Bioresource Technology 249 (2018) 612–619

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Green and sustainable succinic acid production from crude glycerol by T

engineered Yarrowia lipolytica via agricultural residue based in situ fibrous
bed bioreactor
⁎
Chong Lia, Shi Gaoa, Xiaofeng Yangb, Carol Sze Ki Lina,
a
School of Energy and Environment, City University of Hong Kong, Hong Kong
b
Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology,
Guangzhou 510006, People’s Republic of China

G RA P H I C A L AB S T R A C T

A R T I C L E I N F O A B S T R A C T

Keywords: In situ fibrous bed bioreactor (isFBB) for efficient succinic acid (SA) production by Yarrowia lipolytica was firstly
Agricultural residues developed in our former study. In this study, agricultural residues including wheat straw, corn stalk and su-
Crude glycerol garcane bagasse were investigated for the improvement of isFBB, and sugarcane bagasse was demonstrated to be
In situ fibrous bed bioreactor the best immobilization material. With crude glycerol as the sole carbon source, optimization for isFBB batch
Succinic acid
fermentation was carried out. Under the optimal conditions of 20 g sugarcane bagasse as immobilization ma-
Yarrowia lipolytica
terial, 120 g L−1 crude glycerol as carbon source and 4 L min−1 of aeration rate, the resultant SA concentration
was 53.6 g L−1 with an average productivity of 1.45 g L−1 h−1 and a SA yield of 0.45 g g−1. By feeding crude
glycerol, SA titer up to 209.7 g L−1 was obtained from fed batch fermentation, which was the highest value that
ever reported.

1. Introduction industries (Yan et al., 2014). Currently, the massive production of SA

mainly relies on the petrochemical process, but environmental issues
As an important platform chemical, succinic acid (SA) has attracted associate with the volatile crude oil price have shifted people’s atten-
much interest due to its wide applications as a precursor of many im- tion toward the development and commercialization of bio-based SA
portant chemicals in surfactant, ion chelator, food, and pharmaceutical (Mazière et al., 2017). In this context, the market is expected to explode

⁎
Corresponding author.
E-mail address: carollin@cityu.edu.hk (C.S.K. Lin).

