Single batch cultivation of R. marinus DSM 16675
A previous study, focusing on shake flask cultivations in marine broth (MB) supplemented with different mono- and disaccharides, showed that the highest growth of R. marinus DSM 16675 (0.75 g/L) was reached using supplementation with maltose (MBmalt) (Sardari et al. 2017). Therefore, in this study, maltose was chosen as a supplementary carbon source and was added to cultivation of R. marinus DSM 16675, run in complex media (LB or MB) using a stirred tank bioreactor with controlled aeration and pH (Fig. 1). Growth of R. marinus DSM 16675 was initiated after a lag phase of 6 and 4 h in LBmalt and MBmalt, respectively, followed by an exponential growth phase lasting until 10 h cultivation time, reaching a maximum specific growth rate (μmax) of 0.42 h−1 and 0.22 h−1 in LBmalt and MBmalt, respectively. After 10 h, the cell culture entered the stationary phase, as monitored by cease of growth. Consumption of maltose in cultivations using LBmalt was detected between 6 and 10 h cultivation time at an exponential rate, which decreased significantly after 10 h. In the MBmalt cultivation, maltose consumption started after 4 h (indicating start of the exponential growth phase) with a fast consumption rate until 10 h, followed by a period with gradually decreasing maltose concentration until the end of cultivation. It should be noted that accumulation of glucose was seen in both media from the start of the exponential phase until the end of the cultivation, meaning that not all of the maltose was taken up and consumed, but rather enzymatically degraded to its monosaccharide constituents (Fig. 1). Moreover, the obtained glucose in LB medium was consumed for 2 h during the stationary phase without display of cell growth and thus might be used for cells’ maintenance (Navarro Llorens et al. 2010). The maximum OD620 in LBmalt was 6.6 after 13 h which was significantly higher than the OD observed in MBmalt where a maximum OD620 of 1.71 was observed after 24 h.
In the previous study (Sardari et al. 2017) with shake flask cultivations, it was shown that glucose supplementation in MB (MBglu) resulted in a slight increase in the cell growth of R. marinus DSM 16675 and EPSs production. Hence, cultivation of R. marinus DSM 16675 was performed to investigate the growth behavior using LB medium supplemented with 10 g/L glucose (LBglu) at controlled aeration and pH in the bioreactor (Fig. 2). A bioreactor cultivation with LB (without supplementation) was also run in parallel. The cultivation data displayed that glucose supplementation had no effect on the cell growth and only low (if any) uptake of glucose could be detected during the experiment. A maximum OD620 of 2.7 was reached after 16 h in the bioreactor cultivation using LBglu, which was less than that of the cultivation using LB with no additional carbon source (OD620 of 3.6 after 16 h).
Product formation kinetics in single batch cultivations
R. marinus DSM 16675 has been shown to form two products of potential application interest: carotenoids (Ron et al. 2017) and exopolysaccharides (Sardari et al. 2017). To increase the understanding of the formation of these products, the relationship between cell growth and the production of carotenoids and EPSs in single batch cultivations in a pH-controlled bioreactor using LBmalt and MBmalt was studied (Fig. 3). It should be noted that LB and MB (except for the added maltose) contain very low concentrations of sugars, as previously reported by Sezonov et al. (Sezonov et al. 2007) which were precipitated and hydrolyzed together with the EPSs from the medium (Fig. 1 and 2, supplementary materials). The reduction in those sugars was observed between 6 and 10 h cultivation in LBmalt (Fig. 3a) and between 2 and 6 h in MBmalt (Fig. 3b).
As seen in Fig. 3a, the batch cultivation in LBmalt produced carotenoids starting 6 h after inoculation and continued until the end of the exponential phase. The total produced carotenoids profile, represented by absorbance at 450 nm and a final absorbance of 0.061, was best fitted to a second-order polynomial equation using Excel software (Microsoft, 2010) in the exponential phase of the cultivation (Fig. 4a). On the other hand, the changes in EPSs concentration were more difficult to evaluate, with apparent formation only seen from the onset of the stationary phase reaching a final net formation of 5.18 mg/L at the end of cultivation.
In MBmalt a different production pattern of carotenoids and EPSs was observed. Figure 3b depicts that production of both total carotenoids and EPSs started at the beginning of the exponential phase and continued during the stationary phase for both products, reaching a total carotenoids final absorbance at 450 nm of 0.048 and a final EPSs formation of 6.28 mg/L. It can be speculated that the increase in EPSs formation during the stationary phase can be a result of quorum sensing activity that consequently stimulates formation of biofilm (Sengupta et al. 2018). In addition, cell lysis can contribute to biofilm development in which the EPSs are the main components (Bayles 2007; Flemming et al. 2007). Bayles (Bayles 2007) described that the DNA released from the cells during cell lysis at the stationary phase is a structural component of the biofilm matrix. However, in our case the rate of cell lysis seems to be low since the OD was not decreased at the stationary phase.
The total produced carotenoids profile, also, was fitted to a second-order polynomial equation for exponential phase and stationary phase of the batch cultivation (Fig. 4b).
The ratio of EPSs to cell growth (OD) was 1.41 and 4.18 for LBmalt and MBmalt, respectively. This indicates a strong competition between cell growth and EPS production as previously described (Sardari et al 2017). In addition, analysis of the monosaccharide composition of the EPSs hydrolysate from the cultivations in both media was performed to quantify the total EPSs. It showed that the monosaccharides of the produced EPSs from LBmalt consisted of arabinose, galactose, glucose, and mannose. For the EPSs in the culture grown in MBmalt the same monosaccharides were found, but with the addition of xylose, which is in accordance with the previously published data on the EPSs composition from R marinus grown in this medium (Sardari et al. 2017) (Fig. 3, supplementary materials).
