for crude glycerol tolerance
Adaptive laboratory evolution (ALE) is widely used to obtain robust
microbes with high tolerance for different environmental stresses.
However, the achievement of a stably improved phenotype which can fully
withstand a specific stress factor or inhibitor usually requires a
long-term ALE with great efforts (Zhang
et al., 2019 ; Alves et al., 2021 ).
In order to increase the efficiency, long-term ALE of C.
pasteurianum C8 was conducted in a home-made automatic evolutionary
system integrated with the real-time monitoring and control of cell
growth in this study.
As shown in Fig. 1 , the
OD600 of the strain during the repeated batch
cultivations was automatically controlled between 0.5 and 1.5 by the ALE
system. This was realized by adding of fresh medium into the cultivation
bottle whenever the OD600 reached 1.5 and the addition
was stopped when the OD600 reached 0.5 again. There are
many advantages to keep the biomass sufficiently low in the long-term
automatic ALE experiment. First, lower biomass can prevent bio-fouling
in the tube connecting the cultivation bottle and the OD detector.
Second, the pH of the culture can be maintained at 6.3 to 6.5 without
external pH control owing to the negligible acids production by the
limited biomass. It is well known that pH lower than 5.0 leads to
significant growth inhibition to C. pasteurianum (Dabrock
et al., 1992 ), which may affect the efficiency of the ALE experiment.
However, the integration of pH control with probes and peristaltic pumps
for acid / base solution requires a bigger size of bioreactor, and thus
increases the working volume and medium consumption for the long-term
ALE experiment. Thus the strategy employed here makes the system as
simple as possible and highly efficient as well. Last but most
importantly, the time needed for 1 adaptation cycle (OD600 from 0.5 to
1.5) is significantly reduced compared to those reported in the manual
ALE experiment (>12 h for 1 cycle)
(Liang et al., 2020 ;Zhang et al., 2019 ;Alves et al., 2021 ). An example of the
automatic ALE process for 30 g/L crude glycerol is presented in Fig.1 . Totally 10 adaptation cycles were
conducted within 80 h. The immediate dilution of the culture with fresh
medium when the OD600 reached 1.5 not only reduced the
time interval between two adaptation cycles, but also ensured that the
next adaptation cycle was initiated with cells in the exponential growth
phase. Undoubtedly, inoculum in good growth state is beneficial to
avoiding lag phase, reducing infection risk and therefore increasing the
number of adaptation cycles in the automatic ALE experiment. Based on
the above-mentioned advantages, C. pasteurianum C8 was
successfully adapted to each concentration of crude glycerol (30-120
g/L), and finally adapted to 120 g/L crude glycerol after more than 40
days of automatic ALE with 106 adaptation cycles. The △T for one
adaptation cycle progressively decreased during the repeated
cultivations, and a reduction to 4 h which was the same with pure
glycerol, was achieved in the last cycle with 120 g/L (Fig.1 ). Further increase of crude glycerol
concentration to 130 g/L resulted in a significantly longer △T
>10 h, and the cell growth failed to recover even after 20
adaptation cycles. This might be due to the growth inhibition caused by
the impurities and the osmotic pressure from 130 g/L crude glycerol, the
cells were then kept in the automatic ALE system for another 10 repeated
cultivations with 120 g/L crude glycerol for growth stabilization. The
end-point adapted strain obtained from the automatic ALE system was
named as C. pasteurianum G8 and used for characterizing the crude
glycerol tolerance in the subsequent fed-batch fermentations.
As shown in Fig. 4 and Table1, fed-batch fermentations of crude
glycerol by C. pasteurianum G8 were performed with two feeding
strategies (continuous feeding with the feed rate of 40 g/L/h vs. pulse
feeding with 80 g per feed). Meanwhile, the initial glycerol
concentration was set at 80 g/L, which was the same as that in the pure
glycerol fermentation. As expected, C. pasteurianum G8 showed
excellent improved tolerance against the high initial concentration of
crude glycerol. No lag phase was observed after inoculation and the
µmax (0.52 ± 0.02 h-1 in pulse
fed-batch and 0.59 ± 0.02 h-1 in continuous fed-batch)
reached the same level as that in the pure glycerol fermentation. It
should be noted that the same initial concentration of crude glycerol
caused a long lag phase of over 10 h when the parental strain C8 was
used (data not shown). Furthermore, the maximum glycerol consumption
(22.19 g/L/h with pulse and 21.44 g/L/h with continuous feeding) and PDO
production rate (8.74 g/L/h with pulse and 10.18 g/L/h with continuous
feeding) also recovered to the same level as those achieved in pure
glycerol fermentation owing to the increased utilization of crude
glycerol and the fast feeding strategy. The final PDO concentration and
overall productivity reached 71.47 g/L and 3.97 g/L/h with the pulse
feeding (Fig. 4A), and with continuous
feeding (Fig. 4B), 74.23 g/L and 5.30
g/L/h, respectively. These results clearly indicate that the automatic
long-term ALE can effectively promote the production level of PDO in the
crude glycerol fermentation by C. pasteurianum. Despite the
similar PDO titer achieved with both feeding strategies, the yield of
PDO from crude glycerol in the pulsed fed-batch fermentation was about
6% lower than that in the continuous fed-batch fermentation (0.49 vs.
0.52 gPDO/gGly). This is probably due to
the stress response of the strain caused by multiple instant exposure to
a large amount of crude glycerol impurities during the pulse feeding
period, leading to a reduced acetate / butyrate ratio at the end of the
fermentation (1.53 with pulse and 2.17 with continuous feeding).
Similarly, further increasing the initial crude glycerol concentration
to 120 g/L (Fig. 4C) also resulted in
marked decrease of acetate / butyrate ratio to 0.81, even with
continuous feeding strategy. As a result, the titer, productivity and
yield of PDO were all negatively affected (Table1). Nevertheless, C.
pasteurianum G8 exhibited good growth even with 120 g/L initial crude
glycerol (µmax, 0.27 ± 0.01 h-1) and
produced very less butanol (<1.5 g/L) in all the fed-batch
fermentations, indicating much enhanced and stable tolerance to crude
glycerol than that of the parental strain.
Since the fed-batch fermentation with 80 g/L initial crude glycerol
concentration and with a continuous feeding of 50% crude glycerol at 40
g/L/h resulted in the best PDO production, the same strategy and
conditions were used for a scale-up fermentation in 1
m3 bioreactor. As shown in Fig4D and Table1 , the highly crude glycerol-tolerant
strain C. pasteurianum G8 was capable of producing 81.21 g/L PDO
within 19 h, along with an overall productivity of 4.27 g/L/h and a
yield of 0.49 gPDO/gGly in the 1
m3 scale non-sterile crude glycerol fed-batch
fermentation without using N2 aeration and yeast
extract. It should be noted that more glycerol supply with a longer
feeding time resulted in a slightly higher PDO titer in the 1
m3 scale fermentation, but it also triggered higher
butanol production (2.47 g/L) due to the stress induced by both high PDO
concentration and crude glycerol impurities. Butanol production can be
avoided and a significantly higher PDO yield of 0.53
gPDO/gGly can be reached by simply
stopping the fermentation at an early processing time of 14 h (Fig4D and Table1 ). To further increase the PDO titer,
automatic long-term adaptation to a high PDO concentration could be
considered in future work. In general, the results above indicate thatC. pasteurianum G8 has the best potential for achieving efficient
and low-cost industrial bio-production of PDO among the reported
non-pathogenic natural producers so far in terms of PDO titer, yield,
productivity and process economy (Table2 ).