duetz-microtiter plate system combined with FTIR spectroscopy and multivariate analysis, is a feasible approach for high-throughput screening of lipid production in Mucoromycota fungi. Several promising strains have been identified by this method for the production of high-value PUFA and biodiesel.

Keywords: Biodiesel; FTIR; Filamentous fungi; High-throughput screening; Mucoromycota; PUFA; Single cell oil.

Fig. 1

a, b Variety of Mucoromycota fungi morphologies grown under lipid accumulation conditions in Duetz-MTPS (small-big pellets, dispersed, wall-growth), c Mucor mucedo UBOCC-A-101353, d Mucor hiemalis UBOCC-A-101359

Fig. 2

Different microscopic morphologies of oleaginous mycelium of Mucoromycota fungi. a Mucor racemosus FRR 3336, b Mucor circinelloides CCM 8328 (single cell form), c Mucor hiemalis UBOCC-A-101359, d Rhizopus oryzae CCM 8075, e Umbelopsis isabellina UBOCC-A-101350, f Umbelopsis ramanniana CCM F-622, g Umbelopsis vinacea UBOCC-A-101347, h Umbelopsis vinacea CCM F-539, i Absidia coerulea CCM 8230, j Cunninghamella blakesleeana VKM F-993, k Mortierella zonata UBOCC-A-101348, l Mortierella hyalina VKM F-1854

Fig. 3

a Fatty acid profile (%), b total lipid content of biomass (%) and biomass concentration (g/L) of Amylomyces rouxii and Mucor fungi

Fig. 4

a Fatty acid profile (%), b total lipid content of biomass (%) and biomass concentration (g/L) of Rhizopus (1), Umbelopsis (2), Absidia/Lichtheimia (3), Cunninghamella (4) fungi

Fig. 5

a Fatty acid profile (%), b total lipid content of biomass (%) and biomass concentration (g/L) of Mortierella fungi

Fig. 6

Main fermentation parameters for the tested Mucoromycota genera. a Biomass concentration (g/L), b glucose consumption (g/L), c total lipid content of biomass (%), d lipid concentration (g/L medium), e biomass- and f lipid yield on glucose (g/g), g unsaturation indices (−)

Fig. 7

a Scores plot of GC fatty acid data. Numbers in the scores plot refer to strains in Table 1, while letters refer to biological replicates (3 biological replicates: a, b, c or 5 biological replicates: a, b, c, d, e for M. circinelloides strains). b Loadings plot of GC fatty acid data. Fatty acid data was autoscaled before PCA

Fig. 8

(EMSC corrected) FTIR spectra of Mucoromycota fungi with low, intermediate, and high total lipid content. Peak assignments can be found in Additional file 1: Table S3

Fig. 9

a Scores and b loadings (PC1-2) plots of FTIR data (EMSC corrected). The explained variances for the first five PCs are 78, 9, 5, 3 and 2%. Numbers in the scores plot refers to strains in Table 1, while letters refer to biological replicates (3 biological replicates: a, b, c or 5 biological replicates: a, b, c, d, e for M. circinelloides strains). Peak assignments can be found in Additional file 1: Table S3