http://dx.doi.org/10.1016/j.biortech.2017.10.011
Received 24 July 2017; Received in revised form 1 October 2017; Accepted 4 October 2017
Available online 12 October 2017
0960-8524/ © 2017 Elsevier Ltd. All rights reserved.
C. Li et al.
in the following years (Research and Markets, 2014). Nevertheless, the
high cost and long production period make the synthesis of bio-based
SA far from satisfactory. In order to enhance the industrial competi-
tiveness of bio-based SA, the most effective way is to develop a process
that is highly efficient, environmental friendly and with cost-effec-
tiveness.
Proper use of microorganisms is an effective way to increase fer-
mentative SA production efficiency. Traditionally, microbial produc-
tion of SA was achieved using bacteria (Lee et al., 2003; Liu et al., 2008;
Sanchez et al., 2005), but these microorganisms are sensitive towards
high acidity or product concentration. Some are even potentially pa-
thogenic (Wang et al., 2011). These limit their industrial applications.
Recently, increasing attention has been devoted in the use of Yarrowia
lipolytica as a robust SA producer for utilising a wide range of substrates
(Kamzolova et al., 2009; Kamzolova et al., 2014; Yang et al., 2017) with
high cell density, product yield and productivity (Gao et al., 2016; Li
et al., 2017), or even in low pH after gene modification (Yuzbashev
et al., 2010). All of the above features make this genetically regard as
safe strain (Liu et al., 2015) as a suitable SA producer.
Fibrous bed bioreactor (FBB), with improved productivity and high
operation stability, is another way to further increase the SA production
efficiency (Yan et al., 2014). Normally, cotton fiber is used as embed-
ding materials in FBB (Suwannakham and Yang, 2005) and impressive
results in SA production have been reported in our previous study (Li
et al., 2017). However, the rolled-up cotton fiber has low porosity,
small specific surface area and poor water retention capacity, which
would be unfavorable in terms of cell immobilization efficiency. In this
regard, agricultural residues might be suitable alternatives due to their
high fiber content, water retention capacity, environmental benefit and
low price (Yu et al., 2010) when considering the high cost for bio-SA
production.
Fermentation medium is another influential factor when con-
sidering the cost of fermentative SA production. Normally, glucose (Liu
et al., 2008; Okino et al., 2008) and glycerol (Lee et al., 2001) are used
as feedstock in SA production, but their high prices drive the devel-
opment of cheaper substrates. In this study, the utilization of crude
glycerol from biodiesel industry for SA production not only solves the
environmental problem from waste stream of biodiesel production, but
also lowers the production cost effectively (Behr et al., 2008).
As a continuation of our former study in fermentative SA production
via isFBB by Y. lipolytica, the present study aims to improve the process
using renewable feedstock in a green and sustainable approach. In
this study, several agricultural residues were firstly screened for their
suitability as immobilization materials in isFBB. Crucial parameters of
isFBB fermentation including initial crude glycerol concentration,
loading quantity and particle size of sugarcane bagasse, and aeration
rate were then optimized for higher SA production. Afterwards, fed
batch fermentation strategy was employed to improve SA production
further under optimized conditions. Finally, the potential applications
of the fermented biomass and agricultural residues as insect feed and
compost were investigated.
2. Materials and methods
2.1. Microorganism and fermentation medium
The SA producing strain Y. lipolytica PGC01003 used in this study
was modified from Y. lipolytica Po1f by deleting the YLsdh5 gene for
inactivation of succinate dehydrogenase. The storage and activation of
the strain, seed cultivation and YPG medium (pure glycerol
10–100 g L−1) preparation were detailed in our previous study (Li
et al., 2017). Fermentation medium used in this study is essentially YPG
except that the pure glycerol is replaced by appropriate concentrations
of crude glycerol from 60 to 160 g L−1. Nitrogen and carbon sources
were sterilized separately and then mixed under aseptic conditions.
While crude glycerol was filtrated (0.22 μm, Sartorius) for sterilization,
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Bioresource Technology 249 (2018) 612–619
nitrogen source was autoclaved at 121 °C for 15 min. Crude glycerol
concentration in this study refers to the actual glycerol concentration
that was quantified by high performance liquid chromatography
(HPLC).
All chemicals used in this study are analytical reagent grade and
purchased from Acros Organics (NJ, USA) and Sigma-Aldrich (MO,
USA). Crude glycerol was provided by ASB Biodiesel (Hong Kong) Ltd.,
which mainly contains glycerol, water (67.1 wt% and 17.2 wt%, re-
spectively), trace amount of methanol (0.13 wt%) and remaining in-
soluble impurities. Sugarcane bagasse and corn stalk were collected
from street market at Sham Shun Po in Hong Kong, while wheat straw
was collected from countryside of Shandong Province in China.
2.2. Construction of in-situ fibrous bed bioreactor
The isFBB was set up in a 2.5-L bench-top fermenter (Sartorius
Biostat B, Germany). After the pretreatment of washing, sterilization,
drying, crushing and sieving, selected residues were put into silk
stockings and stitched onto a piece of stainless steel wire mesh. The
mesh was then fixed to the baffle of fermenter, and the setup of isFBB
was completed (Li et al., 2017).
2.3. Effect of methanol on the growth and SA production of Y. lipolytica
Fermentation was carried out in 250 mL shaking flasks with 50 mL
YPG medium (25 g L−1 pure glycerol) supplemented with 0, 1, 5, 10,
20, and 30 g L−1 methanol respectively. The experiment was conducted
for 96 h with an initial pH 6.0 at 28 °C and 250 rpm agitation rate re-
spectively.
2.4. Free-cell batch fermentation in fermenter
About 50 mL seed culture was inoculated into 1 L fermentation
