Upgrading Major Waste Streams Derived from the Biodiesel Industry and Olive Mills via Microbial Bioprocessing with Non-Conventional Yarrowia ...
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fermentation
Article
Upgrading Major Waste Streams Derived from the Biodiesel
Industry and Olive Mills via Microbial Bioprocessing with
Non-Conventional Yarrowia lipolytica Strains
Dimitris Sarris 1, * , Erminta Tsouko 1 , Maria Kothri 1 , Maria Anagnostou 1 , Eleni Karageorgiou 1
and Seraphim Papanikolaou 2
1 Laboratory of Physico-Chemical & Biotechnological Valorization of Food By-Products, Department of Food,
Science & Nutrition, School of the Environment, University of the Aegean, Leoforos Dimokratias 66, Lemnos,
81400 Myrina, Greece
2 Laboratory of Food Microbiology & Biotechnology, Department of Food Science & Human Nutrition,
Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece
* Correspondence: dsarris@aegean.gr
Abstract: This study reports the development of a bioprocess involving the valorization of biodiesel-
derived glycerol as the main carbon source for cell proliferation of Yarrowia lipolytica strains and
production of metabolic compounds, i.e., citric acid (Cit), polyols, and other bio-metabolites, the
substitution of process tap water with olive mill wastewater (OMW) in batch fermentations, and
partial detoxification of OMW (up to 31.1% decolorization). Increasing initial phenolics (Phen)
of OMW-glycerol blends led to substantial Cit secretion. Maximum Cit values, varying between
64.1–65.1 g/L, combined with high yield (YCit/S = 0.682–0.690 g Cit/g carbon sources) and produc-
tivity (0.335–0.344 g/L/h) were achieved in the presence of Phen = 3 g/L. The notable accumulation
of endopolysaccharides (EPs) on the produced biomass was determined when Y. lipolytica LMBF
Y-46 (51.9%) and ACA-YC 5033 (61.5%) were cultivated on glycerol-based media. Blending with
Citation: Sarris, D.; Tsouko, E.; various amounts of OMW negatively affected EPs and polyols biosynthesis. The ratio of manni-
Kothri, M.; Anagnostou, M.; tol:arabitol:erythritol was significantly affected (p < 0.05) by the fermentation media. Erythritol
Karageorgiou, E.; Papanikolaou, S. was the major polyol in the absence of OMW (53.5–62.32%), while blends of OMW-glycerol (with
Upgrading Major Waste Streams Phen = 1–3 g/L) promoted mannitol production (54.5–76.6%). Nitrogen-limited conditions did not
Derived from the Biodiesel Industry favor the production of cellular lipids (up to 16.6%). This study addressed sustainable management
and Olive Mills via Microbial
and resource efficiency enabling the bioconversion of high-organic-load and toxic waste streams into
Bioprocessing with Non-
valuable products within a circular bioeconomy approach.
Conventional Yarrowia lipolytica
Strains. Fermentation 2023, 9, 251.
Keywords: crude glycerol; olive mill wastewater; value-added metabolites; sustainable management;
https://doi.org/10.3390/
fermentation9030251
resource efficiency; bioremediation
Academic Editor: Diomi Mamma
Received: 18 January 2023
Revised: 24 February 2023 1. Introduction
Accepted: 1 March 2023 Sustainable development is a top priority for the United Nations, while shared
Published: 3 March 2023 blueprints, including the 2030 Agenda for Sustainable Development for human and envi-
ronmental propensity [1,2] and the ‘Fit for 55’ EU policy for climate neutrality [3], have
already been introduced. The long-term targets include decoupling economic growth from
newly extracted resources, zero net emissions, and decarbonization of production processes.
Copyright: © 2023 by the authors.
Industries of food-, edible oil- and lignocellulosic biomass processing, as well as biodiesel
Licensee MDPI, Basel, Switzerland.
This article is an open access article
production, generate substantial volumes of carbon-laden water waste streams that must
distributed under the terms and
be properly managed to deviate from severe environmental and economic effects [4]. The
conditions of the Creative Commons efficient management of such feedstock via green manufacturing and microbial biocon-
Attribution (CC BY) license (https:// version to obtain value-added products is of utmost importance to facilitate the world’s
creativecommons.org/licenses/by/ transition towards sustainability, renewability, and circular bioeconomy concepts [5].
4.0/).
Fermentation 2023, 9, 251. https://doi.org/10.3390/fermentation9030251 https://www.mdpi.com/journal/fermentationFermentation 2023, 9, 251 2 of 17
Olive oil processing produces around 30 million m3 /per year of olive mill wastewater
(OMW) globally [6]. These high quantities of OMW are toxic since they are conventionally
deposited directly in aquatic ecosystems, leading to the increased organic charge and phy-
totoxicity of air and water. It has been reported that OWM could create 200–400 times more
pollution than urban wastewater [4]. Crude glycerol (mostly from the biodiesel industry)
production is estimated at around 41.9 billion, with 66% of it being generated from the
biodiesel industry [7]. It constitutes the most severe obstacle to the effective development of
biodiesel as an alternative biofuel. Several strategies have been explored for its value-added
valorization [8], with microbial bioconversion being among the most promising.
Organic acids constitute significant building blocks that have a prominent contribution
to the biotechnological production of commodity chemicals. The accumulation of most of
the organic acids, i.e., citric acid (Cit), occurs via both catabolic and anabolic activities in
several microbial strains [9]. The global Cit market is anticipated to reach USD 3.83 billion
by 2025, at a CAGR of 4.9%, while the food and beverages applications of Cit are the
main factors that drive its increasing market [10]. Cit is a “generally recognized as safe”
tricarboxylic acid, and it is widely applied as a flavoring, emulsifying, stabilizing and
texturing agent in food-related applications [11]. Numerous feedstocks have been explored
for Cit production via fermentation with yeasts such as Yarrowia lipolytica. Y. lipolytica can
utilize several hydrophilic and hydrophobic fermentation substrates, including commercial
C5 and C6 sugars, cellulose- and hemicellulose-based hydrolysates, glycerol, and fatty
compounds [12,13].
