International Biodeterioration & Biodegradation 143 (2019) 104720

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International Biodeterioration & Biodegradation

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Accelerated lipid production from distillery wastewater by Rhodosporidium T

toruloides using an open-bubble-column reactor under non-aseptic
conditions
Jiayin Linga,b, Yanbin Xua,c,∗, Chuansheng Lua, Peigen Hea, Jinliang Chena, Li Zhenga,
Manjunatha P. Talawara, Guangyan Xiea, Qingping Dua
a
School of Environmental Science and Engineering, Guangdong University of Technology, Guangzhou, 510006, China
b
School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou, 510006, China
c
Analysis and Test Center, Guangdong University of Technology, Guangzhou, 510006, China

ARTICLE INFO ABSTRACT

Keywords: It is an ideal and sustainable approach to recover renewable energy like biodiesel and to remove organic matters
Lipid from wastewater simultaneously. However, the common demands of aseptic conditions and long reaction time
Distillery wastewater limit the practical applications of this technology. In this study, lipid production by Rhodosporidium toruloides
Oleaginous yeast was investigated using distillery wastewater with an initial soluble chemical oxygen demand (SCOD) of
Non-aseptic
28,080–17,120 mg/L, an initial cell density of 0.5–2 × 108 cells/mL, and an aeration rate of 0.9–4.5 vvm (air
Open-bubble-column reactor
volume/liquid volume/minute) in an open-bubble-column reactor at room temperature. The lipid production
reached its peak of 1.92 ± 0.24 g/L in 12 h with a maximum lipid productivity of 160 mg/(L• h) achieved under
the conditions: initial SCOD 20,315 mg/L, initial cell density 2 × 108 cells/mL and aeration rate 4 vvm, without
external nutrients and sterilization. The associated removal efficiencies for SCOD, total phosphorus (TP) and
total nitrogen (TN) were 71.82 ± 0.52%, 79.53 ± 1.77% and 54.39 ± 6.45%, respectively. Both con-
taminating bacteria and fungi were negligible throughout the cultivation period, suggesting that the studied
process has good adaptability to full scale practical applications.

1. Introduction Most relative studies were conducted under sterile conditions.

Food industry wastewater such as distillery wastewater can cause
many environmental issues for its large generation amount with high
levels of organics and nutrients, but it is also useful as a substrate for
microbial lipid production (Li et al., 2016; Chowdhary et al., 2018;
Chuppa-Tostain et al., 2018). Distillery wastewater contains plenty of
reduced sugars, proteins, organic acids and polysaccharides that can be
served as carbon and nitrogen sources for the growth of oleaginous
microorganisms (Pant and Adholeya, 2007; Chowdhary et al., 2018;
Chuppa-Tostain et al., 2018). Oleaginous yeast is a good feedstock for
lipid production from various low cost carbon sources with rapid
growth rate and short production cycle compared to plants (Kot et al.,
2015). Utilization of wastewater as a substrate for oleaginous micro-
organisms could not only recover organics and nutrients from waste-
water, but also produce valuable compounds such as microbial lipids
which can be converted to biodiesel or food additives (Bellou et al.,
2016; Dourou et al., 2016; Miao et al., 2016).
However, food industry wastewater has a high organic load and is also
suitable for the growth of other microorganisms. The sterilization or
microfiltration of distillery wastewater to create sterile conditions for
this process is impractical, considering the large volume and the
stickiness of wastewater. Therefore, maintaining oleaginous yeasts as
the dominant species under non-aseptic conditions was one of the major
challenges faced by this technology (Vasconcelos et al., 2019). Yet,
there are relatively few studies devoted to the process performed in
non-aseptic conditions using real wastewater and reactors.
On the other hand, most current processes are aerobic and the re-
action time (4–6 days, Gonzalez-Garcia et al., 2013; Arous et al., 2016;
Wang et al., 2017; Islam et al., 2018) is long, even when compared to
the conventional anaerobic treatment process. For example, hydraulic
retention time (HRT) of most anaerobic treatment processes for dis-
tillery wastewater was reported to be 4 days (Pant and Adholeya,
2007). Meanwhile, operational expenses for aerobic processes are
usually higher than for anaerobic process, because of the energy

∗
Corresponding author. School of Environmental Science and Engineering, Guangdong University of Technology, Guangzhou, 510006, China.
E-mail addresses: hopeybxu@163.com, hopeybxu@gdut.edu.cn (Y. Xu).

