Lipid production by oleaginous yeast

Lipomyces starkeyi InaCC Y604

in the presence of furfural and 5-
hydroxymethylfurfural as the inhibitory
chemical compounds
Cite as: AIP Conference Proceedings 2606, 020016 (2023); https://doi.org/10.1063/5.0118890
Published Online: 04 January 2023

Ario Betha Juanssilfero, Eva Agustriana, Ahmad, et al.

AIP Conference Proceedings 2606, 020016 (2023); https://doi.org/10.1063/5.0118890 2606, 020016

© 2023 Author(s).
 Lipid Production by Oleaginous Yeast Lipomyces starkeyi
InaCC Y604 in the Presence of Furfural and 5-
hydroxymethylfurfural as the Inhibitory Chemical
Compounds
Ario Betha Juanssilfero1, a), Eva Agustriana1, b), Ahmad2, c), Nurul Izzati2, d), Urip
Perwitasari1, e), Fahrurrozi1, f), Agus Budiawan Naro Putra1, g), Puspita Lisdiyanti1, h),
Yopi1, i)
1
Research Center for Biotechnology, Research Organization of Life Science, National Research and Innovation
Agency (BRIN), Jl Raya Jakarta Bogor Km 46,Cibinong,West Java 16911, Indonesia
2
Technobiology Study Program, Faculty of Technobiology, Sumbawa University of Technology, Sumbawa, West
Nusa Tenggara, Indonesia
a)
Corresponding author: ario003@brin.go.id
b)
eva.agustriana@brin.go.id
c)
ahmadftb16@gmail.com
d)
nurul.izzati@uts.ac.id
e)
urip.perwitasari@brin.go.id
f)
fahrurrozi@brin.go.id
g)
agus135@brin.go.id
h)
puspita.lisdiyanti@brin.go.id
i)
yopi@bsn.go.id

Abstract. Microbial lipid produced using yeast fermentation with in-expensive carbon sources such as hydrolysate from
lignocellulosic may be an opportunity feedstock for biodiesel manufacturing. Furfural and 5-hydroxymethylfurfural (5-
HMF) that can be engendered during acid hydrolysis of lignocellulose had been brought merely or concurrently into a
culture medium comprising a mixture of two sugars (glucose and xylose) as carbon sources to investigate the sole
inhibitory actions and their synergic enforcement on the growth and lipid production of Lipomyces starkeyi InaCC Y604.
The used strain exhibited a particular resistance level against each of the archetypal inhibitor compounds. When
fermented using medium-contained furfural as a sole inhibitor compound, L. starkeyi InaCC Y604 accumulated the
highest microbial lipid (67.06%). While, when a mixture of furfural and 5-HMF was used as the inhibitor compounds in
the fermentation medium, the microbial lipid production was achieved at 66.9%. Meanwhile, microbial lipid production
was achieved at 63.68% when 5-HMF was used as the sole inhibitor in the fermentation medium. L. starkeyi InaCC Y604
showed the dexterity to tolerate inhibitors (furfural and 5-HMF) and the combination of fatty acid methyl esters (FAMEs)
was unchanged by the existence of inhibitor compounds. The considerable FAMEs comply with oleic, palmitic, stearic,
palmitoleic, and linoleic acids.

INTRODUCTION
Oleaginous microorganisms capable of metabolizing lignocellulose-derived sugars could be a viable and cost-
effective alternative [1]. Oleaginous yeasts create bio-oils for oleochemicals and nutritional oils that are renewable
and sustainable. The potential of oleaginous yeasts to collect between 20 and 70 percent intracellular lipid, primarily
triacylglycerols (TAGs), has been described [2-3]. Oleaginous yeasts produce lipids that are congruent in

Proceedings of the 8th International Symposium of Innovative Bioproduction Indonesia on Biotechnology and Bioengineering 2021
AIP Conf. Proc. 2606, 020016-1–020016-7; https://doi.org/10.1063/5.0118890
Published by AIP Publishing. 978-0-7354-4305-1/$30.00