Natural sweeteners, i.e., mannitol, xylitol, arabitol and erythritol, are gaining a tremen-
dous market share as alternatives of added sugars (sucrose, fructose, glucose, syrups)
in novel formulas as they have been approved as food additives in the EU and US [14].
The worldwide market of polyols was US$26 billion in 2019, while it is projected to reach
US$34 billion by 2024 [15]. This increase is mainly attributed to the increasing need for
polyols in sectors of food, pharmaceuticals, polymers (building blocks for polyurethane
production), and chemicals. Polyols are low-metabolizable sugar alcohols with a strong
sweetening capacity, low caloric and glycemic profile, and several health-promoting prop-
erties related mostly to diabetes (i.e., reduced insulin response), obesity and non-cariogenic
activity. Diabetes was related to 4 million deaths and costs of USD$727 billion for health
care in 2017 [16]. Industrial production of polyols is carried out mostly by the catalytic
reduction of sugars with hydrogen under elevated pressure and temperature, while the
whole process requires highly pure sugars as the initiating material and high-cost chro-
matographic purification steps. Recently scientific research has focused on biotechnological
approaches to improve the production efficiency (yield, productivity, cost) of polyols val-
orizing unconventional carbohydrate-rich feedstock that is, hemicellulosic hydrolysate
derived from corn cob and rapeseed straw, fruit juices, oilseed meals, sugarcane bagasse,
molasses, straws of wheat and rapeseed, residue of soybean extract, and glycerol, em-
ploying lactic acid bacteria or yeast strains i.e., Debaryomyces hansenii, Candida tropicalis,
Kluyveromyces marxianus, and Yarrowia lipolytica etc. [14,16,17]
In the current study, two non-conventional yeast strains, namely Y. lipolytica LMBF
Y-46 and Y. lipolytica ACA-YC 5033, were investigated for their potential to grow and to
produce Cit in various blends of crude glycerol-OMW in shake flask batch fermentations.
Their metabolic profile, including the co-production of EPs, microbial oil and polyols, was
also monitored. The inhibitory effect of initial phenolic compounds (Phen) found in OMW
was thoroughly investigated. The detoxification potential of both strains, as far as the
decolorization of OMW was considered, was determined. The fatty acid profile of cellular
lipids was reported under all the performed fermentation conditions.
The main novelty of this study is the fact that principles of circular bioeconomy were
implemented, including microbial bioprocessing, to produce bioproducts of remarkable
commercial interest, such as Cit and polyols. Microbial biomass rich in EPs and unsaturated
microbial oil could also be considered as a co-product of high nutritional value with targeted
end-uses. The whole fermentation process resulted in a substantial bioremediation of OMWFermentation 2023, 9, 251 3 of 17
via decolorization. In this way, a sustainable and circular process could be developed for
the efficient valorization of major pollutants from the biodiesel industry and olive mills.
2. Materials and Methods
2.1. Microorganisms and Raw Materials
The yeast strains Y. lipolytica ACA-YC 5033 (isolated from traditional Greek wheat
sourdough) [18] and Y. lipolytica LMBF Y-46 (isolated from gilt-head (sea) bream, Sparus
aurata, fish) [19] were used to produce Cit. The strains were maintained on potato dextrose
agar (39 g/L) at 6 ◦ C, and they were regularly sub-cultured to ensure cells’ viability.
The OMWs were obtained from an olive mill in the Perichora region (Corinthia,
Greece), equipped with a three-phase decanter. OMWs were centrifuged (9000 rpm, 15 min,
4 ◦ C; Hettich-Universal 320R, Germany centrifuge) to remove any solids while the super-
natant was collected and stored at −20 ◦ C for further use. Crude glycerol (purity of 85%,
6% potassium and sodium salts, 1% lipids, 5% water,Fermentation 2023, 9, 251 4 of 17
uid phase was separated by filtration and neutralized with 10 mL of 2 M NaOH, and
the carbon sources were determined via a 3,5-dinitrosalicylic acid assay as described by
Miller [23]. The absorbance was measured at 540 nm, and the EPs were expressed as
glucose (g/L) equivalents.
The free amino nitrogen (FAN) concentration of the fermentation broth was deter-
mined according to the ninhydrin colorimetric method [24], and the absorbance was
measured at 570 nm.
Glycerol, mannitol, arabitol, erythritol, and Cit were determined by high-performance
liquid chromatography analysis (Waters Association 600E) equipped with a UV (Waters
486) and RI detector (Waters 410). The column was Aminex HPX-87H (Biorad; 300 mm
length × 7.8 mm internal diameter). The mobile phase was 5 mM H2 SO4 aqueous solution
with 0.6 mL/min flow rate at 65 ◦ C.
Fatty acids were transformed into fatty acid methyl esters (FAMEs) using sodium
methoxide followed by methanol with HCl as a catalyst. FAMEs were determined via
gas chromatography Fisons 8060 equipped with a Chrompack column (60 m × 0.32 mm,
Chrompack; CP-Wax 52 CB GC column-Agilent) and a flame ionization detector (FID)
using He as the carrier gas (2 mL/min). The injection volume was equal to 1 µL while
the GC was run in a splitless mode. The oven program was initiated at 50 ◦ C, heated
to 200 ◦ C with a ratio of 25 ◦ C/min (1 min), then increased with a ratio of 3 ◦ C/min up
to 240 ◦ C, and increased to 250 ◦ C with a ratio of 25 ◦ C/min and maintained for 3 min.
The detector temperature was 250 ◦ C. FAMEs were identified by reference to a standard
(Supelco® 37 Component FAME Mix, 10 mg/mL in CH2 Cl2 , 47885-U, Merck, Rahway,
NJ, USA) [25].