https://doi.org/10.1016/j.ibiod.2019.104720
Received 4 April 2019; Received in revised form 5 May 2019; Accepted 18 June 2019
Available online 27 June 2019
0964-8305/ © 2019 Elsevier Ltd. All rights reserved.
J. Ling, et al.
consumption required to maintain aeration conditions while providing
the same HRT. The high expense for fermentation processes such as
aeration and agitation, coupled with relative low lipid productivity
have limited the economic competitiveness of this technology (Koutinas
et al., 2014).
This study intended to develop a microbial lipid production process
from real distillery wastewater under non-aseptic conditions using an
open-bubble-column reactor to accelerate the process, shorten the re-
action time and improve the lipid productivity. The influence of initial
SCOD (soluble chemical oxygen demand), initial cell density and
aeration rate on the removal efficiencies of organic matters (COD) and
nutrients (nitrogen and phosphorus), and the production of biomass
and lipid were studied and optimized. The amounts of contaminating
microorganisms at different cultivation stages were also assessed.
2. Materials and methods
2.1. Microorganism, medium, and wastewater
The oleaginous yeast strain R. toruloides AS 2.1389 was obtained
from the China General Microbiological Culture Collection Center, sub-
cultured and maintained on the distillery wastewater agar medium
slants at 4 °C. The distillery wastewater agar medium was made of rice
wine distillery wastewater (vinasses) and 20 g/L of agar with pH ad-
justed to 5.5 by NaOH. The YPD medium was used for seed culture
(Zhao et al., 2011). Both media were sterilized at 121 °C for 20 min
before use. The rice wine distillery wastewater was collected from the
S1 distillery in Foshan city, Guangdong, China. It contained SCOD
64,500–65,075 mg/L, TP 881–1443 mg/L, TN 2000–2460 mg/L and
ammonia nitrogen (NH3–N) 479–954 mg/L with pH of 3.3–3.4. Do-
mestic wastewater samples were collected from a local wastewater
treatment plant with SCOD 139 mg/L, TN 22 mg/L, TP 9.4 mg/L and pH
8.3. The wastewater samples were filtered through 3 layers of filter
paper (10–15 μm pore size cotton-fiber) and then stored at 4 °C before
use.
The oleaginous yeast R. toruloides strain grown on the distillery
wastewater medium slant was transferred to a 250-mL flask containing
45 mL of YPD medium, cultured at 30 °C and 200 rpm for 36 h. Then,
the culture was centrifuged at 4000 rpm for 10 min to obtain a cell
density of 4 × 109 cells/mL (Ling et al., 2013), and used as seed for the
wastewater experiment. Cell density was measured using cell counting
chamber.
2.2. Experimental setup
The eight identical open-bubble-column reactors used in this study
were in the shape of a cylinder, 42 cm in height with an internal dia-
meter of 6 cm, equipped with an aerator and an air distributor (sparger)
in the bottom. The total volume was 1 L with a working volume of
500 mL. Since characteristics of wastewater have significant influence
on the growth of microorganisms and lipid production (Sun et al., 2015;
Chi et al., 2018), initial SCOD (dilution factor) was investigated prior to
other factors. For the dilution effect experiment, five hundred milliliters
of real distillery wastewater (without sterilization and pH adjustment)
were mixed with distilled water at ratios of 1:1, 1:1.5, 1:2 and 1:3
(dilution factor 2, 2.5, 3 and 4, initial SCOD 28080-17120 mg/L) and
was filled in the bubble column reactors. The oleaginous yeast was
inoculated in the wastewater with initial cell density to 1 × 108 cells/
mL and then incubated at room temperature (23–30 °C) with an aera-
tion rate of 1.8 vvm (air volume/liquid volume/min) for 5 days. Silicon
antifoam was added at about 0.1% (v/v) to control foaming. Fifty
milliliters of the mixed liquid samples were withdrawn from the reactor
every 24 h for analysis. After each sampling, the aeration of each re-
actor was adjusted according to the volume change in all experiments.
Domestic wastewater, as the most common low concentration
wastewater source, was also used as diluent to compare with distilled
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International Biodeterioration & Biodegradation 143 (2019) 104720
water in this study. Forty milliliters of unpasteurized distillery and
domestic or distilled (1:3) mixed wastewater was added in a 250-mL
flask. The oleaginous yeast inoculated into it to an initial cell density of
2 × 107 cells/mL and then incubated at 30 °C, 200 rpm for 65 h.
In the experiment for effects of initial cell density, 500 mL diluted
distillery wastewater (dilution factor of 3) with initial SCOD
20,163 mg/L was added to the open-bubble-column reactor, and then
inoculated with yeast to an initial cell density of 0.5 × 108, 1 × 108,
1.5 × 108 and 2 × 108 cells/mL and cultured at room temperature
(23–30 °C) for 5 days with aeration rates of 0.9 vvm, 1.8 vvm, 2.7 vvm
and 3.6 vvm, respectively.
For the aeration effect experiment, five hundred milliliters of di-
luted distillery wastewater with a dilution factor of 3 and initial SCOD
20,315 mg/L was inoculated with yeast to an initial cell density of
2 × 108 cells/mL in the open-bubble-column reactor. Aeration was
provided at rates of 3, 3.5, 4 and 4.5 vvm. Samples were taken every
12 h and aeration was adjusted accordingly based on the volume
change after sampling to maintain constant aeration rate as designed.
All the experiments were conducted in replicates.
2.3. Analytical methods
After incubation, samples were centrifuged at 4000 rpm for
10 min at ambient temperature (Zhou et al., 2013). Since contaminating