medium (60 g L−1 crude glycerol) to start the free cell fermentation in a
2.5-L benchtop fermenter. The fermentation was conducted at 28 °C,
600 rpm agitation speed and 3 L min−1 aeration rate. The pH was au-
tomatically controlled at 6.0 by adding 5 M NaOH, and anti-foam was
added when necessary. The inoculation procedure of fermentation as
described in our previous publication (Li et al., 2017).
2.5. isFBB batch fermentation
The isFBB batch fermentation is typically a two-stage process, which
consists of immobilization stage and fermentation stage (Li et al.,
2017). Wheat straw, corn stalk and sugarcane bagasse were the agri-
cultural residues used for cell immobilization in isFBB fermentation. For
each type of agricultural residues, a total weight of 15 g with particle
size of 1.5–3.5 mm was investigated as immobilization materials. The
effects of fermentation parameters including types of agricultural re-
sidue as immobilization materials, initial crude glycerol concentrations
(60–160 g L−1), particle size (1.0–8.0 mm) and loading quantity
(15–25 g) of the selected residue and aeration rates (1–4 L min−1) on
SA production were investigated.
2.6. Fed-batch fermentation for SA production with isFBB
Fed-batch fermentation was carried out in isFBB under the opti-
mized conditions with the initial crude glycerol concentration of
120 g L−1. When the residual glycerol in fermentation broth was below
15 g L−1, about 80–100 mL of 700 g L−1 crude glycerol was fed to
supplement the carbon source.
2.7. Potential applications of fermented yeast biomass and agricultural
residues
To study the feasibility of using fermented yeast biomass as insect
C. Li et al.
feed, about 100 three-day-old Hermetia illucens larvae were placed di-
rectly in 200 g of fermented yeast biomass for cultivation at room
temperature. After 10 days, the larvae were weighed to determine their
overall weight gain.
After fermentation, fermented agricultural residues were dried at
60 °C overnight and pulverized to powder. Then about 2.0–3.0 mg
powder was taken for carbon and nitrogen content measurement by
CHNS/O Analyser (PerkinElmer 2400, USA) to evaluate their possible
utilization as compost.
2.8. Scanning electronic microscope (SEM)
Fresh residues were oven-dried and gold sputtered prior to the SEM
analysis. The preparation procedure of fermented agricultural residues
with immobilized yeasts and free yeast cells recovered from fermenta-
tion broth for SEM were the same, except that pretreatment of free cells
was carried out by filtration using 0.1 μm polycarbonate membrane
(Whatman, Belgium). Procedure for SEM sample preparation was de-
scribed in our previous publication (Li et al., 2017).
2.9. Analytical techniques
The biomass from fermentation broth was calculated according to
the optical density (OD600), which has a linear correlation with the dry
cell weight (DCW). Biomass holdup of isFBB refers to the dry weight of
yeasts attached to the residues. The total biomass of isFBB fermentation
was calculated by adding the total quantity of DCW and biomass
holdup. Concentrations of fermentation substrate and products were
determined by HPLC (Waters, USA).
3. Results and discussion
3.1. Feasibility study
3.1.1. SEM study
Agricultural residues including wheat straw, corn stalk and su-
garcane bagasse were observed by SEM, before and after the isFBB
fermentation, to investigate the relationship between the micro-
structure of these residues and their cell immobilization capacities.
Some parts of the corn stalk were rough that was suitable for attach-
ment of yeast cells (Liao et al., 2011) while another parts were smooth
and unfavorable for cell immobilization (see Supplementary materials).
This is because the structure of corn stalk is heterogeneous, while the
inner parts are fluffy and porous, the outer parts are hard with high
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Bioresource Technology 249 (2018) 612–619
Fig. 1. Influence of methanol on cell growth and SA production of Y.
lipolytica.
density materials. Similar observation was recorded in wheat straw
(Han et al., 2001). In contrast, the bagasse was rough on surface and
homogeneous in microstructure. Accordingly, higher quantity of yeast
cells were immobilized to bagasse as compared to the other two ma-
terials. Moreover, the fluffy structure of bagasse made it in an ad-
vantage when comparing the immobilization efficiency based on
weight. The overall amounts of immobilized cells were 8.6 g, 12.1 g and
27.6 g for every 15 g of corn stalk, wheat straw and sugarcane bagasse,
respectively.
The morphology of the yeast cells from free cell and isFBB fer-
mentations were then examined by SEM, because it was reported that
morphological change in bacteria during FBB fermentation can increase
the specific surface area of cells and improve the substrate uptake and
products excretion (Chen et al., 2012; Suwannakham and Yang, 2005).
Higher percentage of filamentous yeasts were observed from isFBB
fermentation broth than that from free cell fermentation (see
Supplementary materials), which might result from the oxygen transfer
limitation inside the immobilized materials. Owing to the low oxygen-
transfer efficiency of the immobilized materials, the semi-anoxic micro-
environment was created in the immobilized materials, and the im-
mobilized cells would become filamentous. The filamentous cells have
low cell activity and would drop off from the immobilized materials
into the broth during the fermentation (Szabo, 1999; Herrera and
Sentandreu, 2002; Timoumi et al., 2017).
Results from this morphological study shows that no abnormal ap-
pearance except dimorphism was observed, which demonstrates high
stability of Y. lipolytica in FBB fermentation as compared to bacterial
fermentation (Krishnan et al., 2001). Moreover, according to Jost’s
study, the semi-anoxic condition in FBB fermentation favors the cell
growth and SA production in Y. lipolytica fermentation (Jost et al.,
2015).
3.1.2. Influence of methanol on cell growth and SA production during Y.
lipolytica fermentation
