E. coli BR Operation
During E. coli BR operation, two major CER peaks could be observed (Fig. 1A), which is consistent with a prior E. coli study (Sauvageau et al. 2010). During the current study, nitrogen, calcium, iron, and yeast extract were not shown to limit the growth. Little glucose was consumed during the late stages of BR and it was not depleted by the end of BR (Fig. 1B). The restricted growth was most likely due to the accumulation of organic acids (e.g., acetic acid), resulting from bacterial Crabtree effect (Mustea and Muresian 1967), rather than the depletion of a limiting nutrient. Excessive organic acids might be produced from rapid growth in the early stages of BR, partly inhibiting the subsequent stages of growth and preventing the full consumption of glucose. Some future investigations can be carried out to identify the mechanisms of this inhibition and eliminate barriers towards complete consumption of glucose.
SCF Long Cycle and Short Cycle Operation
Much like the case for E. coli BR operation, E. coli SCF long cycles did not exhaust glucose when CER flattened (cycling condition) (Fig. 2B). This was not the case for S. cerevisiae undergoing SCF operation, wherein glucose depletion was observed at the end of BR and long cycles (Fig. 3B and 3C). As discussed earlier, the accumulation of organic acids might have an inhibitory effect on E. coli growth. Previous work investigating E. coli undergoing SCF long cycles (Sauvageau et al. 2010) found that glucose was totally consumed by the time CER stopped decreasing; however, while that study was performed with similar nutrient conditions, it used a different strain of E. coli (ATCC 11303) (Sauvageau et al. 2010).
The improvement in production of E. coli biomass was significant once SCF operation was adjusted to a short cycle scheme; 1.8-fold and 2.7-fold increases in yield and volumetric productivity, respectively (Table 1). In the previous study investigating SCF long cycles with E. coli ATCC 11303 (Sauvageau et al. 2010) the yield was found to be 0.23 L∙g glucose-1, and the productivity of E. coli cells was 0.28 h-1 (also shown in Table 1; calculated based on an original figure (Sauvageau et al. 2010) using Eq. 1 and 2). In comparison, the yield and volumetric productivity during the current short cycle operation also prevailed. As for S. cerevisiae cells grown in SCF operation, short cycles led to a 1.6-fold increase in volumetric productivity while providing a similar yield to long cycles (Table 1). It was also noted that the average glucose consumption rate and average CER were enhanced during short cycles of both E. coli and S. cerevisiae, despite lower cell density (Fig. 2 and 3) – meaning more glucose was consumed per cell and more CO2 was released per cell. That is to say, cellular activity was generally more intense during SCF short cycles.
Table 1 Yield and volumetric productivity in cellular biomass production during SCF long and short cycle operation
||Yield of Cells (L∙g glucose-1)
||Volumetric Productivity of Cells (h-1)
|E. coli long cycle operation (this study)
|E. coli long cycle operation (2010) (Sauvageau et al. 2010)
|E. coli short cycle operation (this study)
|S. cerevisiae long cycle operation (this study)
|S. cerevisiae short cycle operation (this study)
a values were calculated using data from (Sauvageau et al. 2010) and Eq. 1 and 2.
Significant improvements in yield, productivity, and metabolic activity highlight the advantages of the SCF short cycle scheme as compared to the long cycle counterpart. Yield and productivity would likely be further improved if the SCF short cycle strategy were used in the context of recent E. coli and S. cerevisiae SCF studies, such as E. coli biomass production (Sauvageau et al. 2010), bacteriophage production (Sauvageau and Cooper 2010; Storms et al. 2014), recombinant protein production (Storms et al. 2012), ethanol fermentation (Wang et al. 2017, 2020), and shikimic acid production (Agustin 2015). Instantaneous specific productivity of bacteriophage, recombinant protein, and shikimic acid was found to be optimal near the completion of synchronized cell replication (corresponding to a maximum in CER) during SCF long cycle operation (Storms et al. 2012, 2014; Agustin 2015).