microorganisms were mainly bacteria, which were much lighter than
yeast cells and presented as a thin white layer on the top of the red
biomass of R. toruloides after centrifugation at 4000 rpm for 10 min, the
top white layer of cell pellets was re-suspended carefully by mild
shaking until the white layer of biomass become invisible. The super-
natant with contaminating microorganisms was transferred to a new
pre-weighted centrifuge tube and centrifuged at 10,000 rpm for 10min.
The red cell pellets in the bottom were washed with distilled water
once. Both cell pellets were dried at 105 °C overnight until the weight
remained constant, and the dry cell weight was estimated as biomass
production of yeast and contaminating microorganisms, respectively.
The sum of the dry weight for yeast mass and biomass of contaminating
microorganisms was calculated as the total biomass production.
Supernatants of samples were taken for COD, TP, TN, and NH3–N
analysis using Hach's reagents with Hach reactor DRB200 and spec-
trophotometer DR3900, following the HACH methods (Hach Company,
Colorado) and the Standard Methods (APHA, 2005). The DO (dissolved
oxygen) and pH were measured using dissolved oxygen transmitter and
pH meter, respectively.
The total lipids were analyzed according to Folch et al. (1957) with
modifications described by Li (2001) and Zhou et al. (2013). The ratio
between productions of lipid and total biomass was calculated as the
lipid content of dry biomass. The lipid extracted was transmethylated
by H2SO4 followed Zhou et al. (2013). The fatty acid composition was
analyzed by GC-MS (Agilent Technologies 7890A-5975C system) ac-
cording to Shun and Yun (2011).
For potential contaminating fungi analysis, domestic wastewater
was used as diluent, and the biomass produced under optimal condi-
tions from the previous step and control reactors (without inoculation
of oleaginous yeast) was collected at the 60th hour via centrifuging
(10,000 rpm, 10 min, at 4 °C) for ITS (Internal Transcribed Space) DNA
analysis. The ITS gene analysis was performed consistent with Bai et al.
(2018). The IBM SPSS Statistics19 software utility was used for statis-
tical analysis.
3. Results and discussion
3.1. Optimization of the initial SCOD
3.1.1. Initial SCOD on COD removal efficiency and pH
Generally, the COD removal efficiency in distillery wastewater in-
creased along with decreases in the initial SCOD of the wastewater as
J. Ling, et al.
Fig. 1. Effects of initial SCOD (28,080–17,120 mg/L, dilution factor 2–4) on COD removal, pH, total biomass and lipid production in open-bubble-column reactor.
well as the quality of effluent (Fig. 1a). There was no significant dif-
ference in COD removal efficiency when initial SCOD of distillery
wastewater changed from 28,080 to 25,457 mg/L (dilution factor 2 to
2.5) with ANOVA analysis, p > 0.05. However, COD removal effi-
ciency increased significantly (p < 0.05) when initial SCOD decreased
from 25,457 to 22,736 mg/L (dilution factor 2.5 to 3), but did not in-
crease further when initial SCOD changed from 22,736 to 17,120 mg/L
(dilution factor 3 to 4) in the first two days of cultivation. Besides, the
biomass grew in groups with a dilution factor 2.5 and 2 (initial SCOD
28,080 and 25,457) showed different colors with other groups, which
will be further discussed in Part 3.1.2.
The speed of increase of pH in wastewater was consistent with that
of COD removal efficiency and biomass production (Fig. 1b and c). The
pH in wastewater samples with initial SCOD 22,736 and 17,120 mg/L
(dilution factor 3 and 4) increased faster than those with initial SCOD
28,080 and 25,457 mg/L (factor 2 and 2.5). This could be attributed to
the degradation of organic acids contained in wastewater by the growth
and lipid generation of R. toruloides (Saayman and Viljoen-Bloom, 2006;
Huang et al., 2016). The more rapidly yeasts grew, the more rapidly
organic acids degraded, leading to a rapid increase in pH level.
3.1.2. Initial SCOD on biomass and lipid production
In general, higher initial SCOD resulted in higher biomass produc-
tion (Fig. 1c). However, the biomass production for samples with an
initial SCOD of 22,736 mg/L (dilution factor 3) was similar to initial
SCOD 28,080 and 25,457 mg/L (dilution factor of 2 and 2.5) in the first
two days of cultivation. This indicates that the microorganisms grew
faster or entered the stationary phase earlier in wastewater with rela-
tively low initial SCOD (high dilution factor), though their maximum
biomass production was smaller than samples with higher initial SCOD.
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On the other hand, relatively high and stable lipid production and
lipid content was not obtained in the group with the highest initial
SCOD, but in samples with initial SCOD 22,736 mg/L (dilution factor of
3) on the 2nd day of cultivation with values of 0.80 ± 0.10 g/L and
93 ± 14 mg/g dry biomass, respectively (Fig. 1d). The associated lipid
productivity was 17 mg/(L•h). Furthermore, the standard deviations in
lipid production enlarged on the 4th day, which implied that lipid
production in the late stationary phase or early death phase of growth
was less stable than in the exponential phase and early stationary phase.
Biomass grown in wastewater with initial SCOD 25,457 and
28,080 mg/L (dilution factor 2 and 2.5) displayed a dark gray color,
while those in samples with initial SCOD 22,736 to 17,120 mg/L (di-
lution factor of 3 and 4) displayed a red color (the color of R. toruloides).
This suggests that toxic compounds such as phenolic compounds and
furfural, and organic acids such as propionic acid and acetic acid con-
