3. Results
3.1.
Effects
of O/S ratios on SLs morphology and sedimentation
Under different O/S ratio
conditions, SLs would exhibit different morphologies. When the O/S value
was higher than 0.04 g/g, the SLs presented noncrystalline
characteristics, definition of oily SLs. The microscopic results showed
that biomass and broth were enclosed by the mixture of rapeseed oil and
SLs due to their strong hydrophobicity. Under the action of gravity, SLs
began to gradually precipitate, and the fermentation broth showed
obvious stratification (Fig. 2). With the decrease of O/S ratio, the
fermentation broth began to be in an emulsified state, and SLs no longer
stratified with fermentation broth, thus presenting a relatively uniform
state (Fig. 2). With the further decrease of O/S ratio (< 0.02
g/g), crystalline SLs gradually appeared in the broth (Fig. 2). By
comparing the structure compositions of SLs in different forms, it could
be found that under high O/S ratio condition (> 0.04 g/g),
the proportion of deposited SLs,
which was mainly a mixture of lactone- and acidic-forms SLs, accounted
for 92% of the total SLs, while the supernatant only contained a small
portion of lactone- and acidic- form SLs. In contrast, when the O/S
ratio was low
(<
0.02 g/g), the crystalline SLs composed of lactone-form SLs accounted
for 38% of the total SLs, whereas the broth consisted of lactone- and
acidic-form SLs (Table S1). These results were in agreed with the
conclusion that acidic-form SLs has stronger hydrophilicity and
lactone-form SLs has stronger hydrophobicity. Notably, the crystalline
SLs could not settle on its own, but as the O/S ratio increased, it
could be found through the microscope that the crystalline SLs would be
surrounded by oily particles to form oily SLs, and then began to
precipitate (Fig. S1). Therefore, it was further inferred that the
settleability of SLs was dependent on their morphologies, which could be
contributed to the hydrophobicity of lactone-form SLs as well as the
rapeseed oil and SLs contents.
3.2.
The characteristic
of SLs sedimentation
When
the O/S ratio was greater than 0.04 g/g, the sedimentation height was
directly proportional to the concentration of SLs (Fig. 3A), and then
the average sedimentation rate could be obtained by formula (1)-(3).
According to formula (3), although the average sedimentation rate was
related to SLs concentration in the broth, the rapeseed oil content
would significantly affect the settling time, thus exhibiting that O/S
ratio was correlated to the average sedimentation rate (Fig. 3B). With
the increase of O/S ratio, the average of sedimentation rate of SLs was
significantly enhanced, which reached the maximum value of 0.075 cm/s at
0.25 g/g. However, it started to
decrease with the
further
increase of O/S ratio. As the main reason was the increase of rapeseed
oil concentration, the density of the mixture of SLs and rapeseed oil
was lower. Therefore, the average of sedimentation rate of SLs was
decreased. Further increase of O/S ratio can achieve temporary
suspension, but SLs would be re-sedimented by standing still.
It was found that the O/S ratio was the key factor affecting the
morphology and subsequent settling of SLs. In general, the sedimentation
rate of mixture (SLs and rapeseed oil) particles was mainly determined
by the radius, density, and viscosity. Analysis of SLs particle size
showed that when SLs was oily, with the increased of O/S ratio, the
particle size of the mixture gradually increased, and the maximum radius
was about 3.0 mm (Fig. 3C). Therefore, the particle size of mixture may
be an important factor affecting on the SLs settling. In the early stage
of fermentation, the viscosity of supernatant had marginal changes. When
the SLs concentration was higher than 250 g/L, the supernatant viscosity
was sharply increased (Fig. 3D). The high viscosity would affect the
mixing and mass transfer, and reduce the production efficiency of SLs in
the late fermentation. Therefore, when SLs concentration was around
200-220 g/L, the in-situ separation would be carried out in this
study, thus viscosity had limited influence on SLs precipitation. By the
density analysis, it was shown that after mixing SLs and rapeseed oil,
the density decreased with the increase of O/S ratio (Fig. 3E). However,
1.0 g SLs was found to be bound to a maximum of 0.3 g rapeseed oil (Fig.