In specific, the results observed in this study for S. cerevisiae SCF long cycle operation generally agree with a preceding study (Tan et al. 2022). Growing the engineered yeast strain in SCF long cycles had substantially improved shikimic acid yield and productivity compared to BR (Tan et al. 2022). Hence, it may be possible to further enhance shikimic acid production by implementing the SCF short cycle strategy. Based on the improved productivity of cellular biomass (Table 1), the production rate of the primary metabolite will highly likely be further reinforced in SCF short cycles.
The incomplete depletion of glucose during short cycles might be the only concern for the implementation of this SCF scheme. To address this, recycling of the carbon source might be considered in future studies.
A close link between CER maximum and the completion of synchronized cell division can be established. Firstly, the cycle time of short cycles – from start-of-cycle to CER maximum – allowed one generation of complete cell doubling. Considering the SCF cycling process is based on the replacement of exactly one-half of the working volume of the fermenter, if E. coli and S. cerevisiae cells had not completed one round of cell replication within each cycle, washout would have occurred and the CER profile would have been unrepeatable. The short cycles, however, were stable and repeatable. Secondly, during previous SCF studies based on long cycles with E. coli (ATCC 11303 (Sauvageau et al. 2010) and CY15050 (Storms et al. 2012)) and the engineered S. cerevisiae (CEN.PK 113-1A Matα (Agustin 2015)), step-wise doublings in cell count were observed at the CER maxima. This suggested that cell replication was in unison, and synchronized cell doubling was completed when CER reached its maximum. Thirdly, significant up-regulation of DNA replication-related genes and selected cyclin genes (CLN1, CLN2, CLB3, CLB1, and CLB2) were observed only during the first-half of S. cerevisiae SCF long cycles (Tan et al. 2022). It is very likely that there was little replication activity after the maximum in CER. Fourthly, the cycle time of SCF long cycles was significantly longer than the expected doubling time in the same nutrient conditions. In contrast, the duration of short cycles was very close to those doubling times.
As to S. cerevisiae SCF short cycles, the expression profiles of the investigated cyclin genes provided valuable information (Fig. 4). Firstly, the amplitudes and sequential changes in up-regulation suggested that a certain level of cell synchrony was established. Cyclical changes in glucose concentration established by the mode of operation provided a forcing function to induce the entrainment effect required for cell synchrony; greater glucose availability early in the SCF cycles preferentially favored some stages of the cell cycle. It was also noted that, compared to SCF long cycles, a decrease in up-regulation amplitudes was observed during short cycles (Fig. 4B and Tan et al. 2022). This was likely due to the incomplete utilization of glucose, resulting in a tamer entrainment effect during short cycles. Secondly, the sequence of cyclin genes expression suggested that the replication of partially synchronized cells started from the middle of the short cycles and was completed at the same point in the subsequent cycle. CLN1 and CLN2 were expressed later than CLB1 and CLB2 during SCF short cycles (Fig. 4B) – an inverse sequence compared to the standard yeast cell cycle (Fitch et al. 1992; Cho et al. 1998). This unexpected, distinct, cycle-spanning cell replication pattern in short cycles was likely caused by other driving forces apart from the oscillation of glucose concentration, as the nutrient cycle itself is expected to lead to an alignment between the starts of SCF and cell cycles.
This leads to another question – might synchronized yeast cell replication also present a cycle-spanning pattern during SCF long cycles, starting from the middle of a long cycle and ending at the same point in the succeeding long cycle? The answer should be no. One reason is that there was no significant expression of selected cyclin genes during the second-half of long cycles (Tan et al. 2022). Hence, there was hardly any replication activity during the late stages of long cycles. Moreover, the onsets of SCF long cycles and the yeast cell cycle were aligned, as suggested by the congruent expression sequence of the cyclin genes during the first half of long cycles and the standard yeast cell cycle. Furthermore, the cycle time of long cycles was more than twice the doubling time of S. cerevisiae in the same nutrient conditions. It is unlikely that one round of cell replication occurred throughout the whole cycle time of long cycles.