2.4. Nomenclature
X (TDW; g/L): total yeast dry biomass; S (g/L): crude glycerol; EPs: total endo-
polysaccharides (g/L); Phen (g/L): total initial phenolic compounds; Cit (g/L): citric acid;
Man (g/L): mannitol; Ara (g/L): arabitol; Ery (g/L): erythritol; ΣPol (g/L): total polyols;
FAN (mg/L): free amino nitrogen; YX/S (g/g): yield of biomass on initial glycerol (g of
produced total dry weight per g of initial glycerol); YCit/S (g/g): yield of citric acid on
available carbon sources (g of produced citric acid per g of initial glycerol plus the amount
of consumed polyols); YΣPol/S (g/g): yield of total polyols on initial glycerol (g of total
polyols produced per g of initial glycerol); YL/X (%, g/g): yield of cellular lipids on the
biomass produced (g of lipids per g of produced total dry weight); Productivity Cit (g/L/h):
citric acid productivity. All yields were calculated when the maximum values of each target
product were obtained throughout fermentations.
2.5. Statistical Analysis
Statgraphics was used for statistical analysis. The data were compared using analysis
of variance (ANOVA) and Pearson’s linear correlation at a 5% significance level. Significant
differences between means were determined by the Honest Significant Difference (HSD-
Tukey test) method at a level of p < 0.05. Data were reported as mean values ± standard
deviation of three independent replicates (p < 0.05, 95%).
3. Results
The yeast strains Y. lipolytica LMBF Y-46 and ACA-YC 5033 were investigated on OMW-
glycerol blends of different initial phenolic concentrations (Phen) for cell proliferation and
production of metabolic compounds (Table 1). Kinetics studies were also performed in
fermentation media in which OMW was not involved while glycerol was used as the sole
carbon source (control experiment). The total dry weight (TDW, X), EPs and total polyols
(ΣPol) produced by both Y. lipolytica strains were decreased at statistically significant levels
in the presence of OMW, compared to the control experiments (p < 0.05). The maximum
TDW of 9.5 g/L was observed at Phen = 0 g/L with a YX/S = 0.124 g/g initial carbon
sources and a YEPs/X of 51.9% w/w in the case of Y. lipolytica LMBF Y-46, while respectiveFermentation 2023, 9, 251 5 of 17
values for Y. lipolytica ACA-YC 5033 were 10.7 g/L, YX/S = 0.139 g/g initial carbon sources
and YEPs/X =61.5% w/w.
Table 1. Fermentation efficiency of Y. lipolytica LMBF Y-46 and Y. lipolytica ACA-YC 5033 cultivated
on blends of olive mill wastewater (OMW) and crude glycerol under nitrogen-limited conditions in
shake-flask fermentations. The blends of OMW-glycerol were properly formulated to have different
initial phenolics concentrations (Phen) of ca. 1 g/L, 2 g/L and 3 g/L. Different superscript letters
within the same column (with respect to the same yeast strain) indicate statistically significant
differences (p < 0.05).
Phen Time YCit/S YΣPol/S Productivity
X (g/L) EPs (g/L) Cit (g/L) Man (g/L) Ara (g/L) Ery (g/L) ΣPol (g/L) YX/S (g/g)
(g/L) (h) (g/g) (g/g) Cit (g/L/h)
Y. lipolytica LMBF Y-46
191 c 9.0 ± 0.3 4.4 ± 0.6 29.3 ± 1.9 8.4 ± 0.3 2.0 ± 0.2 17.3 ± 0.9 27.8 ± 1.1 a 0.360
0
240 a, b 9.5 ± 0.4 a 4.9 ± 0.2 a 40.1 ± 1.9 a 9.4 ± 0.7 2.5 ± 0.2 1.2 ± 0.1 13.1 ± 0.6 0.124 0.437 0.167
121 a, c 8.2 ± 0.6 b 2.5 ± 0.2 b 35.1 ± 1.7 13.1 ± 0.8 1.2 ± 0.1 8.3 ± 0.4 22.6 ± 1.5 b 0.106 0.292
≈1
215 b 7.0 ± 0.4 1.5 ± 0.1 58.0 ± 2 b 4.1 ± 0.2 - - 4.1 ± 0.2 0.605 0.27
121 a, c 8.1 ± 0.5 b 2.4 ± 0.1 b 23.5 ± 1.5 13.1 ± 0.6 - 9.0 ± 0.4 22.0 ± 1.1 b 0.106 0.288
≈2
215 b 7.3 ± 0.6 2.1 ± 0.1 60.3 ± 3.1 c 2.3 ± 0.1 - - 2.0 ± 0.2 0.626 0.281
121 a, c 8.1 ± 0.4 b 1.6 ± 0.2 c 40.4 ± 1.9 2.6 ± 0.1 7.9 ± 0.4 5.1 ± 0.4 15.6 ± 0.6 c 0.103 0.199
≈3
191 b 7.9 ± 0.8 1.1 ± 0.1 64.1 ± 3.1 dFermentation 2023, 9, 251 6 of 17
ACA-YC 5033 showed slightly higher values of productivity (0.344 g/L/h) and YCit/S
(0.690 g/g carbon sources) compared to Y. lipolytica LMBF Y-46 (Table 1).
The production of microbial oil was quite moderate in all the examined cases
(0.54–1.44 g/L). YL/X values were negligible, varying within 5.8–9.6% w/w even though
nitrogen-limited conditions prevailed in the growth media (initial FAN concentration
≈85.1 ± 5.1 mg/L, after 48–72 h of fermentation FAN < 9.5 ± 0.75 mg/L) and glycerol was
consumed at satisfying levels, suggesting a non-typical oleaginous behavior for the yeasts.
When Y. lipolytica LMBF Y-46 was cultivated on OMW-glycerol blends containing 2 g/L,
YL/X reached up to 16.6% (after 121 h of fermentation), while a YL/X =14.6% (after 165 h
of fermentation) was determined on blends containing 3 g/L when Y. lipolytica ACA-YC
5033 was used. The fatty acid profile of microbial oil produced by both Y. lipolytica strains
was analyzed. In all the examined cases, the primary fatty acid was oleic acid (C18:1;
>50%), followed by palmitic acid (C16:0; >15%) and linoleic acid (C18:2; Table 2). As can
be observed in Table 2, there is not any particular fluctuation pattern of fatty acids when
different fermentation media (control and blends of OMW-glycerol) were applied.