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composition to vegetable oils for human habitation and biodiesel. Lipomyces starkeyi, Rhodotorula glutinis,
Yarrowia lipolytica, Cryptococcus curvatus, and Rhodosporidium toruloides are among the most common
oleaginous yeast [4-6].
When cultured on hydrolyzed lignocellulosic materials like forestry waste and other agricultural residues, some
oleaginous yeasts can develop and accumulate TAGs [7]. These types of oleaginous yeast show traits that are
beneficial for research and industrial implementations, including its deployment for capacious lignocellulosic
biomass or its tolerance to inhibitors produced during lignocellulosic biomass pretreatment [4,7]. Distension of these
studies to a larger group of oleaginous yeast species would typically be useful for the advancement of more vigorous
industrial yeast strains allowed to exploit a vaster range of nutrients and bear with upper-levels of inhibitors.
Glucose and xylose are the most typically sugars incepted from lignocellulosic biomass. As a result, oleaginous
yeast strains that consume both sugars (glucose and xylose) are preferable, as are those that can ingest minor sugars
such as mannose, galactose, or arabinose [8-10]. Dilute acid hydrolysis has been confirmed to be a rapid and
affordable method for generating sugars from lignocellulosic biomass [7]. It has the leverage of besides being
solubilizing hemicelluloses but additionally converting them to fermentable sugars. However, one obstacle related to
dilute acid hydrolysis is the conformation of toxic compounds such as aldehyde derivatives, aliphatic acids, and
phenolic compounds, which all impede microorganism growth significantly [4,7]. Consequently, an apperception of
the above-mentioned inhibitors and how to alleviate their adverse effects is required.
Lipomyces starkeyi is an emerging species amid the renowned oleaginous yeasts that have gained a lot of
attention because of its incredible biotechnology potential. With either or both glucose and xylose as a carbon source
in batch or fed-batch fermentation, the microbial lipid accumulations of this yeast have been well investigated [4,11-
13]. In this study, Lipomyces starkeyi InaCC Y604 was employed as indigenous yeast belonging to the Indonesian
Culture Collection (InaCC).
Our previous study demonstrated the potential of L. starkeyi InaCC Y604 to accumulate microbial lipid in
various synthetic carbon sources and showed promising findings with high-level microbial lipid appropriate to 70%
w/w [13]. In spite of that, in industrial applications, oleaginous yeast should be liable to accumulating high
concentration of intracellular lipid and be tolerant to inhibitory chemical compounds. Therefore, this study was
aimed at examining the capability of L. starkeyi InaCC Y604 to accumulate microbial lipid and elucidating its
tolerance with primary inhibitors generally settled on in lignocellulosic hydrolysates.

MATERIALS AND METHODS

Yeast Inoculum Preparation

Lipomyces starkeyi InaCC Y604 strain was supplied by the Indonesian Culture Collection (InaCC). A potato
dextrose agar (PDA) plate was used to recover the isolate while a yeast extract-malt extract-peptone-glucose
(YMPG) agar plate was used for culture maintenance. The inoculum was prepared by inoculating a single colony
from a YMPG agar plate to a pre-culture medium which contained of YMPG broth in an Erlenmeyer flask. The
details of inoculum preparation are described elsewhere [4,11,13]. For microbial lipid production, a nitrogen-limited
mineral medium (-NMM) broth was employed..
A proper volume of 1M H2SO4 was added to alter the pH of the medium to 5.5. After that, medium was sterilized
by filtration prior the inoculation. The media proportion used in this study were described other-where [11]. The -
NMM medium, which contained glucose (50 g/L) and xylose (50g/L) as a mixture of carbon sources, was added
with furfural (12mM) and 5-HMF (2mM) either individually or a mixture to evaluate microbial lipid accumulation
in the company of inhibitory substances. Four typical –NMM medium were used in this study: i) –NMM medium
contained glucose and xylose as the control medium, and from this point onward named –NMM GX; ii) –NMM GX
supplemented with furfural (-NMM GX+furfural); iii) –NMM GX supplemented with 5-HMF (-NMM GX+5-
HMF); iv) and –NMM GX supplemented with a mixture of furfural dan 5-HMF (-NMM GX+furfural+5-HMF; v).
All chemicals used were of analytical grade unless stated otherwise.