S2). Therefore, the minimum density of the mixture of SLs and rapeseed
oil was 1.10 g/cm3. In contrast, the density of the
supernatant was mainly determined by glucose concentration, which was
less than 100 g/L in real fermentation process, so the density of the
supernatant was much lower than the mixture of SLs and rapeseed oil
(Fig. 3F), and density difference was the main reason determining the
SLs settling or floating. When the O/S ratio was low (< 0.04
g/g), crystallized or emulsified SLs would not settle by itself. When
the O/S ratio was high (> 0.04 g/g), an oily mixture was
formed and began to settle. With the further increase of O/S ratio, the
hydrophobicity of the mixture was strengthened and the particle size
increased, thus the settling rate was accelerated under gravity action.
In the settling process, the hydrophobic mixture formed by SLs and
rapeseed oil was the key to settling (Fig. S3). The stronger
hydrophobicity, the larger the particle size, the faster sedimentation
rate will be. However, with the further increased of O/S ratio, density
became the main influencing factor, so that the sedimentation rate of
mixture decreased or suspension. In terms of sedimentation rate, it was
mainly dependent on the particle size of mixture and density difference.
To improve the efficiency of SLs separation and reduce the loss of
substances during in-situ separation process, low glucose
concentration and appropriate O/S ratio should be adopted.
3.3.
Enhancement of
sedimentation efficiency by UEST
Although
SLs sedimentation could be achieved by adjusting the O/S ratio in the
broth, it was common to result in some losses of biomass, glucose, and
rapeseed oil during the in-situ separation process. Especially
for rapeseed oil, its loss and SLs sedimentation efficiency always
presented a contradiction, and it would be lost a lot at high O/S ratio
of 0.25 g/g, even if SLs could be quickly settled. On the other hand,
under the low O/S ratio condition, the rapeseed oil loss was reduced,
but corresponding SLs sedimentation rate also slowed down. Therefore,
UEST was introduced to accelerate the sedimentation rate of SLs and
simultaneously reduce the loss of substrate and biomass. Ultrasound not
only accelerates the aggregation of SLs particles, enhancing the gravity
force, but also rapidly removes air bubbles, reducing the interference
of air bubbles on SLs precipitation
(Fig.
S4 A). By comparing the effect of ultrasonic on particle size, it can be
found that ultrasonic enlarged the particle size of mixture, increasing
by more than 37.3% (Fig. S4 B). Moreover, it could be found that after
the treatment of ultrasound, the cell viability and SLs production
capability would not be affected (Fig. S4 C and D). Therefore,
ultrasound can accelerate the deposition of SLs by reducing bubbles and
increasing particle size, and has no significant effect on the growth of
strain and SLs synthesis.
The average sedimentation rate of SLs enhanced with the increase of the
ultrasonic time and power (Fig. 4A, B and C). The maximum sedimentation
rate increases by 46.9% to 485.4% with UEST from high to low O/S
ratios (Fig. 4 D). When the O/S was 0.10 g/g, the sedimentation rate
reached 0.0165 cm/s, independent on ultrasonic power. Since low O/S
ratio leaded to less loss in in-situ separation process, UEST
could not only reduces the influence of rapeseed oil on viscosity, but
also further reduces the loss of rapeseed oil and accelerates SLs
separation under low O/S ratio condition. In the following experiments,
the ultrasonic power of 100 W and the time of 10 min (the height of
separation device was 10 cm and the sedimentation rate was 0.0165 cm/s)
were adopted to achieve effective sedimentation.
3.4.
Semi-continuous
fermentation of SLs production by in-situ separation strategy with
UEST
The SLs sedimentation was
regulated by the S/O ratio. Therefore, before in-situ separation
of SLs, the O/S ratio was adjusted to 0.10-0.12 g/g, and the glucose
concentration was controlled at approximately 30 g/L. The whole
semi-continuous fermentation cycle lasted 378 h, during which 4 times ofin-situ separation of SLs were conducted with UEST once the SLs
concentration in the broth reached 200-220 g/L, and finally the total of
2039.9 g SLs was produced with the consumption of 1545.7 g rapeseed oil
and 1979.3 g glucose respectively
(Fig. 5A and B). Through the
analysis of SLs productivity and yield during different phases, it was
found that the cell activity has been maintained at a high level
throughout the whole fermentation process and the average SLs
productivity and yield reached 2.15 g/L/h and 0.58 g/g respectively
(Fig. 5C). Moreover, UEST in-situ separation could reduce the
losses of biomass, glucose and rapeseed oil by 68.2%, 16.2%, and
65.5%, respectively (Table 1), in comparison to direct in-situseparation strategy without UEST, thus achieving efficient SLs
separation under low O/S ratio condition. Correspondingly, thein-situseparation efficiency and SLs separation rate improved by 34.5%% and
26.4%, respectively (Table 1).