tained in distillery wastewater (Ioannou et al., 2015; Chowdhary et al.,
2018) inhibited the growth of this red yeast strain, and proper dilution
could help to reduce the concentration of these compounds and relieve
their inhibitory effects (Zhao et al., 2012; Fortela et al., 2016). Taking
acetic acid as an example, it can inhibit the growth and lipid accumu-
lation of R. toruloides when its concentration is higher than 5 g/L (Zhao
et al., 2012). Adding the synergistic effects of acetic acid and furfural as
they co-existed in distillery wastewater could dramatically decrease
their minimum critical inhibitory concentrations for R. toruloide. While
they could be used as carbon sources for lipid production, if their
concentrations were lower than the minimum critical inhibitory level
(Zhao et al., 2012). Fortela et al. (2016) demonstrated that a total VFAs
(volatile fatty acids) load of 10,000–20,000 mg/L could lead to almost
complete inhibition of the growth of oleaginous microbial consortia.
This could be a possible explanation for the observed phenomenon that
J. Ling, et al.
the highest initial SCOD did not result in the highest lipid production in
this study. Combining distillery wastewater with other wastewater with
less toxic compounds, such as domestic or sugar industry wastewater,
could be a feasible approach to make distillery water more treatable for
lipid production purposes (Fito et al., 2018).
3.1.3. Dilution water type (comparison of domestic wastewater and distilled
water)
The removal efficiencies of COD, TP and TN with domestic waste-
water used as diluent (89.71 ± 0.87%, 95.23 ± 0.73% and
75.79 ± 1.50%) were higher than those with distilled water as diluent
(86.76 ± 1.64%, 90.18 ± 2.50% and 69.84 ± 3.83%). Refers to
productions of biomass and lipid, no significant difference was found
between the two tested groups (p > 0.05), which were 3.93 ± 0.46 g/
L and 474 ± 42 mg/L in domestic wastewater group, and
3.87 ± 0.71 g/L and 524 ± 30 mg/L in distilled water group, re-
spectively. This implies that R. toruloides grew slightly faster in do-
mestic wastewater diluted conditions as compared to distilled water
diluted. This could be attributed in part of organic acids in distillery
wastewater being neutralized by compounds contained in domestic
wastewater when mixed, which had a higher initial pH (3.9) than dis-
tilled water diluted distillery wastewater (3.8). Overall, domestic was-
tewater is suggested to be suitable for use as diluent.
3.2. Optimum inoculum dose of R. toruloides
3.2.1. Initial cell density on COD removal efficiency, DO and pH
The COD in wastewater dropped faster with a smaller standard
deviation in samples with higher initial cell density (Fig. 2a). The
highest COD removal efficiency (90.57 ± 0.25%) was obtained in
samples with initial cell density of 2 × 108 cells/mL, followed by
samples with initial cell density of 1.5 × 108 cells/mL
(90.48 ± 1.02%). Their associated effluent COD values reached their
minimum (1902 ± 51 mg/L and 1920 ± 205 mg/L) within 2 days,
while it took 4 days for those with initial cell density of 1 and
0.5 × 108 cells/mL. COD removal in groups with relative low initial cell
density (1 and 0.5 × 108 cells/mL) was less stable than in groups with
high initial cell density (2 and 1.5 × 108 cells/mL), and larger standard
deviations were observed especially in early stage cultivation.
The tendencies of pH and DO were similar to each other (Fig. 2b and
d). With the growth of microorganisms entering the early and late
stationary phase (Fig. 2c), both pH and DO increased to a certain level,
and high initial cell density contributed to high ascending speed of both
the pH and DO level, which followed the order of groups with initial
cell density of 2, 1.5, 1 and 0.5 × 108 cells/mL. The standard deviation
of DO level was large before the growth of microorganisms entered the
late stationary phase, especially for groups with relatively low initial
cell density (1 × 108 cells/mL on 3rd day and 0.5 × 108 cells/mL on
5th day). Although aeration rates (vvm) provided for samples increased
with initial cell densities as designed, the DO levels were similar among
different groups during lag phase, which were all recorded at low levels
with relatively large fluctuation. This implies that the higher initial cell
density, the more aeration it took to maintain a similar DO level.
Meanwhile, the DO levels in the bioreactor increased when the growth
of yeast entered the stationary phase. It suggests that the growth of R.
toruloides consumed much more oxygen in the lag and exponential
phases than the stationary phase and less aeration would be required to
maintain the DO of 20–50% suggested by previous studies (Chi et al.,
2011; Dourou et al., 2016) for the growth of yeast in stationary phase.
In the later cultivation stage, a fall in DO level was observed. The
growth of contaminating microorganisms increasing the oxygen con-
sumption in the death phase of the yeast culture could be one possible
reason for this.
3.2.2. Initial cell density on biomass and lipid production
High initial cell density resulted in high biomass production, with
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International Biodeterioration & Biodegradation 143 (2019) 104720
short incubation time required to reach the maximum (Fig. 2c). The
maximum biomass production increased with increases in initial cell
density, with the highest value (11.64 ± 0.48 g/L) obtained in samples
with an initial cell density of 2 × 108 cells/mL. The peak of biomass
production for samples with initial cell density of 2 and 1.5 × 108 cells/
mL were both observed on the 2nd day, while those for samples with
