3. Results and Discussion
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 oily characteristics. The
microscopic results showed that biomass and broth were enclosed by the
mixture of 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. 3). 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. 3). With the further
decrease of O/S ratio (< 0.02 g/g), crystalline SLs gradually
appeared in the broth (Fig. 3). 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 number 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 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 oil
and SLs contents.
3.2. The mechanism 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. 4A), 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 oil content would significantly affect the settling time,
thus exhibiting that O/S ratio was correlated to the average
sedimentation rate (Fig. 4B). 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.
Stokes formula is used to
calculate the sedimentation rate of spherical particles under an ideal
condition. Although the SLs sedimentation is a complicated process and
it is impossible to achieve an ideal environmental state, the Stokes
formula can still give important theoretical guidance. According to
formula 4, the sedimentation rate of spherical particles was mainly
determined by the radius, density, and viscosity. Analysis of SLs
particle size showed that when SLs was oily, the radius of SLs particle
increased with the O/S ratio and the maximum radius maintained at 1.5 mm
once the O/S ratio exceeded 0.04 g/g (Fig. 5A). In the real fermentation
process, the O/S ratio was usually above 0.04 g/g, therefore the
particle size of SLs seemed to be less influence on the SLs settling. In
addition, the viscosity of fermentation broth was an important factor
affecting the sedimentation rate of SLs. 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. 5B). 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 oil, the
density decreased with the increase of O/S ratio (Fig. 5C). However, 1.0
g SLs was found to be bound to a maximum of 0.3 g oil (Fig. S2).
Therefore, the minimum density of the mixture of SLs and 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 oil (Fig. 5D), and density difference
was the main reason determining the SLs settling or floating. As the
main reason for suspending SLs was the increase of oil concentration or
bubbles, the density of the mixture of SLs and oil was lower than the
supernatant to achieve temporary suspension, but SLs would be
re-sedimented by standing still.
As mentioned above, the SLs
sedimentation requires the formation of a mixture of SLs and oil. 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 increased of O/S ratio, the hydrophobicity of the mixture was
strengthened, thus the settling rate was accelerated under gravity
action.
In the settling process, the hydrophobic mixture formed by SLs and oil
was the key to settling (Fig. 6). The stronger hydrophobicity, the
faster sedimentation rate will be. In terms of sedimentation rate, it
was mainly dependent on the density difference of supernatant and the
mixture. 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
oil during the in-situ separation process. Especially for 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 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. S3A). Moreover, it could be found that after the
treatment of ultrasound, the cell viability and SLs production
capability would not be affected, demonstrating that it was feasible to
introduce UEST to improve the efficiency of SLs sedimentation. (Fig. S3
B and C).
The average sedimentation rate of SLs enhanced with the increase of the
ultrasonic time and power (Fig. 7A, B and C). The maximum sedimentation
rate increase by 46.9% to 485.4% with UEST from high to low O/S ratios
(Fig. 7 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 oil on viscosity, but also further reduces the
loss of 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, and the sedimentation efficiency was related
to supernatant density dependent on glucose concentration. 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 of in-situ separation of SLs
were conducted with UEST once the SLs concentration in the broth reached
200-220 g/L, and the total of 2039.9 g SLs was produced with the
consumption of 1545.7 g oil and 1979.3 g glucose respectively
(Fig. 8A 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 and 0.58 g/g respectively (Fig.
8C). Moreover, UEST in-situ separation could reduce the losses of
biomass, glucose and oil by 68.2%, 16.2%, and 65.5%, respectively
(Table 1), in comparison to direct in-situ separation 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).
SLs has been regarded as a green
and sustainable biosurfactants, but their high production costs limit
large-scale application and development (Jiménez-Peñalver et al., 2019).
Coupling fermentation on the basis of in-situ product separation
is one of the most effective ways to reduce production costs and improve
productivity (Table 2). Wang et al.
(2020)
designed a simple in-situ separation device coupled with the
bioreactor and adopted high cell density fermentation to achieve
high-efficient production of SLs. After running for 480 h,
the
productivity and yield of SLs were 2.39 g/L/h and 0.73 g/g,
respectively, with an average separation efficiency of 74.3%. Zhang et
al. (2018) developed a new bioreactor with dual ventilation pipes
containing dual sieve plates (DVDSB) for semi-continuous fermentation of
SLs. By regulating the air pressure and different sources of oil
(transgenic or non-transgenic oil), the gravity sedimentation of SLs at
the bottom of bioreactor could be accomplished
with
SLs titer, productivity, and yield of 477 g/L, 1.59 g/L/h and 0.60 g/g,
respectively. Otherwise, Dolman et al. (2017) suggested that the glucose
concentration could adjust the density of fermentation broth thereby
controlling the ups and downs of SLs. The whole fermentation process
lasted 1023 h to produce 623 g/L SLs, and during the SLs in-situseparation process, the recovery rate of SLs was as high as 86.0%,
which was 9 times concentration of previously reported. Apart from SLs
separation by gravity sedimentation, Liu et al. (2019) realized the SLs
separation in the upper layer of the broth through the principle of
froth flotation. Subsequently, part of biomass and glucose could be
recovered by washing the settled SLs, and the final titer and
productivity of SLs were 342 g/L and 1.55 g/L/h, respectively. Though
these strategies have achieved relatively satisfactory results in the
in-situ separation of SLs, the mechanism of SLs separation either by
gravity sedimentation or by froth flotation was still not been clearly
elucidated, which led to a blind separation process and low separation
efficiency. Oil or glucose concentration has been pointed out to possess
a significant impact on the settlement of SLs (Zhang et al., 2018),
however, these researches focused more on the final results and failed
to further analyze the causes of the sedimentation, resulting in big
differences in different studies. Herein, the sedimentation mechanism of
SLs was studied and it was found that the O/S ratio was the key factor
affecting the SLs morphology and subsequent settling. In contrast, the
sedimentation rate was dependent on the hydrophobicity of the oily SLs
as well as the density difference with the broth. These results provided
a solid foundation for rational regulation of SLs deposition. On the
other hand, the UEST can accelerate the particle collision, make the
particle become larger, eliminate the air bubbles, and further reduces
the settling resistance (Luo et al., 2019). Palme et al. (2010) and
Hincapié Gómez et al. (2015) applied ultrasonic enhanced deposition
technology in the separation and recovery of microalgae and yeast cells,
respectively. In this work,
UEST
was introduced to enhance the sedimentation of SLs, simultaneously
greatly reduce the losses of oil, glucose, and biomass duringin-situ separation process. Moreover, semi-continuous
fermentation by in-situ separation of SLs with UEST was
conducted. Compared to the batch fermentation (168 h), the fermentation
cycle was more than 2-fold longer (378 h), and the average productivity
and yield from semi-continuous fermentation were increased by 26.5% and
23.4% respectively. This could be contributed to that the
semi-continuous fermentation could separate out SLs and toxic substances
in time, and allow the cells being in the optimal production
environment, which also proved the feasibility of semi-continuous
fermentation as a model for the efficient production of SLs. In
comparison with other semi-continuous fermentations of SLs, both the
productivity and yield in this work were at high levels, especially for
the index of YSLs/DCW representing the SLs production
yield to the DCW, its value reached 36.1 g/gDCW, which
was 86.1% higher than the highest value in the literatures.
Furthermore, the specific SLs productivity (QP/DCW) also
exhibited a 41.2% higher than the highest value ever reported. It could
be expected to achieve a more ideal production efficiency in combination
with high cell density strategy, laying a foundation for further
industrial application.