Table 2. Intercellular lipid accumulation (YL/X, % w/w) and the fatty acid composition of lipids
(%, w/w, when the maximum lipids were produced in terms of both absolute and relative values)
produced by Y. lipolytica LMBF Y-46 and ACA-YC 5033 on blends of olive mill wastewater (OMW)
and crude glycerol under nitrogen-limited conditions in shake-flask fermentations. The blends of
OMW-glycerol were properly formulated to have different initial phenolics concentrations (Phen) of
ca. 1 g/L, 2 g/L and 3 g/L.
Fatty Acid Methyl Esters (% w/w)
YL/X ∆9 C16:1 ∆9 C18:1 ∆9,12 C18:2
Phen (g/L) Time (h) C16:0 C18:0
(% w/w)
≈0 215 8.8 18.9 4.7 11.9 50.5 14.0
Y. lipolytica ≈1 121 9.6 15.1 5.3 11.2 53.1 15.3
LMBF Y-46 ≈2 121 16.6 23.3 3.2 9.9 51.0 12.6
≈3 121 9.6 17.3 3.6 7.0 51.8 20.2
≈0 -spective Phen of 1 g/L, 2 g/L and 3 g/L. When ACA-YC 5033 was cultivated on the control
media, the Cit production rationally passed to a stationary phase after 169 h, while the
polyols consumption for its synthesis was not observed. In the OMW-glycerol blends, a
very similar pattern to the aforementioned was monitored (Figure 2). ΣPol were entirely
consumed when maximum values of Cit were obtained in blends of OMW-glycerol with
Fermentation 2023, 9, 251 2 g/L and 3 g/L of Phen, while when 1 g/L was applied, ΣPol depletion was determined 7 of 17
equal to 60.1%. The yeast strain LMBF Y-46 mostly catabolized erythritol in the absence
of OMW, while in blends of OMW-glycerol with 2 g/L and 3 g/L of Phen, mannitol, arabi-
tol and erythritol were the most consumable polyols. At Phen = 1 g/L, the ΣPol decrease
were
was found
mainly to be intothe
attributed therange of 0.06–0.08
consumption h−mannitol
of both 1 (data not
and shown).
erythritol,Therefore,
with similarsuggesting that
observations
potential microbial inhibitory phenomena due and
made for ACA-YC 5033 at Phen = 1 g/L 2 g/L
to the (Figuresof1(Ab–Db)
presence phenolicand
compounds did
2(Ab–Db)).
not occur.
A
14 80 32
a 28 b
12
10 60 24
Polyols (g/L)
X, EPs (g/L)
S, Cit (g/L)
20
8
40 16
6
12
4
20 8
2 4
0 0 0
0 48 96 144 192 240 288 0 48 96 144 192 240 288
Fermentation time (h) Fermentation time (h)
B 10 80 28
a b
24
8
60 20
Polyols (g/L)
X, EPs (g/L)
S, Cit (g/L)
6 16
40
4 12
8
20
2
4
Fermentation 2023, 9, x FOR PEER REVIEW 0 0 0 9 of 19
0 48 96 144 192 240 288 0 48 96 144 192 240 288
Fermentation time (h) Fermentation time (g/L)
C 10 80 28
a b
24
8
60 20
Polyols (g/L)
X, EPs (g/L)
S, Cit (g/L)
6 16
40
4 12
20 8
2
4
0 0 0
0 48 96 144 192 240 288 0 48 96 144 192 240 288
Fermentation time (h) Fermentation time (h)
D 10 100
a 16 b
8 80
Polyols (g/L)
12
X, EPs (g/L)
S, Cit (g/L)
6 60
8
4 40
2 20 4
0 0 0
0 48 96 144 192 240 288 0 48 96 144 192 240 288
Fermentation time (h) Fermentation time (h)
Figure1. 1.
Figure Kinetics
Kinetics ofbiomass
of (a) (a) biomass
(X, g/L,(X,
▲)g/L, N) and endopolysaccharides
and endopolysaccharides (EPs, g/L, ■)(EPs, ) and citric acid
g/L,acid
and citric
(Cit,g/L,
(Cit, g/L, ◆)u) production,
production, as as
as well wellglycerol (S, g/L, ○)
as glycerol g/L, #) consumption
(S, consumption and (b) total and (b)(g/L,
polyols total◼),
polyols (g/L, n),
mannitol
mannitol(g/L, △),4
(g/L, arabitol
), arabitol ◇), erythritol
(g/L,(g/L, (g/L, ○)
3), erythritol production
(g/L, during shake-flask
#) production fermenta- fermentations
during shake-flask
tions of Y. lipolytica LMBF Y-46 when 0 g/L (A), 1 g/L (B), 2 g/L (C), 3 g/L (D) of phenolic compounds
of Y.applied
were lipolytica LMBF Y-46
in shake-flask when 0 g/L (A),
nitrogen-limited media.1 g/L (B), 2 g/L
The culture (C), 3 were:
conditions g/L (D) of phenolic
250 mL Erlen- compounds
were applied
meyer in shake-flask
flasks filled up to 50 mL at nitrogen-limited
180 rpm, initial pH media. Theranging
= 6.0, pH culturebetween
conditions were:
4.8 and 250 mL Erlenmeyer
6.0, incu-
bation
flaskstemperature
filled up to T =5028mL°C, initial
at 180glycerol concentration
rpm, initial pH = of 77.9
6.0, pH± 0.52 g/L. between 4.8 and 6.0, incubation
ranging
temperature T = 28 ◦ C, initial glycerol concentration of 77.9 ± 0.52 g/L.
A 14 80 32
a 28 b
12
10 60 24
lyols (g/L)
, EPs (g/L)
, Cit (g/L)
20
8
40 16
6
12Figure 1. Kinetics of (a) biomass (X, g/L, ▲) and endopolysaccharides (EPs, g/L, ■) and citric acid
(Cit, g/L, ◆) production, as well as glycerol (S, g/L, ○) consumption and (b) total polyols (g/L, ◼),
mannitol (g/L, △), arabitol (g/L, ◇), erythritol (g/L, ○) production during shake-flask fermenta-
tions of Y. lipolytica LMBF Y-46 when 0 g/L (A), 1 g/L (B), 2 g/L (C), 3 g/L (D) of phenolic compounds
were applied in shake-flask nitrogen-limited media. The culture conditions were: 250 mL Erlen-
Fermentation 2023, 9, 251 8 of 17
meyer flasks filled up to 50 mL at 180 rpm, initial pH = 6.0, pH ranging between 4.8 and 6.0, incu-
bation temperature T = 28 °C, initial glycerol concentration of 77.9 ± 0.52 g/L.