Methanol is toxic to a number of microorganisms (Celik et al.,
2008), and its presence at any concentration would have a negative
influence on bacterial growth (Salakkam and Webb, 2015). In this
study, the effect of methanol on cell growth and SA production during
Y. lipolytica fermentation was investigated.
As shown in Fig. 1, SA titer was 10.3 g L−1 when 1 g L−1 methanol
was supplemented, which was 1.3 times higher than the control group
(0 g L−1 methanol). When the initial methanol concentration was
5 g L−1, the resultant SA titer decreased to 8.3 g L−1, which was in the
same level as the control group. Then, SA titer decreased with the rise of
C. Li et al.
methanol concentration, and it was only 1.4 g L−1 when methanol was
30 g L−1. Similar trends were observed in DCW and SA yield. The DCW
and SA titer were the highest at 5.6 g L−1 and 0.38 g g−1, respectively
when the initial methanol concentration was 1 g L−1, but decreased to
0.9 g L−1 and 0.21 g g−1 when the initial methanol concentration was
30 g L−1. The glycerol consumption was also negatively influenced by
methanol. These results reveal that methanol inhibits the cell growth
and SA production during Y. lipolytica fermentation when the initial
methanol concentration in fermentation medium was over 5 g L−1.
3.2. Optimization of fermentation parameters for improved SA production
3.2.1. Effect of agricultural residues on SA production
To select the most suitable immobilization material for isFBB, batch
fermentation was carried out using agricultural residues and crude
glycerol (60 g L−1) was used as carbon source. Free cell fermentation
was conducted as the control group.
As shown in Fig. 2, the overall fermentation time in free cell fer-
mentation was longer than the isFBB fermentations. Compared to the
fermentation using isFBB, this fermentation resulted in a similar SA titer
of 20.6 g L−1. SA productivities of the isFBB fermentations were higher
than the free cell fermentation, which were 0.81, 0.84, and
0.90 g L−1 h−1 when loaded with corn stalk (CS), wheat straw (WS)
and sugarcane bagasse (SB), respectively. The use of sugarcane bagasse
as immobilization materials resulted in the highest biomass holdup of
27.6 g. These demonstrate that the higher biomass holdup using su-
garcane bagasse as cell immobilization material would lead to shorter
lag time and higher SA productivity.
In summary, the residues examined in this study were suitable for
improved SA production via isFBB fermentation, and bagasse was
identified to be the most suitable immobilization material. This agrees
with the SEM study that the surface structure of bagasse enables yeast
cell attachment.
3.2.2. Effect of initial crude glycerol concentration on SA production
In order to determine the optimal glycerol concentration for the cell
growth and SA production by Y. lipolytica, isFBB fermentation with
various concentrations of crude glycerol (60–160 g L−1) and bagasse
(15 g, 1.5–3.0 mm) was performed.
According to Fig. 3, glycerol was consumed completely for all fer-
mentations, but prolonged lag phase was observed with the rise of in-
itial crude glycerol concentration. The lag time was about 3 h when the
initial glycerol concentration was 120 g L−1, but increased to 6 and 9 h
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Bioresource Technology 249 (2018) 612–619
Fig. 2. SA production from free cell fermentation and isFBB
fermentation with different agricultural residues.
when the initial glycerol concentrations were 140 and 160 g L−1, re-
spectively. The fermentation time increased from 50 h to 62.5 hours
when the initial glycerol concentration increased from 120 g L−1 to
160 g L−1, respectively. Similarly, SA titer also increased with the rise
of initial crude glycerol concentration, which reached at 55.3 g L−1
when the glycerol concentration was 160 g L−1 (Fig. 3B). The highest
SA productivity and yield reached 0.97 g L−1 h−1 and 0.41 g g−1, re-
spectively when the initial glycerol concentration was 120 g L−1.
Whereas slightly lower SA yields were obtained (0.38 g g−1 and
0.35 g g−1) for those fermentation with an initial glycerol concentra-
tions of 140 g L−1 and 160 g L−1, respectively.
The highest DCW reached was 60.0 g L−1 when the initial crude
glycerol of 160 g L−1 was used in fermentation medium (Fig. 3D), while
the biomass holdup was 30.4 g. This contributed to a high total biomass
accumulation, which reveals the great potential of Y. lipolytica for high
cell-density fermentation. The final acetic acid (AA) concentration at
high glycerol concentrations were at low levels (Fig. 3C), which in-
dicates AA was not a major by-product in fermentation with high initial
glycerol concentration. This would also benefit the subsequent SA re-
covery step.
3.2.3. Effect of the particle size of sugarcane bagasse on SA production
As an important factor which can affect the specific surface area, the
influence of the particle size (1.0–8.0 mm) of bagasse (15 g) on SA
production from crude glycerol (120 g L−1) was investigated.
As shown in Table 1, the highest SA titer was 51.4 g L−1 when
1.0–1.5 mm bagasse was loaded. This relatively high SA titer combined
with a short fermentation time contributed to the highest SA pro-
ductivity at 1.13 g L−1 h−1. The same trend was observed for the total
biomass and yield. A total biomass of 83.8 g was obtained when
1.0–1.5 mm bagasse was used as immobilization material, which was
much higher than that from fermentations with other particle sizes
examined. The highest SA yield reached was 0.43 g g−1 at this particle
size. Therefore, the smaller particle size would favor higher im-
mobilization efficiency and improved SA production, especially in
terms of SA productivity.
3.2.4. Effect of the loading quantity of sugarcane bagasse in SA
fermentation
The influence of bagasse loading quantity on biomass accumulation
and SA production was investigated with bagasse size from 15 to 25 g
(1.0–1.5 mm) in this study. As shown in Table 2, SA titers were
51.6 g L−1, 52.6 g L−1 and 53.8 g L−1 for fermentations with bagasse
C. Li et al. Bioresource Technology 249 (2018) 612–619