Analysis of Sugars
The sugars concentrations through fermentation were measured using a high-performance liquid chromatography
(HPLC) system (Shimadzu, Kyoto, Japan). The column temperature was set at 80 oC, where An ICSep Coregel® -
87H column (300 x 7.8 mm Transgenomic, USA) was used. Sulfuric acid with concentration 5mM was used as a

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mobile phase at a flow rate of 0.6 mL/min. The cell mass and insoluble components were eliminated by
centrifugation and then filtered over a 0.22 mm filter prior analysis.

Total Lipid Measurement

The Folch method [11,14] was used to reckon microbial lipid production by gravimetric analysis. A Mixer Mill
(Retsch, Germany) was used for cells comminution. Subsequent to communited fracture, cells were then centrifuged
and the supernatant was desolated. The cell pellets were dehydrated to a consistent mass in a drying oven at 60 oC.
The weight difference was used to calculate the microbial lipid content, which was represented as a percentage of
dry cell weight (DCW).

Total FAMEs Measurement

The transesterification was carried out according to methods described elsewhere [11-13]. Supelco 37
component component FAME (Sigma-Aldrich) were used to identified FAME by differentiating their retention
times and peak areas. Tridecanoic acid (C13:0) was used as the internal standard and included in each particular
sample. Finally, the percentage of each single fatty acid was calculated as the percentage (%) to the total number of
fatty acids formed over fermentation.

RESULTS AND DISCUSSION

Fermentation Profile of Lipomyces starkeyi InaCC Y604

Two-stage cultivations of L. starkeyi InaCC Y604 were used to produce microbial lipids. The first stage was
performed by the preparation of seed culture at OD600nm 2.0-3.0 to initiate the cell growth. The seed was then
inoculated into four different –NMM mediums (with or without inhibitory chemical compounds) to initiate the lipid
production stage by L. starkeyi InaCC Y604.
The fermentation pattern, along with the sugars assimilation and cell growth (in OD600nm) from distinct typical
mediums for microbial lipid productions of L. starkeyi InaCC Y604, are presented in Fig. 1. The time course of
carbon sources consumed varied during 3-5 days of incubation in all mediums used (Fig. 1). Generally, in all four
typical mediums, L. starkeyi InaCC Y604 requisite three days to completely consume glucose, whereas for xylose
reached 4-5 days subjected to medium compositions.
Figure 1a demonstrates that L. starkeyi InaCC Y604 may practically concurrently assimilate a glucose-xylose
mixture as the culture control. The rate of xylose consumption appeared to be lower than that of glucose on the first
and second days. Nevertheless, the xylose assimilation was remarkably elevated immediately when glucose in the
medium descended to a range from 3-5 g/L. Glucose consumption rates were greater than xylose in the early stages
of cultivation, but the two sugars were consumed at the identical rate in the late stages of fermentation. When
unveiled to a mixture of glucose and xylose, most microorganisms at the first stage normally metabolize preferred
sugars, mainly glucose. However, glucose can inhibit the utilization of other sugars through a catabolic inhibition
mechanism or allosteric contention for sugar bearer [15,16].
Figure 1b, shows the fermentation profile of L. starkeyi InaCC Y604 in the presence of furfural in the
fermentation medium. It was shown the slight inhibition to the glucose assimilation at the first to second days
compared to the control mediums. The glucose concentration in the medium contained furfural was exhausted after
3 days fermentation which has a similar period with the control medium. On the other side, the xylose assimilation
was significantly obstructed by the presence of furfural especially at the first to second day’s fermentation. The
xylose concentration was depleted after five days of fermentation, which was one day slower than the control
medium.