A 14 80 32
a 28 b
12
10 60 24
Polyols (g/L)
X, EPs (g/L)
S, Cit (g/L)
20
8
40 16
6
12
4 8
20
2 4
Fermentation 2023, 9, x FOR PEER REVIEW 0 0 0 10 of 19
0 48 96 144 192 240 288 0 48 96 144 192 240 288
Fermentation time (h) Fermentation time (h)
B 10 80 28
a b
24
8
60 20
Polyols (g/L)
X, EPs (g/L)
S, Cit (g/L)
6 16
40
4 12
20 8
2
4
0 0 0
0 48 96 144 192 240 288 0 48 96 144 192 240 288
Fermentation time (h) Fermentation time (h)
C 10 80 28
a b
24
8
60 20
Polyols (g/L)
X, EPs (g/L)
S, Cit (g/L)
6 16
40
4 12
8
20
2
4
0 0 0
0 48 96 144 192 240 288 0 48 96 144 192 240 288
Fermentation time (h) Fermentation time (h)
D 10 100
a 16 b
8 80
Polyols (g/L)
12
X, EPs (g/L)
S, Cit (g/L)
6 60
8
4 40
2 20 4
0 0 0
0 48 96 144 192 240 288 0 48 96 144 192 240 288
Fermentation time (h) Fermentation time (h)
Kinetics
Figure2.2.Kinetics
Figure ofbiomass
of (a) (a) biomass
(X, g/L,(X,
▲)g/L, N) and endopolysaccharides
and endopolysaccharides (EPs, g/L, ■) (EPs, ) and citric
g/L,acid
and citric
acidg/L,
(Cit, (Cit,◆)g/L, u) production,
production, as well as as well (S,
glycerol as g/L,
glycerol (S, g/L, #)and
○) consumption consumption and(g/L,
(b) total polyols (b) ◼),
total polyols
mannitol
(g/L, n),(g/L, △), arabitol
mannitol (g/L,(g/L, ◇), erythritol
4), arabitol 3), ○)
(g/L,(g/L, production
erythritol during
(g/L, shake-flask fermenta-
#) production during shake-flask
tions of Y. lipolytica ACA-YC 5033 when 0 g/L (A), 1 g/L (B), 2 g/L (C), 3 g/L (D) g/L of phenolic
fermentations of Y. lipolytica ACA-YC 5033 when 0 g/L (A), 1 g/L (B), 2 g/L (C), 3 g/L (D) g/L of
compounds were applied in shake-flask nitrogen-limited media. The culture conditions were: 250
phenolic compounds
mL Erlenmeyer were
flasks filled upapplied
to 50 mLinatshake-flask nitrogen-limited
180 rpm, initial media.
pH = 6.0, pH ranging The culture
between 4.8 andconditions
were: 250 mL Erlenmeyer flasks filled up to 50 mL at 180 rpm, initial pH
6.0, incubation temperature T = 28 °C, initial glycerol concentration of 77.9 ± 0.52 g/L. = 6.0, pH ranging between
4.8 and 6.0, incubation temperature T = 28 ◦ C, initial glycerol concentration of 77.9 ± 0.52 g/L.
Table 3 depicts the evaluation of color removal that was attained by both yeast strains
on blends of OMW-crude glycerol. In all fermentations, maximum decolorization values
were achieved at a more prolonged fermentation time by LMBF Y-46, compared to ACA-
YC 5033. In the case of LMBF Y-46, its ability to decolorize OMW-glycerol blends was
significantly reduced (p < 0.05) with increasing Phen concentration while the maximum
value of 31.1% (after 121 h) was observed at Phen = 1 g/L. In the case of ACA-YC 5033, the
highest color removal of 25.9% was monitored at Phen = 3 g/L after 96 h of fermentation.Fermentation 2023, 9, 251 9 of 17
The secondary anabolic activity of Cit synthesis occurred when the yeast strains en-
tered the so-called idiophase, which is related to nitrogen exhaustion from the culture
media. More specifically, FAN was almost depleted from the media after 48 h of fermenta-
tion, being lower than 12.5 ± 0.91 mg/L (initial FAN concentration ≈85.1 ± 5.1 mg/L) in
blends of OMW-glycerol in Y46 (1–3 g/L Phen) and ACA-YC 5033 (1–2 g/L Phen). This
event directly triggered Cit production at 48 h of fermentation apart from the experiment
carried out on OMW-glycerol blends with Phen = 3 g/L using Y. lipolytica ACA-YC 5033,
that Cit production initiated at 72 h (in this case, FAN was almost depleted at 72 h, reach-
ing 17.5 ± 1.32 mg/L). Cit showed a considerable and prolonged production rate, up to
165–215 h (Figure 1(Ba–Da) and Figure 2(Ba–Da)). Surprisingly, the FAN uptake during
the control experiments was slower, as it was almost consumed (13.7 ± 1.93 mg/L) after
96 h. In each fermentation, after reaching its maximum value, Cit secretion plateaued, with
slight variations thereafter (Figures 1 and 2).
Glycerol was fastly catabolized by both strains during all fermentations. More specifi-
cally, when Y. lipolytica Y-46 was used, glycerol was consumed by (w/w) 96.1% after 191 h
in the control experiment, and by 94.4%, 77.7%, and 89.5% on OMW-glycerol blends with
Phen of 1 g/L, 2 g/L, and 3 g/L, respectively, after 121 h of fermentation (Figure 1(Aa–Da)).