initial cell density of 1 and 0.5 × 108 cells/mL were in the 2nd to 3rd,
and 4th day, respectively. On the other hand, relatively low initial cell
density (0.5 and 1 × 108 cells/mL) showed larger standard deviation in
biomass production than groups with higher initial cell density (1.5 and
2 × 108 cells/mL), especially in the later cultivation stage (4th and 5th
day) when the growth of yeast entered the late stationary and death
phases. It suggests that high initial cell density improved the system
stability in the aspect of biomass production.
The highest, and relatively stable, lipid production and lipid content
were both obtained in samples with initial cell density of 2 × 108 cells/
mL after cultivation for one day with values of 1.19 ± 0.02 g/L and
119 ± 1 mg/g dry biomass, respectively (Fig. 2e). The tested initial
cell density ranges were high compared to that of traditional processes
(about 0.315–1.05 × 107 cells/mL) which were estimated based on cell
density of seed culture (Gonzalez-Garcia et al., 2013; Ling et al., 2013).
Further increasing the initial cell density and the aeration rate to meet
the associated oxygen demand for growth would both increase the cost
of the process and the requirement of the facility to produce large
amounts of seed cells under sterile condition. Therefore, 2 × 108 cells/
mL was chosen as the optimal initial cell density in this study without
further increasing the test range of initial cell density.
3.3. Selection of the optimum aeration rate
3.3.1. Aeration rate on R. toruloides biomass and lipid production
Biomass production for samples with an aeration rate of 3.5 or 4
vvm (initial DO about 50–70% air saturation) was over 1 g/L higher
than samples with aeration rates of 3 and 4.5 vvm (Fig. 3a). Meanwhile,
they both reached the stationary phase within 12 h, while samples with
aeration rates of 3 and 4.5 vvm were still increasing from 12 h to 24 h
and entered the stationary phase after 24 h of cultivation. This suggests
that both insufficient aeration and excess aeration could impair the
growth of yeast. Deesuth et al. (2016) and Yen and Chang (2015) also
demonstrated that the number of yeast cells with aeration were higher
than those without aeration in an ethanol fermentation and lipid pro-
duction process from rice straw hydrolysate. Meanwhile, aeration af-
fects the growth of microorganisms by both DO and shear force. Dif-
ferent types of air distributor could lead to differing shear forces even
with the same DO level. Excess aeration may contribute to excess
shedding of yeast cells, resulting in the premature drop for newly split
cells in this study.
The highest and relatively stable lipid production (1.92 ± 0.24 g/
L) and lipid content of dry biomass (164 ± 8 mg/g) were obtained
from samples with an aeration rate of 4 vvm at 12 h, which means 12 h
was enough for biomass and lipid production under this condition
(Fig. 3b). Also, there was no significant change in lipid production after
the growth of yeast entered the stationary phase (12–48 h). However,
there was a small rise in biomass and a notable drop in lipid production
at 60 h in all samples. This suggests that the growth of yeast entered late
stationary or death phase while the contaminating microorganisms
were between the lag and exponential growth phases. Oleaginous yeasts
consume their own intracellular lipids during their late stationary
growth, especially when the carbon source in the substrate becomes
insufficient, and this could be another important reason for this phe-
nomenon (Papanikolaou and Aggelis, 2010; Dourou et al., 2018). A
similar phenomenon was also observed when R. toruloides was cultured
in glycerol-based media under sterile conditions (Papanikolaou et al.,
2017). Accumulations of potential competitive metabolic products such
as citric acid, which could also contribute to the outflow of carbon
towards metabolic pathways competitive to lipogenesis and the
J. Ling, et al.
Fig. 2. Effects of initial cell density on changes in COD, pH and DO of wastewater and productions of biomass and lipid from distillery wastewater (initial SCOD
20,163 mg/L, dilution factor 3) in open-bubble-column reactor.
decrease of lipid production in the late stationary growth phase
(Dourou et al., 2018).
3.3.2. Aeration rate on COD and nutrient removal
Removal efficiencies of COD, TP and TN in all samples reached
about 72%, 87% and 54%, respectively, within 24 h, but those of
samples with aeration rates of 3.5 and 4 vvm increased to the same
level about 12 h earlier than samples with aeration rates of 3 and 4.5
vvm (Fig. 4a, b, and c). COD, TP and TN in the group with an aeration
rate of 4 vvm dropped fastest among all tested groups, with removal
efficiencies of 71.82 ± 0.52%, 79.53 ± 1.77% and 54.39 ± 6.45%,
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respectively. The removal of COD, TP and TN in diluted distillery
wastewater in all tested groups slowed down significantly from 12 h to
60 h, with increases in removal efficiency of about 9%, 5% and 13%,
respectively.
Ammonia nitrogen concentration in diluted distillery wastewater
descended from 214 mg/L to about 33 mg/L (groups with aeration rate
3, 3.5 and 4 vvm) −51 mg/L (group with aeration rate 4.5 vvm) in the
first 12–24 h and then ascended to about 187 mg/L until the end of the
experimental period (Fig. 4d). It suggests that R. toruloides used am-
monia nitrogen for growth, and also degraded other nitrogen sources in
the wastewater while converting to ammonia at the same time.
J. Ling, et al. International Biodeterioration & Biodegradation 143 (2019) 104720