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 FIGURE 1. Time course for carbon source assimilation and cell growth of L. starkeyi InaCC Y604 in four typical mediums
namely: a. –NMM GX (control); b. –NMM GX+furfural; c. –NMM GX+5-HMF; d. –NMM GX+furfural+5-HMF. The closed
square refers to cell growth (DCW, g/L); closed triangle plots symbolize xylose consumption; and the closed diamond symbolize
glucose assimilation.

The fermentation profile in the presence of 5-HMF (Figure 1c) has a similar pattern with the previously
mentioned above (Fig. 1b), where the glucose and xylose assimilation were inhibited during the first and second
days of fermentation. Nevertheless, the glucose consumption rate on the first day in the fermentation medium
containing 5-HMF was slightly higher than occurred in the fermentation profile by the presence of furfural (Fig. 1b).
The fermentation profile of L. starkeyi InaCC Y604 in the presence of furfural and 5-HMF mixture is shown in
Fig. 1d. Glucose assimilation during 2 days of fermentation was seemed weak compared to other conditions.
However, the glucose assimilation rate was significantly increased after two days of fermentation and exhausted in 3
days. Longer fermentation occurred for xylose assimilation, and the existence of 5-HMF in the fermentation medium
resulted in insufficient assimilation of xylose at the late stage of fermentation. This condition was indicated by the
existence of a residual amount of xylose that is not assimilated during cultivation.
Two furan aldehydes derivatives (furfural, and 5-hydroxymethylfurfural) were employed in this study to
demonstrate the irrepressible of pentose and hexose sugars generated throughout the pretreatment of lignocellulosic
biomass. The hydrolysate of lignocellulosic biomass normally contains more than one inhibitory compound [4,7,17].
Therefore, two typical inhibitory compounds were used as a cocktail mixture to evaluate the effect of inhibitory
compounds on L. starkeyi InaCC Y604 fermentation to produce microbial lipid. The concentration of inhibitor were

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selected to represents the range often found in hydrolysates [4]. The concentration of inhibitors varied significantly
depending on the feedstock and the pretreatment technique [18]. Many yeast strains exhibited delayed carbon
sources assimilation in the presence of inhibitory compounds during fermentation process [4,7,17,18]. Furfural was
found less toxic to L. starkeyi followed by 5-HMF [4]. There’s an unknown interrelationship within the
hydrophobicity and toxicity of these two furan aldehydes. The aldehydes hydrophobicity notwithstanding,
determinant of its toxicity [4,19,20].

Effect of Inhibitor Compounds on Microbial Lipid Production and FAME Composition

The presence of inhibitor compounds in the cultivation medium apart from being inhibited cell growth and
reduced the carbon source consumption rate also resulted in a decrease in microbial lipid production. The lipid
production for L. starkeyi InaCC Y604 in the four typical mediums is compared in Table 1. The microbial lipid
productions by this strain were achieved up to 67% (w/w) even though in the presence of inhibitory compounds. In
our previous study using L. starkeyi NBRC107654, microbial lipid production was achieved at 77% (w/w) when
fermented in more complex inhibitory compounds [4]. Nevertheless, the potentiality of oleaginous yeasts to produce
lipid is strain’s specific. Generally, the biomass production, and lipid accumulation were concise in the presence of
inhibitory compounds in the cultivation medium. In the presence of furfural, the lipid content was condensed from
73.9% (w/w) while cultivated in –NMM GX as the control medium to 67.1% (w/w) in –NMM GX+furfural; in the
presence of 5-HMF lipid content dropped to 63.7% (w/w); and 61.9% (w/w) in the presence of furfural+5-HMF.
In spite of the fact that the microbial lipid produced by L. starkeyi InaCC Y604 was reduced in the presence of
furfural and 5-HMF, this strain's ability to accumulate lipid was greater than 60% (w/w), indicating that it could
detoxify furfural and 5-HMF. Both of these inhibitor compounds were converted toward less toxic resembling
alcohols (furfuryl alcohol and HMF alcohol, respectively), then toward less toxic corresponding acid (furoic acid
and HMF acid) [17]. The partial conversion into less toxic alcohols and acids of furfural and HMF provided strong
support for its tolerance since microbial tolerance is generally understood as the biodegradation capability into less
toxic derivatives [21,22]. Despite the fact that many previous research have exhibited that yeast could metabolize
furans [7,17], 5-HMF [4] the exact mechanism incorporated remains unknown in most cases and ought to be
explicated in future studies. Our data designated that L. starkeyi InaCC Y604 tends to be more sensitive to 5-HMF
than to furfural at the represented range found in the hydrolysate.