Thereafter, since glycerol was available in negligible quantities in the fermentation media,
Cit biosynthesis was further prolonged since the yeast strains started to metabolize the
so-far-produced polyols, valorizing them as the carbon source (Figure 1(Ab–Db)). The
polyols consumption reached 27.7 g/L–13.1 g/L when the maxim Cit production was
observed in the control fermentation. Similarly, ΣPol was consumed by 81.8%, 90.9%
and 97.6% when maximum values of Cit were obtained in blends of OMW-glycerol with
respective Phen of 1 g/L, 2 g/L and 3 g/L. When ACA-YC 5033 was cultivated on the
control media, the Cit production rationally passed to a stationary phase after 169 h, while
the polyols consumption for its synthesis was not observed. In the OMW-glycerol blends, a
very similar pattern to the aforementioned was monitored (Figure 2). ΣPol were entirely
consumed when maximum values of Cit were obtained in blends of OMW-glycerol with
2 g/L and 3 g/L of Phen, while when 1 g/L was applied, ΣPol depletion was determined
equal to 60.1%. The yeast strain LMBF Y-46 mostly catabolized erythritol in the absence of
OMW, while in blends of OMW-glycerol with 2 g/L and 3 g/L of Phen, mannitol, arabitol
and erythritol were the most consumable polyols. At Phen = 1 g/L, the ΣPol decrease
was mainly attributed to the consumption of both mannitol and erythritol, with similar
observations made for ACA-YC 5033 at Phen = 1 g/L and 2 g/L (Figure 1(Ab–Db) and
Figure 2(Ab–Db)).
Table 3 depicts the evaluation of color removal that was attained by both yeast
strains on blends of OMW-crude glycerol. In all fermentations, maximum decolorization
values were achieved at a more prolonged fermentation time by LMBF Y-46, compared
to ACA-YC 5033. In the case of LMBF Y-46, its ability to decolorize OMW-glycerol
blends was significantly reduced (p < 0.05) with increasing Phen concentration while
the maximum value of 31.1% (after 121 h) was observed at Phen = 1 g/L. In the case of
ACA-YC 5033, the highest color removal of 25.9% was monitored at Phen = 3 g/L after
96 h of fermentation. This strain’s capacity to decolorize OMW-glycerol blends was not
affected by the different Phen.Fermentation 2023, 9, 251 10 of 17
Table 3. Decolorization (%) of the fermentation media achieved by Y. lipolytica LMBF Y-46 and
ACA-YC 5033 on blends of olive mill wastewater (OMW) and crude glycerol under nitrogen-limited
conditions in shake-flask fermentations. The blends of OMW-glycerol were properly formulated
to have different initial phenolics concentrations (Phen) of ca. 1 g/L, 2 g/L, and 3 g/L. Different
letters within the same column (with respect to the same yeast strain) indicate statistically significant
differences (p < 0.05).
Phen (g/L) Time (h) Decolorization (%)
≈1 121 31.1 ± 1.91 a
Y. lipolytica LMBF
≈2 145 28.1 ± 1.12 b
Y-46
≈3 145 19.5 ± 0.77 c
≈1 48 25.8 ± 1.53 a
Y. lipolytica ACA-YC
≈2 72 22.2 ± 0.98 a
5033
≈3 96 25.9 ± 1.03 a
4. Discussion
The current practice for olive oil production is the three-phase milling method that
generates solid and liquid wastes in significant quantities. The liquid effluent, which is
called OMW, is toxic for terrestrial and water bodies due to its high chemical and biochemi-
cal oxygen demand, heavy metals, strong acidity, dark color and phenolic components [26].
Indicatively, it has been reported that 1 m3 of OMW can cause similar environmental
damage as 200 m3 of domestic sewage. The efficient management of OMW in industrial
facilities is quite challenging due to large amounts being produced annually. Several lab-
scale methods, including biological, chemical, and physical approaches, have been reported
while physicochemical approaches have failed due to their high cost and generation of large
slush effluents [27]. This study reported a sustainable strategy to valorize OMW, including
microbial bioprocessing to produce biobased products of exceptional commercial interest,
such as Cit and polyols. Microbial biomass rich in EPs and unsaturated microbial oil could
also be considered a co-product of high nutritional value with targeted applications. Basic
principles of circular bioeconomy were implemented while the whole fermentation process
resulted in a substantial bioremediation of OMW via decolorization.
The formulation of the fermentation media included OMW blended with crude glyc-
erol derived from the biodiesel industry. The OMW was dually considered as the fermen-
tation media as well as the process water. This strategy is directly in line with the sixth
sustainable development goal that ‘requires availability and sustainable management of
water and sanitation for all. More specifically, it addresses Target 6.6, which sets bind-
ings for the protection and restoration of water-related ecosystems, including mountains,
forests, wetlands, rivers, aquifers, and lakes [1,2]. The economic viability of the whole
approach could be increased since crude glycerol was utilized as the sole carbon source.
Glycerol is mostly generated from the biodiesel production process, and it contains several
impurities such as methanol, soap, free fatty acids, salts, di- and tri-glycerol and water. A
potential stockpile of over 40 billion liters of crude glycerol could be available annually as
the starting material for value-added end-uses [7]. The valorization of crude glycerol in
fields of pharmaceuticals, food and cosmetics requires combined purification and refining
methods that are cost and energy intensive. The price of reformed glycerol is five- to 10-fold
higher than that of crude glycerol (3–20 cents/lb.), but still, many biodiesel manufacturers
treat crude glycerol as a waste rather than purifying it for commercial applications [28,29].
High-added value alternatives, integrated within concepts of environmental circularity
and bio-economy, could provide long-term and sustainable valorization models of crude
glycerol. Especially the development of a crude-glycerol-based biorefinery could lead to
the production of a spectrum of marketable products paving the way towards economically
viable industrial bioprocesses, simultaneously increasing revenues for the existing biodiesel
industry. The bioconversion of crude glycerol using robust microorganisms such as Y.