Identical trends and amplitudes of cyclin gene expression during BR late-log phase were identified not only between replicate experiments in the present study (Fig. 4A and S2A) but also amongst current qPCR results, previous qPCR results, and previous RNA-Seq results (Tan et al. 2022). This great alignment of the gene expression profiles across different studies and analytical techniques significantly increases confidence in the trends observed. Consequently, relative quantification results were considered to truly reflect transcriptional changes during SCF short cycles (Fig. 4B).
An Overview of Characteristic Events in SCF
Significant differences in the occurrence of some SCF key events can be observed among SCF studies with different microorganisms implemented. These characteristic events include: (1) the time at which the limiting nutrient was depleted or reached a plateau, (2) the characteristic values of control parameters, and (3) the completion of one generation of synchronized cell division.
Nitrogen or carbon sources are frequently set as the limiting nutrients dictating the cycling of SCF operation. Control parameters used to establish cycling conditions have included dissolved oxygen (DO), carbon dioxide evolution rate (CER), or oxidation-reduction potential (ORP) (Brown 2001). Mass flow rate of the exit gas has also been used for SCF of S. cerevisiae SuperstartTM producing ethanol (Wang et al. 2020) and was a direct reflection of CER under anaerobic conditions. In studies of phenol degradation using Pseudomonas putida ATCC 12633 (Hughes and Cooper 1996) and Acinetobacter calcoaceticus RAG-1 ATCC 31012 grown on hexadecane (van Walsum and Cooper 1993), CER patterns were found to mirror DO patterns, and CER maximum aligned with DO minimum. Under aerobic conditions, this relationship between CER and DO would generally be true (cautions on rare exceptions). In another study investigating toluene removal using P. putida ATCC 12633 undergoing SCF (Brown et al. 2000), the inflection point of ORP was observed near the concurrence of CER maximum and DO minimum. However, ORP patterns during SCF operation are generally more complex than other parameters. An increasing trend in ORP was observed in the toluene removal study using P. putida ATCC 12633 (Brown et al. 2000), while a decreasing trend was shown in the removal of oxidized nitrogen using Pseudomonas denitrificans ATCC 13867 (Brown et al. 1999). The presence and absence of oxygen in these two studies were likely responsible for these diverging patterns. In contrast, DO and CER generally present similar patterns amongst various studies and hence have been more often applied as the control parameter. Overall, it is illustrated that DO minimum would coincide with CER maximum during SCF operation, and the inflection point of ORP is likely close to this point. In the present study, the time at which this event occurs is referred to as the control parameters’ “characteristic values” or “characteristic points”.
In many SCF studies published before 2010, co-occurrence was always identified for the depletion of a limiting nutrient and the characteristic values of control parameters. SCF cycling was triggered upon this concurrence unless an extended cycle strategy was applied. In antibiotic production using Streptomyces aureofaciens ATCC 12416c (Zenaitis and Cooper 1994), phenol degradation using P. putida ATCC 12633 (Hughes and Cooper 1996), and cultivating Bacillus subtilis ATCC 21332 (Sheppard and Cooper 1991), DO minimum occurred concomitantly with nitrogen depletion or the complete removal of phenol. Moreover, the co-occurrence of the completion of cell replication with the aforementioned two key events was observed in a wealth of SCF studies, and this is summarized as Trend A in Fig. 5A. The first SCF upgrade from continuous phasing identified that the depletion of nitrogen, DO minimum, and the doubling endpoint of optical density (OD) took place at the same moment (Sheppard and Cooper 1990). In sophorolipid production using Candida bombicola ATCC 22214 (McCaffrey and Cooper 1995) and citric acid production using Candida lipolytica ATCC 20390 (Wentworth and Cooper 1996), cell count doubled within a narrow time window near the minimum in DO, concomitant with the exhaustion of the nitrogen source. In A. calcoaceticus RAG-1 ATCC 31012 grown on ethanol (Brown and Cooper 1991) and the degradation of aromatic compounds using P. putida ATCC 12633 (Sarkis and Cooper 1994), the completion of cell doubling co-occurred with carbon source exhaustion and DO minimum. Similarly, in oxidized nitrogen removal using P. denitrificans ATCC 13867 (Brown et al. 1999), the end of doubling of cell dry weight corresponded to the inflection point of ORP and nitrogen depletion.