Fig. 3. IsFBB fermentation for SA production from various concentrations of crude glycerol with sugarcane bagasse as immobilization material. (A-Glycerol, B-SA titer, C-Acetic acid, D-
DCW).

Table 1
isFBB fermentation for SA production with various sizes of sugarcane bagasse.

Particle size (mm) Time (h) SA titer (g L−1) SA productivity (g L−1 h−1) SA yield (g g−1) DCW (g L−1) Biomass holdup (g)

1.0–1.5 45.5 ± 1.0 51.4 ± 0.3 1.13 ± 0.02 0.43 ± 0.02 51.6 ± 1.0 32.2 ± 0.5
1.5–3.5 45.0 ± 1.0 47.8 ± 0.3 1.06 ± 0.01 0.40 ± 0.01 41.5 ± 1.0 30.4 ± 1.0
3.5–8.0 50.0 ± 1.0 48.2 ± 0.5 0.97 ± 0.01 0.41 ± 0.01 40.3 ± 1.5 30.2 ± 0.5

Table 2
isFBB fermentation for SA production with various quantities of sugarcane bagasse.

Loading quantity (g) Time (h) SA titer (g L−1) SA productivity (g L−1 h−1) SA yield (g g−1) Biomass holdup (g) DCW (g)

15 45.5 ± 0.5 51.6 ± 0.3 1.13 ± 0.02 0.43 ± 0.02 32.2 ± 0.5 51.6 ± 1.0
20 39.0 ± 0.5 52.6 ± 0.5 1.35 ± 0.02 0.44 ± 0.01 56.5 ± 1.0 55.4 ± 1.5
25 54.0 ± 1.0 53.8 ± 0.5 1.00 ± 0.01 0.45 ± 0.01 75.2 ± 1.5 57.4 ± 1.5

Table 3
isFBB fermentation for SA production with various aeration rates.

Aeration rate (L min−1) Time (h) SA titer (g L−1) SA productivity (g L−1 h−1) SA yield (g g−1) DCW (g L−1) Biomass holdup (g)

1 43.5 ± 1.0 53.2 ± 1.0 1.22 ± 0.01 0.44 ± 0.01 53.6 ± 2.0 55.3 ± 2.5
2 41.0 ± 1.0 51.8 ± 1.0 1.26 ± 0.01 0.43 ± 0.02 50.2 ± 1.5 51.9 ± 2.0
3 37.5 ± 0.5 51.6 ± 1.0 1.38 ± 0.02 0.43 ± 0.02 52.1 ± 1.5 53.8 ± 2.0
4 37.0 ± 0.5 53.6 ± 1.0 1.45 ± 0.02 0.45 ± 0.01 51.7 ± 2.0 53.2 ± 2.0

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Table 4
Summary of values from every optimization step and the corresponding improvements.