Fig. 3. Influence of aeration rate (vvm, air volume/liquid volume/min) on total biomass production and contaminating microorganisms (a), and lipid production (b)
from distillery wastewater (initial SCOD 20,315 mg/L, dilution factor 3) in open-bubble-column reactor. Initial cell density was 2 × 108 cells/mL. (c) Relative
abundances of fungi in samples collected at 60th hour in ITS DNA analysis. (d) and (e) were optical microscope observations of the yeast R. toruloides samples taken
on 36th and 48th hour of cultivation in the open-bubble-column reactor (magnification 400), respectively.

However, the utilization speed of NH3–N was smaller than the gen-
eration speed, which led to the upward trend of NH3–N in the later
stage of cultivation (Ling et al., 2013).
3.3.3. Aeration rate on pH and DO
The tendency of pH to change in the wastewater corresponded to
the decrease in COD and the increase in ammonia nitrogen (Fig. 4e). It
rose faster in samples with aeration rates of 3.5 and 4 vvm, compared to
those with aeration rates of 3 and 4.5 vvm, and then slowed in all
samples after 24 h. This could be attributed to the degradation of or-
ganic acids contained in wastewater as mentioned before (Saayman and
Viljoen-Bloom, 2006; Huang et al., 2016), and the increase in NH3–N in
the later cultivation stage could also contribute to the rise in the pH of
wastewater (Ling et al., 2013).
DO of wastewater in samples with aeration rates of 3, 3.5, 4 and 4.5
vvm were 2–60%, 40–60%, 50–70% and 83–103% air saturation at
time zero, respectively (Fig. 4f). DO level was very unstable when the
aeration rate was lower than 3.5 vvm. DO of samples with an aeration
rate of 4.5 vvm was significantly higher than other groups. Rapid tur-
bulent flow in the reactor, stickiness of the distillery wastewater, and
frequent attachment of bubbles on the detective electrode all con-
tributed to such high and unstable DO readings. There were notable
drops in DO of samples with an aeration rate of 3 vvm at 12 h, sug-
gesting that yeast in the exponential growth phase consumed more
oxygen than in the lag phase. A similar phenomenon was also observed
by Saran et al. (2017) in glucose based medium under sterile condi-
tions. This phenomenon did not occur in samples with aeration rates of
3.5 and 4 vvm, and this could be attributed to the growth of yeast
passing the exponential growth phase and entering the stationary phase
within 12 h (Fig. 3a). DO of samples changed to 80–100% air saturation
and remained constant after the growth of R. toruloides went into the
stationary phase (24–48 h) in all samples (Figs. 4f and 3a). Then, they
dropped dramatically again after 48 h concurrent with a slight rise in
biomass and fall in lipid production, which implies that the growth of
contaminating microorganisms (Figs. 4f, 3a and 3b) in this stage also
consumed high levels of oxygen.
3.4. Monitoring of contaminating microorganisms during lipid production
The dry weight of biomass for contaminating microorganisms (the
white part) was very small compared to the red biomass of R. toruloides
and the total biomass during the whole process (Figs. 2c and 3a), with a
maximum of only 1.2% of the total biomass under the optimal condi-
tions (Fig. 3a). The amounts of contaminating microorganisms (mainly
bacteria) were lower than the detection limit until 48 h without any
white biomass visible after centrifugation, or notable bacterial con-
tamination under microscope (Fig. 3a, d, and e). In control samples
(samples without inoculation of R. toruloides), the dry weight of in-
digenous biomass (white biomass, mainly yeast) was lower than 0.05 g/
L (detection limit) until the 36th hour. Meanwhile, no notable COD
change in wastewater was observed until 36th hour (data not shown).
In ITS DNA analysis for fungi, the relative abundance of Rhodotorula
was as high as 99.98% in samples with R. toruloides inoculation, while it
was only 0.005% in control samples (Fig. 3c). Other fungal genera