TABLE 1. Lipid production comparison of L. starkeyi InaCC Y604 grown in four typical mediums
Medium Cell biomass Lipid content
g/Lb % (w/w)c
-NMM GX (control) 34.5±0.1 73.9±1.3
-NMM GX+furfural 28.8±0.7 67.1±3.4
-NMM GX+5-HMF 25.3±0.3 63.7±2.1
-NMM GX+furfural+5-HMF 22.7±0.9 61.9±3.1
aThe values are given as mean ±SD of triplicate determinations
bFinal cellular biomass at the end of cultivation time
cLipid content determined by gravimetric

Table 2 shows the fatty acid methyl esters (FAMEs) composition of L. starkeyi InaCC Y604 cultivated in four
different mediums. Holistically, FAME profiles were no significant variations between the control medium and the
medium containing inhibitor compounds. The FAMEs were unchanged by the presence of furfural and/or 5-HMF,
which displays that inhibitory compounds insignificantly alter the FAME biosynthetic pathway. Furthermore, some
traces fatty acid in the presence of furfural+5-HMF were slightly increased (data not shown). The major fatty acids
produced in all cultivation conditions were oleic acid (C18:1), palmitic acid (C16:0), stearic acid (C18:0),
palmitoleic acid (C16:1), and linoleic acid (C18:2). The profiles of FAMEs produced by L. starkeyi InaCC Y604 in
four typical medium compositions are similar to those of vegetable oils. In this study, oleic acids were discovered to
be the most abundant fatty acid generated by L. starkeyi InaCC Y604 (more than 45 percent). The use of a large
amount of oleic acid as a raw material for biofuels synthesis can increase biodiesel's oxidative stability and cold
flow qualities. Furthermore, numerous renewable polymers, elastomers, lubricants, and other oleochemicals have
used high oleic acid as an input [4,23,24].

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 TABLE 2. FAMEs profiles of L. starkeyi InaCC Y604 grown on four typical mediumsa
FAMEs compositions (%)
Trace
Medium
C16:0 C16:1 C18:0 C18:1 C18:2 fatty
acidsb
-NMM GX (control) 32.5±1.0 3.1±1.0 6.9±0.8 53.2±1.5 1.6±0.4 2.7
-NMM GX+furfural 37.3±3.0 1.9±1.2 4.9±2.8 48.7±3.5 1.9±0.7 2.4
-NMM GX+5-HMF 39.5±4.5 3.7±2.1 5.1±3.8 45.2±2.7 2.6±0.8 2.9
-NMM GX+furfural+5-HMF 35.3±1.1 4.6±1.0 9.2±0.8 46.8±1.9 2.4±1.4 2.2
aThe values are given as mean ± SD of duplicate determination.
bThe percentage of individual FAME below 1% were categorized as trace elements

CONCLUSION
This study exhibited that supplementary to obstructing growth, the existence of inhibitor compounds
corresponded with diminished microbial lipid accumulation. This corolarry is diversified over different inhibitors: 5-
HMF usually reduces both growth and lipid content more extremely than furfural and it is also strain’s specific.
Alternative for diminishing these effects comprise usage of of hydrolysates with detoxification of hydrolysate,
decreased inhibitor concentrations, or preference of yeast strains which have naturally resistant to the inhibitors.
This study dispenses reinforcement for those desiring to pursue the third option. The process development will be
aided by further evaluation of the effects of crucial inhibitors on biomass and microbial lipid accumulation by a
prospective yeast expectant.

ACKNOWLEDGMENTS
This study was financed by DIPA-PN Biorefinery 2019 under grant 3403.001 and Research Center for
Biotechnology- National Research and Innovation Agency (BRIN).

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