The reliability of Trend A (Fig. 5A) had been considered universal. For example, in studies tackling hydrocarbon degradation using A. calcoaceticus RAG-1 ATCC 31012 (Brown and Cooper 1992) and cultivating B. subtilis ATCC 21332 (Sheppard and Cooper 1991), the authors directly took the equivalence of SCF cycle time and cell doubling time as a default. However, this was only true when synchronized cell division was accomplished upon initiating SCF cycling. Also, it should be noted that the end point of the doubling of OD or dry weight does not necessarily represent the doubling endpoint of cell number. These can be decoupled and display different trends in synchronized populations – the cell count increases in a step-wise manner, while OD or dry weight present a continuous, near-linear increase regardless of the completion of synchronized cell division (Marchessault and Sheppard 1997; Storms et al. 2012).
Moreover, during polyhydroxybutyrate (PHB) production using Alcaligenes eutrophus DSM 545 (Marchessault and Sheppard 1997) and the growth of B. subtilis ATCC 10774 (Sheppard 1993), while the minimum in DO and the nitrogen source depletion coincided, synchronized cell replication was completed much earlier – in the middle of the SCF cycles. As the timing of the cell cycle end point showed a significant difference, these studies serve as representations for Trend B in Fig. 5B. In summary, for most of the microbial systems used in earlier SCF studies, the depletion of a limiting nutrient and characteristic values of control parameters (DO minimum, CER maximum, or ORP inflection point) occurred concurrently at the end of each SCF cycle (Trends A and B in Fig. 5A and 5B). One round of synchronous cell doubling was completed at the same time (Trend A in Fig. 5A) or, in some instances, in the middle of the cycles (Trend B in Fig. 5B).
Compared to Trends A and B, the scenario observed in a study investigating biosurfactant production using Corynebacterium alkanolyticum ATCC 21511 growing on hexadecane in SCF (Crosman et al. 2002) was substantially different. DO minimum and the completion of synchronized cell division occurred concomitantly, but a considerable amount of carbon source was left over. Recent SCF works using E. coli ATCC 11303 (Sauvageau et al. 2010), E. coli CY15050 (Storms et al. 2012), and engineered S. cerevisiae CEN.PK 113-1A Matα (Agustin 2015) depicted an identical trend – cell count doubled step-wise at the maximum in CER (at the cycle midpoint), but glucose, the limiting nutrient, was only exhausted once the decrease in CER flattened (at the end of the cycles). In ethanol fermentation using S. cerevisiae SuperstartTM undergoing SCF (Wang et al. 2020), glucose was depleted upon the time when CER flattened (reflected by exit gas mass flow rate in anerobic conditions), though cell counts were not reported due to clumping of the yeast cells. As mentioned earlier, the same trend was observed in the present study when cultivating E. coli MG1655 or engineered S. cerevisiae CEN.PK 113-1A Matα (except that, for E. coli undergoing SCF long cycles, the end of cycle was due to an inhibitory effect rather than glucose depletion). Trend C in Fig. 5C is used to describe the pattern observed in these studies.
Moreover, transcriptional evidence during S. cerevisiae SCF short cycles presented in this study revealed a likely cell replication pattern in short cycles – partially synchronized cell cycle starting and ending in the middle of short cycles. This cycle-spanning mode of cell replication is presented by the black dashed line in Fig. 5C. Overall, in studies displaying Trend C, the flattening of CER decrease or DO increase coincided with the depletion or a plateau of the limiting nutrient at the end of SCF long cycles, but synchronized cell replication was completed in the middle of the long cycles, corresponding to CER maximum or DO minimum. SCF short cycles ended at CER maximum or DO minimum, but partially synchronized cell replication likely started and ended in the middle of the short cycles. The limiting nutrients were not depleted by the end of the SCF short cycles.