6
J. Ling, et al. International Biodeterioration & Biodegradation 143 (2019) 104720

Fig. 4. Effects of aeration rate (vvm, air volume/liquid volume/min) on changes in COD, total phosphorus (TP), total nitrogen (TN), ammonia nitrogen (NH3–N), pH
and DO of wastewater in open-bubble-column reactor. Initial SCOD was 20,315 mg/L (dilution factor 3) and initial cell density was 2 × 108 cells/mL.

including Candida, Trichosporon, Mucor, and Kazachstania provided only
0.02% of the relative abundance. It is in agreement with the observa-
tion that the mass of red oleaginous yeast R. toruloides constituted the
major proportion of total biomass (Fig. 3a) under optimal conditions,
even at the end of cultivation (60th hour).
Contaminating biomass increased slowly at the end of experiment
period (Figs. 2c and 3a). Coincidentally, pH of distillery wastewater
also increased from about 3.5 to 7–8.4 during the first 48 h of cultiva-
tion. This implies that low pH could be one of the reasons for low de-
tection of bacterial contaminating microorganisms in the early stage of
cultivation, and a high inoculum dose allows R. toruloides to grow much
faster than indigenous microbes (mainly yeast) and dominate the
wastewater. These results are in agreement with previous studies (Yen
et al., 2015; Dourou et al., 2016; Taskin et al., 2016), where both low
pH and high inoculum size were reported to be beneficial to the pre-
vention of undesired contaminations in non-sterile whey medium, sugar
beet molasses, crude glycerol, olive mill wastewater enriched with
glycerol, as well as low temperature (Taskin et al., 2015). Furthermore,
relatively high carbon source concentration could contribute to the
dominance of yeasts, inhibiting the growth of other microorganisms
(Taskin et al., 2016). However, the growth and lipid accumulation of
oleaginous yeast could also be inhibited when carbon source con-
centration was too high (Back et al., 2016), especially when the carbon
source in wastewater contained toxic compounds like phenolic com-
pounds and furfural as discussed in Part 3.1.2.
In spite of the potential increase in the material cost and the possible
negative effects on the treatability of the effluent generated in the
process, the use of antibacterial agent was an effective approach for

7
J. Ling, et al. International Biodeterioration & Biodegradation 143 (2019) 104720

preventing bacterial contamination in microbial lipid production pro-

Fei et al. (2016)

Lu et al. (2009)
cesses under non-aseptic conditions. Thyme essential oil could con-

Papanikolaou
et al. (2017)

Soccol et al.
Saran et al.
Zhao et al.,

Zhou et al.
Yang et al.
tribute to high lipid production by Zygomycetes under non-aseptic

This work
Reference

conditions in medium with glycerol as the carbon source (Moustogianni

(2014)

(2017)

(2017)

(2013)
2011
et al., 2014). Sodium chloride was reported as not only a good anti-
bacterial agent when R. toruloides was cultured in non-sterile glucose
fatty acid (%)

based medium, but also stimulated lipid accumulation (Tchakouteu

Unsaturated

et al., 2017). As Sarris et al. (2017) and Dourou et al. (2016) suggested,
56.74

65.23

73.52
the use of olive mill wastewater containing high concentration of
59.7

45.6

54.9

71.7

75.8

74.9
phenol as both antibacterial agent and supplemental nutrition in glu-
cose based medium or as substrate could also be a better choice than
acid (C26:0)
Hexacosanic

other commercial solutions for inhibiting bacterial contamination

during a non-aseptic process.

3.6

0.4
–

–
3.5. Fatty acid profile of R. toruloides and comparison with the literature
acid (C24:0)
Lignoceric

Table 1 shows that the fatty acids produced from rice wine distillery
1.14

wastewater by R. toruloides AS 2.1389 were mainly oleic acid (C18:1)

0.7
–

–
(39.74%), linoleic acid (C18:2) (33.09%), palmitic acid (C16:0)
(19.31%) and stearic acid (C18:0) (5.07%), which together provided
acid (C23:0)
Tricosanoic

about 97% of all the fatty acid detected. These results were similar to
Jatropha oil (Table 1 Line No. 9, Lu et al., 2009), suggesting that the
0.05

microbial lipids produced in this process are suitable for biodiesel

–

production due to a high proportion of long chain fatty acids, such as

Behenic

(C22:0)

C16 and C18 series compounds, and a high unsaturated ratio (73.52%)
0.34
acid

0.3

1.5

(Ashraful et al., 2014; Patel et al., 2017).

–

As shown in Table 1, oleic acid was the largest proportion of the

Linolenic

lipids produced by Rhodosporidium toruloides, with a higher percentage

(C18:3)

of unsaturated fatty acids than saturated, generally. Palmitic acid, oleic

0.64

0.64
acid

4.1

0.5

0.3

acid, stearic acid, linoleic acid and linolenic acid were the main com-
–

ponents of the lipids produced by R. toruloides regardless of the different

Linoleic

(C18:2)

strains and substrates used. However, the lipid profile of R. toruloides

33.09
13.5

18.1

13.6

38.4
acid

2.8

9.8

2.9

7.6

varied from strain to strain even when using the same carbon source
(Line No. 1 and 3, No. 4 and 5 in Table 1). It seems that the two major
(C18:1)

fatty acids in the lipid were the same when the same R. toruloides strain
45.02

39.74
Oleic

53.3

45.8

28.6

49.6

62.1

49.9

35.3
acid

was used, even from different substrates, though their compositions and
the proportion of unsaturated fatty acids could be different (Line No. 1
(C18:0)
Stearic

and 2, No. 3 and 4, in Table 1).