Experiments SA Titer (g L−1) SA Productivity (g L−1 h−1) SA Yield (g g−1) Total biomass (g)

Value Improvement (%)a Value Improvement (%) Value Improvement (%) Value Improvement (%)

Free cell 20.6 ± 1.0 N/A 0.78 ± 0.02 N/A 0.34 ± 0.01 N/A 24.9 ± 1.0 N/A
Optimization of Residues type 20.4 ± 0.5 0 0.90 ± 0.02 15.4 0.34 ± 0.01 0 51.4 ± 1.0 106.4
isFBB selection
Initial crude 48.2 ± 1.0 136.3 0.97 ± 0.01 7.8 0.41 ± 0.01 20.6 70.9 ± 2.0 37.9
glycerol
Bagasse size 51.4 ± 0.3 6.6 1.13 ± 0.02 16.5 0.43 ± 0.02 4.9 83.8 ± 5.5 18.2
Bagasse 52.6 ± 0.5 2.3 1.35 ± 0.02 19.5 0.44 ± 0.01 2.3 111.9 ± 8.0 33.5
quantity
Aeration rate 53.6 ± 1.0 1.9 1.45 ± 0.02 7.4 0.45 ± 0.01 2.3 104.9 ± 6.0 N/Ab

Improvement as compared to free 160.2 85.9 32.4 321.3

cell fermentation (%)

a
Improvement after every step.
b
Not applicable.

Fig. 4. SA production by fed batch fermentation with

crude glycerol as feedstock.

quantities of 15 g, 20 g and 25 g, respectively. Similar trends were
observed in the biomass holdup and total biomass production. With a
total biomass of 111.9 g, the shortest fermentation time of 39 h was
used in fermentation with 20 g bagasse, in which the highest SA pro-
ductivity reached was 1.35 g L−1 h−1. This value was 19.5% and 35%
higher than those fermentations containing 15 g and 25 g bagasse, re-
spectively, corresponding to total biomass of 83.8 g and 132.6 g, re-
spectively. Although with the highest biomass accumulation, the
longest fermentation time and lowest productivity were recorded in
fermentation with 25 g bagasse. This would be due to the limitation of
free space within bioreactor, as high quantity of bagasse potentially
limits the circulation of oxygen and biomass within the bioreactor.
3.2.5. Effect of the aeration rate on SA production
As an aerobic microorganism, oxygen can influence not only the
growth of Y. lipolytica, but also the metabolites production (Jost et al.,
2015). In this experiment, the influence of aeration rate from 1 to
4 L min−1 on SA production was investigated.
According to Table 3, with the increase of aeration rate from 1 to
4 L min−1 fermentation time declined from 43.5 h to 37.0 h gradually,
which reveals that sufficient oxygen can accelerate the consumption of
glycerol and facilitate the yeast growth. As a result, the highest pro-
ductivity was obtained at 1.45 g L−1 h−1 owing to the shortest fer-
mentation time at aeration rate of 4 L min−1, although SA titer was not
strongly influenced by aeration rate. This value doubled the result from
free cell fermentation with crude glycerol as feedstock (Gao et al.,
2016). SA yield and biomass holdup were not significantly affected by
aeration rate, but they were 7.2% and 222.4% higher than those with
cotton as immobilization material (Li et al., 2017).
To evaluate the performance of isFBB optimization, Table 4 sum-
marizes the values from all optimization step and the corresponding
improvements. With increasing initial crude glycerol concentration
from 60 to 120 g L−1, SA titer increased 2.4 times from 20.6 g L−1 to
48.2 g L−1. Similarly, improvement in the resultant SA titer was ob-
served after the optimization of bagasse size and quantity, as well as
aeration rate. Significant improvement in SA yield was observed by
increasing the initial crude glycerol concentration, of which the value
experienced a 20.6% increase from 0.34 to 0.41 g g−1. The SA yield
was 0.45 g g−1 after all optimization steps, which is higher than our
previous study (Li et al., 2017), and it is equivalent to 70.5% of the
theoretical yield (Yuzbashev et al., 2010).
The utilization of isFBB resulted in higher SA productivity and
biomass accumulation. SA productivity improved by 15.4% from 0.78
to 0.90 g L−1 h−1 when the fermentation mode was shifted from free
cell to isFBB, and finally reached 1.45 g L−1 h−1 after all optimization.
The improvement in biomass accumulation was much higher with the
use of isFBB, of which the value doubled from 24.9 to 51.4 g, and
reached 104.9 g in the end of fermentation. However, there were no
significant improvement in both SA titer and yield in isFBB fermenta-
tion as compared to free cell fermentation mode.