The discrepancies amongst the three major trends in the characteristic events during SCF operation were likely derived from intrinsic differences in the microorganisms and nutrient environments used. A. eutrophus and B. subtilis ATCC 10774 following Trend B, and C. alkanolyticum, E. coli, and S. cerevisiae following Trend C likely sensed nutrient conditions more actively and adopted a feed-forward strategy (Levy and Barkai 2009) – in which cells proactively sensed external changes and regulated gene transcription and expression prior to the alteration of the growth rate (Levy and Barkai 2009). Completing one generation of the synchronized cell cycle but deciding not to continue the proliferation at the expense of the remaining limiting nutrient seemed to be the growth strategy of these microorganisms (Fig. 5B and 5C). On the contrary, for a number of microorganisms following Trend A, all the available limiting nutrient was used in completing the cell doubling (Fig. 5A). The difference between A. eutrophus and B. subtilis ATCC 10774 in Trend B, and C. alkanolyticum, E. coli, and S. cerevisiae in Trend C is expected to lie in the respiratory intensity between the end of the cell cycle and the time at which the limiting nutrient was depleted or reached a plateau. For the former group, the intensity of respiration increased even after synchronized cell replication. Therefore, the characteristic value of the control parameter (DO minimum) co-occurred with the exhaustion of the limiting nutrient but not with the end of cell doubling (Fig. 5B). For microbes displaying Trend C, respiration slowed significantly after synchronized cell replication (during the consumption of residual limiting nutrient), and therefore CER maximum or DO minimum occurred at the completion of synchronized cell doubling but not at the depletion or a plateau of the limiting nutrient (Fig. 5C).
Different nutrient conditions can lead to different physiologies and affect the trends in SCF. For example, implementing different types of limiting nutrients – nitrogen or carbon – in a continuous phased culture tremendously affected where synchronized cell replication of Candida utilis (Y-900) ended when the cycle time was set to 4, 6, 8, and 12 h (Müller and Dawson 1968). Further studies on this topic could lead to more in-depth understanding of the physiological patterns during SCF.
Limiting nutrient depletion has been one of the original premises of SCF, but a broader picture is emerging. The Trend C observed in some studies suggests a deviation from the original description of SCF – SCF does not necessarily require limiting nutrient depletion. Consequently, a novel description of SCF is proposed below, taking into consideration all SCF scenarios presented in Fig. 5. This new SCF definition excludes the requirements of limiting nutrient depletion and joint occurrence of the key events.
SCF is a semi-continuous fermentation approach that allows the completion of one generation of microbial cell replication during each cycle. The cycling procedure comprises harvesting precisely one half of the working volume and then replenishing with the equivalent amount of fresh medium. Cycling is dictated by microbial growth and metabolism and is triggered automatedly based on monitoring one or more growth- and/or metabolism-associated sensing elements (e.g., DO, CER, ORP, exit gas mass flow rate, etc.). SCF cycling takes place directly after the completion of one generation of cell proliferation or with a delay, depending on the microorganism, the initial nutrient conditions, and the control parameter conditions for cycling being implemented. SCF cycling is not necessarily related to the time at which the limiting nutrient is depleted or reaches a plateau. If limiting nutrient depletion or a plateau does not co-occur with the cell cycle completion, we identify SCF operation that cycles in advance of exhaustion or a plateau of the limiting nutrient as “short cycle”, and correspondingly, SCF operation that cycles upon depletion or a plateau of the limiting nutrient as “long cycle” (Fig. 5). “Extended cycle” is generally referred to as SCF operation that cycles beyond exhaustion or a plateau of the limiting nutrient (Fig. 5).