lipolytica, which are tolerant to impurities, is of utmost importance [28,29].Fermentation 2023, 9, 251 11 of 17
The impurities contained in crude glycerol, i.e., potassium and sodium salts and
methanol, could affect microbial proliferation and the biosynthesis of the biobased prod-
ucts. However, several studies have reported that these impurities have not shown any
significant impact on the metabolism of certain Y. lipolytica strains, with similar results
obtained when crude and commercial-grade glycerol were used [30,31]. In the study by
Papanikolaou et al. [32], the maximum biomass formation was slightly higher in the case
of pure glycerol compared to crude glycerol, while polyols production was not affected
by glycerol purity. Additionally, the consumption pattern of both crude and pure glycerol
was very similar. TDW formation was substantial in all fermentations (8.1–10.7 g/L), and it
compares well with literature cited publications (Table 3). In most cases, the onset of new X
production, after nitrogen-limited conditions prevailed in the fermentation media, did not
arise concurrently with the production of storage lipids. In fact, insignificant amounts of
lipids were produced inside the yeast cells without exceeding 20% w/w of cellular lipids in
X, despite the fact that fermentations were performed under nitrogen limitation. This rise
of biomass synthesis that was monitored at the late growth stages (Figure 1(Aa–Da) and
Figure 2(Aa–Da)) suggests EPs accumulation in TDW, in accordance with other scientific
reports [33,34]. The fermentation media that did not contain any phenolic compounds
(control) favored EPs production both in absolute (g/L) and relative (%, w/w) values.
Glycerol has been extensively reported as an efficient and very promising carbon
source for microbial oil production [35]. Additionally, Y. lipolytica is able to accumulate
cellular lipids of more than 30% w/w in dry X when cultivated on glycerol alone or blended
with OMW [36]. OMW has also been reported as a natural inducer of lipogenesis in
several yeast strains [37]. Despite the aforementioned, in this study, the quite moderate
microbial oil production by both Y. lipolytica strains could be attributed to the shift of
metabolism towards the production of EPs and enhanced secretion of Cit into the medium.
The Cit concentration increased with fermentation time, verifying the aforementioned
assumption that the yeast metabolism was shifted towards Cit formation in detriment to
lipids biosynthesis (Figures 1 and 2). When glycerol is used as the fermentation media,
Cit production is triggered by nitrogen-limited conditions. The latter conditions cause
a rapid decrease of intracellular AMP, followed by the deactivation of NAD+ - isocitrate
dehydrogenase. Eventually, Cit is secreted inside the cytosol when critical values are
reached. Yeast strains with non-typical oleaginous behavior (non-capable of accumulating
microbial oil higher than 20% on TDW), such as these used in this study, secrete Cit into
the fermentation environment contrary to oleaginous ones that convert it into cellular
lipids [30]. Despite the low lipid production, blends of OMW-crude glycerol seemed to
somehow enhance the intracellular lipid content of the yeast biomass compared to the
control fermentations, indicating that some compounds contained in OMW might have
created a positive effect on lipids accumulation. The opposite phenomenon was described
by Dourou et al. [38] when Y. lipolytica A6 was cultivated on OMW-based media (with
around 2 g/L Phen) supplemented with 50 g/L glycerol. In this case, YL/X reached 14.9%
while the X production was much lower (5.6 g/L) compared to the yeasts evaluated in
the current study (see Table 1). The fatty acid composition of cellular lipids produced by
both strains was typical of Y. lipolytica lipids, with C18:1 being the predominant fatty acid,
followed by C16:0, C18:0 and C18:2 [31,38]. The highest Phen of 3 g/L seemed to favor
the synthesis of C18:2. Increasing Phen led to a decreasing tendency of C18:0 in the case
of Y. lipolytica LMBF Y-46, while the opposite phenomenon was observed in the case of
Y. lipolytica ACA-YC 5033. The fatty acid composition of lipids produced by Y. lipolytica
is affected by the presence of OMW in the growth media since, in the study by Dourou
et al. [38], the C16:1 and C18:0 content of microbial oil produced on OMW-glycerol media
increased during fermentation while C18:1 remained almost stable. On the other hand,
glucose-based fermentation seemed to result in a decreasing tendency of C16:0 and C18:0
with time [38].
So far, the most commercially efficient Cit producer is Aspergillus niger, while Y. lipoly-
tica strains have been lately investigated as potential candidates [30,38]. The productionFermentation 2023, 9, 251 12 of 17
of Cit employing A. niger strains is continuously growing on an annual basis, while con-
centrations between 150–200 g/L have been reported when fermentation is optimized or
patents are developed [39,40]. Circularity approaches, valorizing green olive processing
wastewaters enriched with sugars from white grape pomace, have reported the production
of 85 g/L with a yield of 0.56 g/g when A. niger B60 was used [41]. In this study, Cit was
effectively produced with both Y. lipolytica strains and valorizing blends of OMW-glycerol,
especially when the highest Phen was applied (64.1–65.1 g/L). The positive effect of OMW
supplementation on Cit production has been previously demonstrated when Y. lipolytica
ACA-DC 5029 was grown in crude glycerol and OMW blends under submerged shake-flask
fermentation [37]. The presence of other organic acids, such as pyruvic, a-ketoglutaric,
or fumaric acid, was not detected in our investigation, while other studies [42] report the
aforementioned acid in traces,Fermentation 2023, 9, 251 13 of 17
Table 4. Cont.
Fermentation YCit/S Productivity
Strain Feedstock Time (h) X (g/L) Cit (g/L) Reference
Mode (g/g) (g/L/h)
Crude glycerol batch flask
ATCC 20 460 142 6.1 27.8 0.78 0.20 [34]
~45 g/L culture
Crude glycerol
SKY7 fed batch 96 12.5 18.74 2.20 0.19 [47]
10–13 g/L
Crude glycerol
batch flask Present
LMBF Y-46 ~77 g/L, OMW 191 7.9 ± 0.8 64.1 ± 3.1 0.682 0.335
culture study
(Phen 3 g/L)
Crude glycerol
ACA-YC batch flask Present
~77 g/L, OMW 189 9.6 ± 0.8 65.1 ± 3.6 0.690 0.344
5033 culture study
(Phen 3 g/L)