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C. Li et al.
In summary, the optimization in isFBB fermentation was conducted
in this study, which led to 2.6, 1.9, 1.3, and 4.2 times increase in SA
titer, productivity, yield and total biomass accumulation, respectively.
3.3. High SA production by fed-batch fermentation in isFBB
As one of the most effective fermentation modes to achieve a high
SA production, the study on fed batch fermentation became one of the
hot features in recent years (Gao et al., 2016; Okino et al., 2008). In this
study, fed batch fermentation strategy combined with isFBB was em-
ployed in fermentative SA production under the optimized conditions.
As shown in Fig. 4, SA titer increased gradually with the feeding of
glycerol. After seven times feeding of concentrated glycerol, SA titer of
209.7 g L−1 was obtained at 322 h, which was the highest value ever
reported. However, with a relatively long fermentation time, the
average SA productivity was 0.65 g L−1 h−1. Although this SA pro-
ductivity is higher than the reported value by Yuzbashev et al. (2010), it
is slightly lower than that using pure glycerol as feeding medium (Li
et al., 2017). This decrease may result from the inhibition of methanol
and other impurities that accumulated from the feeding of concentrated
crude glycerol, or of the higher resultant SA concentration. Never-
theless, the total biomass achieved in this study (110–120 g) was much
higher than the former study with cotton towel as immobilization
material (Li et al., 2017), which indicates that bagasse is a more sui-
table material in isFBB.
3.4. Potential application of fermented by-products
As alternative protein and lipid sources, the biomass of Y. lipolytica
has been used in feeding for animals and impressive results were
achieved in weight gain and DHA production (Hatlen et al., 2012;
Matty and Smith, 1978). In this study, the feasibility of using yeast
biomass as insect feed was conducted on Hermetia illucens larvae. Ac-
cording to the results, the overall weight gain of the larvae was 13.3-
fold increase from 0.003 g to 0.04 g with low mortality, which suggests
that the yeast biomass can be used in insect feed production. While the
larvae can be used as protein-rich insect meal and as protein supple-
ment for animal feed production (Hale, 1973), this study may benefit
the animal husbandry.
On the other hand, although agricultural wastes have been used in
composting, the high C/N ratio led to high resistance to microbial de-
composition and slows down the composting process (Abdulla, 2007).
The nitrogen and carbon contents of the fresh bagasse in the study were
1.1 mg g−1 and 441.3 mg g−1, respectively. This represented a high C/
N ratio at 401.2 and it was unfavorable for composting (Tuomela et al.,
2000). However, the resultant nitrogen content of the bagasse was
11.4 mg g−1, which was increased by 10.6 times and the resultant
carbon content was 367.1 mg g−1 in isFBB fermentation. This implies a
sharp decline in C/N ratio to 34.7 was achieved, which is within the
optimum range for composting (Tuomela et al., 2000). This indicates
that the fermented bagasse is suitable in composting application.
4. Conclusions
This study presents a novel fermentation strategy using a green and
sustainable approach, of which agricultural wastes and crude glycerol
from biodiesel industry were reused in efficient SA production via in-
situ fibrous bed bioreactor fermentation by Yarrowia lipolytica.
Moreover, SA titer of 209.7 g L−1 was achieved by fed batch fermen-
tation under optimized conditions, which is the highest SA production
reported worldwide. Finally, this study demonstrates the feasibility of
reusing the fermented by-products in insect feed application and com-
posting. Therefore, the work presented in this paper will assist the
transition towards an advanced circular economy.
618
Bioresource Technology 249 (2018) 612–619
Acknowledgements
The work described in this study was fully supported by a grant
from the City University of Hong Kong [Project No. CityU 7004694].
Dr. Xiaofeng Yang gratefully acknowledges the China Postdoctoral
Science Foundation [2017M612648] for financial support. Special
thanks to Eco-Nutrient Biotechnology Limited Company for conducting
the investigation of using yeast biomass as insect feed.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.biortech.2017.10.011.
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