17.7

11.2

20.1

16.9

5.07
acid

6.6

4.6

5.6

Our lipid and biomass productions (1.92 ± 0.24 g/L and

11

11.73 ± 2.02 g/L) and COD removal efficiency (71.82 ± 0.52%)

Margaric

were in agreement with previous studies conducted under sterile con-

(C17:0)

ditions, in which lipid yields of 1.62–2.16 g/L, biomass of

0.56
acid

7.61–16.20 g/L and COD removal of 55.05–75.01% were obtained by

–

oleaginous yeast in ethanol fermentation wastewater and diluted oil

acid (C16:1)
Palmitoleic

mill effluent enriched with glycerol (Wang et al., 2017; Islam et al.,
Composition of lipids from Rhodosporidium toruloides and Jatropha oil.

2018). However, to construct and maintain sterile conditions would

1.47

11.2

0.69
2.1

0.7

1.2

increase both the capital and operational cost of the process.

3
–

There were also studies (Gonzalez-Garcia et al., 2013; Ling et al.,

Palmitic

2013; Zhou et al., 2013) on the lipid production via Rhodotorula sp. or
(C16:0)

22.91

19.31
26.8

33.2

35.1

21.5
acid

Rhodosporidium sp. in distillery or bioethanol wastewater with similar

0.9
24

19

or higher lipid production (1.33–3.54 g/L) compared to our results.

However, the lipid productivity (160 mg/(L·h)) of this study was about
Myristic

(C14:0)

3–15 times of those in previous studies (11–49 mg/(L·h)) conducted in a

0.68
acid

1.4

1.5

2.6

1.0

1.4

0.1

shake flask (Gonzalez-Garcia et al., 2013; Ling et al., 2013; Zhou et al.,
–

2013). To achieve similar or higher lipid production than the present

sugarcane juice
synthetic crude
waste glycerol

lignocellulosic

glucose based

glucose based

study, the cultivation time required by shake flask (72–144 h, Gonzalez-

based media

hydrolysate

wastewater

wastewater
bioethanol

Garcia et al., 2013; Ling et al., 2013; Zhou et al., 2013) was about 6–12
Substrate

distillery
medium

medium
glycerol

times that of the bioreactor (12 h) in this study. Long reaction time and
low lipid productivity would increase operational expense and energy
–

consumption.
R. toruloides A29
R. toruloides Y4

R. toruloides AS
R. toruloides Y4

R. toruloides Y2
Microorganism

Due to the short reaction time and no requirement for aseptic

R. toruloides

Jatropha oil
R. toruloides

R. toruloides
DEBB 5533
DSM 4444

DSM 4444

conditions or external nutrient additions, the operational cost and en-

2.1389

ergy consumption for aeration, agitation and sterilization could be re-

duced by the present process. Although both preparation of high cell
Table 1

density seed culture and providing a high aeration rate in the ex-
No.

ponential growth phase may increase the cost to some extent, the

8
J. Ling, et al.
further development of this process to continuous mode, repeat batch
mode (Xu et al., 2015) or fed-batch mode (Saran et al., 2017) could be a
feasible approach to reduce the cost for high cell density seed culture
preparation and promote lipid production in future work.
4. Conclusions
In this study, a process for producing microbial lipids from distillery
wastewater by Rhodosporidium toruloides was developed in an open-
bubble-column reactor under non-aseptic conditions. The optimal
conditions were a dilution factor of 3 for distillery wastewater (initial
SCOD 20,163–22,736 mg/L), initial cell density 2 × 108 cells/mL, and
aeration rate 4 vvm. Under these conditions, the reaction time could be
reduced to 12 h with lipid productivity increased from 17 to 160 mg/(L•
h). This process could reduce the operation cost of the resource re-
covery process from wastewater via reduction of the reaction time and
adaptability to non-aseptic conditions.
Funding
This research was supported by China Postdoctoral Science
Foundation (No. 2016M590761), National Natural Science Foundation
of China (No. 51708131) and Students' Innovation and
Entrepreneurship Training Program of Guangdong Province (No.
201811845158).
Declarations of interest
None.
Conflicts of interest
The authors (Jiayin Ling, Yanbin Xu*, Chuansheng Lu, Peigen He,
Jinliang Chen, Li Zheng, Manjunatha P Talawar, Guangyan Xie,
Qingping Du) of the original research manuscript entitled ‘Accelerated
lipid production from distillery wastewater by Rhodosporidium
toruloides using an open-bubble-column reactor under non-aseptic
conditions’ declare that there are no conflicts of interest.
The authors would like to thank the staffs from distillery and local
wastewater treatment plant, and Mr Jiande Ling for their assistance in
wastewater sampling.
This work was financially supported by China Postdoctoral Science
Foundation (No. 2016M590761), National Natural Science Foundation
of China (No. 51708131) and Students' Innovation and
Entrepreneurship Training Program of Guangdong Province (No.
201811845158).
The authors of this original research manuscript are approved for
the submission of this article to the ‘International Biodeterioration and
Biodegradation’.
This paper has not been published before and it is not submitted to
other journals except ‘International Biodeterioration and
Biodegradation’.
Acknowledgements
The authors would like to thank the staffs from distillery and local
wastewater treatment plant, and Mr Jiande Ling for their assistances in
wastewater sampling.
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