1 S in YCit/S is referred to both carbon sources (crude glycerol and glucose); 2 refers to YX/S.
Nitrogen-limited conditions that prevailed in the fermentative environment at the very
early stages of microbial growth, combined with pH values higher than 4.8, seemed to pri-
marily favor the Cit biosynthesis while mannitol, arabitol and erythritol were present as co-
products. The aforementioned conditions have also been verified by Papanikolaou et al. [48]
that lead to Cit production as the major metabolic compound, besides polyols, when Y. lipoly-
tica strains are employed. Additionally, the limitation of Y. lipolytica growth by biogenic
macro-elements, that is, N, P, or S, has already been proven as an efficient strategy to
regulate Cit production [42]. In other studies, the aforementioned conditions have directed
the carbon catabolism mainly towards the polyols biosynthesis, in detriment to organic
acids production, i.e., low concentrations of Cit 4.8 (maintained constantly) shifted the carbon flow towards the Cit formation
while individual polyols were constantly much lower than Cit concentrations. The fer-
mentation media and the pH values have been reported to affect the ΣPol concentration
as well as the ratio of Man:Ara:Ery [51]. Values of pH lower than 3 lead to enhanced
polyols production. More specifically, ΣPol of 21–38.3 g/L has been achieved in pure
glycerol-based growth media buffered at pH from 2.5 to 2.8 when nine strains of Y. lipolytica
were investigated under batch shake flasks experiments, with erythritol accounting for
more than 82–95%, besides mannitol and arabitol [51]. These results (ΣPol production) are
similar or slightly higher compared to data obtained in our study with pure-glycerol-based
cultures (ΣPol = 27.8–30.3 g/L) with erythritol representing around 53–62% (see Table 1,Fermentation 2023, 9, 251 14 of 17
control experiment). Thus, YΣPol/S was somehow higher in our study, ranging within
0.360–0.394 g/g compared to the study of Tomaszewska et al. [51] (YΣPol/S = 0.21–0.38 g/g).
Bioreactor batch cultures of Y. lipolytica Wratislavia K1 on crude glycerol have reached
up to ΣPol = 80.2 g/L with a ratio of Man:Ara:Ery of 0.14:0.01:1 [51]. YΣPol/S values of
0.30–0.40 g/g [53], 0.48–0.67 g/g [54,55], 0.67–0.69 g/g (repeated-batch bioreactor) [56],
and 0.80–0.82 g/g [57] have been attained under optimized conditions that favor their
synthesis (including bioreactor trials, pH values, C/N ratio etc.), glycerol as the fermenta-
tion feedstock (pure or biodiesel-derived) and/or combined with mutants or genetically
engineered strains of Y. lipolytica. The yeast strains that were evaluated in the current study,
are wild-type isolates, originated from traditional Greek wheat sourdough (ACA-YC5033)
and fish-origin products (LMBF Y-46), that require further optimization of the fermentative
parameters for either enhanced Cit production or targeted polyols accumulation. Based
on Table 1, it can be observed that when OMW was not involved in the formulation of
the fermentation media, erythritol was the major polyol, while the carbon polyols’ flow
shifted towards mannitol production (around 54–76%) when blends of OMW-crude glyc-
erol were implemented. The presence of phenolic compounds found in OMW might have
a major impact on the metabolic processes of Y. lipolytica strains promoting the production
of particular polyols i.e mannitol, as observed in our study. The carbon sources contained
in the growth media can also affect the overproduction of a particular polyol. For example,
growth media supplemented with glucose led to the production of a erythritol to mannitol
ratio of 3.25 while enhanced mannitol production combined with lack of erythritol were
reported when fructose and glycerol were applied using Candida magnoliae [58].
Natural yeasts do not possess the enzymatic complexes (i.e., laccases, lignin peroxi-
dases and manganese peroxidases) [59] to oxidize phenolic compounds that are contained
in OMW or other relative feedstock [60]. Nevertheless, this study reported the partial de-
colorization of OMW-crude glycerol blends by both yeast strains, which is very promising
since OMW remediation difficulties are interlinked with the breakdown of phenolic com-
pounds and, therefore, the removal of color from this dark and toxic effluent [60,61]. More
specifically, the color removal that was achieved by LMBF Y-46 reached up to 31.1%, while
slightly lower values (25.9%) were determined when ACA-YC 5033 was employed (Table 3).
Similar results have been reported in trials with Y. lipolytica ACA-DC 5029, which could
remove the color of OMW- based media up to ~30% w/w [37]. The potential of Yarrowia
strains to reduce the color of OMW is well documented. Still, it should be mentioned
that color intensity is dependent on several physicochemical parameters and, thus, is not
proportional to phenolics concentration [62–64]. Papanikolaou et al. [62] reported a remark-
able potential of several Y. lipolytica strains to decolorize OMW, indicating that the color
reduction of dark waste effluents may be a strain-dependent process. Although the biore-
mediation mechanism employing yeast strains is not clear, it could be assumed that yeast
cells adsorb phenolics by van der Waals interactions which are weak and reversible [65], or
they use phenolic compounds for energy and maintenance [66].
5. Conclusions
Blends of OMW-crude glycerol were efficiently valorized as feedstock to produce
primary Cit, polyols with elevated mannitol concentration, cellular lipids and EPs by the
non-conventional Y. lipolytica LMBF Y-46 and ACA-YC 5033. The utilization of OMW
to substitute (partially or totally) tap water in bioprocesses could facilitate large-scale
process transferability. This study also addressed the bioremediation of waste effluents via
satisfying media decolorization that was achieved by both yeast strains. This approach
suggests that Y. lipolytica strains could be considered as a very attractive biorefinery-
oriented cell factory that may yield a spectrum of potentially marketable products of high
added value leading simultaneously to the reduced environmental impact of the olive
milling industries.Fermentation 2023, 9, 251 15 of 17
Author Contributions: Formal analysis, D.S. and E.T.; investigation, M.A., E.K. and D.S.; data cu-
ration, D.S. and E.T.; writing—original draft preparation, E.T., M.K. and D.S.; writing—review and
editing, E.T., M.K. and D.S.; visualization, D.S. and S.P.; supervision, S.P. and D.S.; project administra-
tion D.S. and S.P.; All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the project “Infrastructure of Microbiome Applications in
Food Systems-FOODBIOMES” (MIS 5047291), which is implemented under the Action “Regional
Excellence in R&D Infrastructures,” funded by the Operational Programme “Competitiveness, En-
trepreneurship and Innovation” (NSRF 2014-2020) and co-financed by Greece and the European
Union (European Regional Development Fund).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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