The cells produced carotenoids in both media, but interestingly, the production of carotenoids continued in the stationary phase when the cells were grown in MBmalt (Fig. 3b). This might be due to accumulation of tricarboxylic acid cycle metabolites with subsequent conversion to other products such as carotenoids (Henke et al. 2017; Tao et al. 2011).
Production of short chain fatty acids (SCFA) was investigated to explain the decrease in pH during the cultivation, and corresponding consumption of NaOH (1 M), approximately 3 mL in LBmalt and 0.5 mL in MBmalt, during the batch cultivations of R. marinus DSM 16675 in both media. However, very little lactic acid, formic acid, and acetic acid were produced. The main reason for the drop in pH might instead be due to the reaction of the produced CO2 with water and formation of carbonic acid in the culture media (Das and Mangwani 2015).
As seen in Fig. 1, both maltose and glucose were left in the culture medium at the end of both cultivations, excluding that the carbon source is the limiting factor for growth. The highest cell densities were reached in the batch cultivations of R. marinus DSM 16675 using LBmalt as growth medium. Hence, LBmalt was selected as growth medium for fed-batch cultivation of R. marinus DSM 16675, aiming at further increasing cell densities, and solve the eventual nutrient limitations occurring towards the end of the batch cultivations.
Fed-batch cultivation of R. marinus DSM 16675
Fed-batch cultivation with stepwise feeding was applied for R. marinus DSM 16675 using LB medium supplemented with 5 g/L of maltose in the bioreactor (Fig. 5).
As seen in Fig. 5a, the cell growth reached the stationary phase after 12 h of cultivation at an OD of 6.73 after consumption of approximately 3 g/L maltose. The first feed, which consisted of 20 mL of feed solution, was added at 12.5 h of cultivation and growth resumed. The maximum OD reached was 7.84 after 14 h, which decreased to 6.4 after 1 h. At that point, the second feed was added at 15 h of cultivation, but growth was not recovered. Instead, the OD dropped unexpectedly. The maximum OD achieved in the fed-batch cultivation did not exceed the OD in the prior batch cultivations. This could theoretically be due to substrate inhibition, since the concentration of maltose reached 12.47 g/L after the second feed addition. However, repeated fed-batch cultivations with maltose concentrations never exceeding 5 g/L also reached the stationary phase at the same cell densities (Fig. 5b).
Other potential explanations for the termination of growth at 14 h could be either inhibition due to accumulation of growth-inhibiting metabolites, or quorum sensing. To distinguish between these alternatives, sequential batch cultivation with cell recycling was selected as a method to circumvent metabolites reaching growth-inhibiting levels.
Sequential batch cultivation of R. marinus DSM 16675 with cell recycling
Since the fed-batch cultivations were not successful in increasing biomass concentration, sequential batch cultivation of R. marinus DSM 16675 with cell recycling in LBmalt and MBmalt was investigated (Fig. 6).
The first batch cultivation, of R. marinus DSM 16675, in the cultivation sequence in the respective medium was started with the addition of a 10% (v/v) inoculum followed by an exponential phase with a duration of 5 h (cultivation time 3–8 h), reaching the stationary phase after 9 h of cultivation, and a final OD620nm of 5.68 and 1.82 in LBmalt and MBmalt, respectively (Fig. 6). At that point, the cultivation was stopped and the next batch was prepared with fresh medium and inoculated with the recycled cells from the previous batch. Between batch 4 and batch 5, a drop in the starting cell density was observed (compared to the final cell density of the previous batch cycle). This must be due to loss of cells during handling between the cycles while removing the old medium and adding fresh medium. After seven cycles using LBmalt, the maximum OD of 20.2 was reached after 35 h cultivation time, resulting in a threefold increase relative to that of the batch and fed-batch trials. To the best of our knowledge, the final OD value of 20.2 is the highest cell density achieved with the genus according to literature (Fig. 6a). Our results are comparable with the results obtained with other similar thermophilic bacteria that were cultivated to reach higher cell densities (Table 1). As can be seen, sequential batch with cell recycling is as effective as other fermentation techniques to produce high cell densities with thermophiles.
The maximum OD of 14.32 was reached over four cycles in the MBmalt medium after 30 h of cultivation, which is an eightfold increase relative to that of the batch trial (Fig. 6b).
The higher cell density in LBmalt medium can be due to the higher concentration of yeast extract in LBmalt (5 g/L) compared to that in MBmalt (1 g/L). Yeast extract generates high cell densities for Gram-negative bacteria as described by Gray (Gray et al. 2008). Also, LBmalt has twice as much of tryptone (casein peptone) (10 g/L) as peptone in MBmalt (5 g/L), which can explain the higher cell densities in LBmalt compared to MBmalt Another difference is the salt composition and concentration, which seems to slightly hinder growth of R. marinus DSM 16675 in MBmalt. Marine broth emulates open seawater salt composition and has therefore a higher NaCl (2% (w/v)) concentration compared to LB (1% (w/v)).
In each cultivation cycle, the stationary phase was reached before depletion of maltose, which is in line with results from the prior batch cultivations, most likely due to depletion of other nutrients or accumulation of growth-inhibiting products.
The maximum specific growth rate (μmax) was 0.42 h−1 for the first batch cultivation in LBmalt and 0.26 h−1 for the first batch cultivation in MBmalt, which is in accordance with the specific growth rates observed in the single batch cultivations (described in Sect. 3.1.1). However, the specific growth rate decreased gradually in the later batches until the end of cultivation. This shows that the limitation in growth in the fed-batch (described in Sect. 3.2) may have been caused by accumulation of growth-inhibiting metabolites, alleviated by the change of medium in the sequential batch. However, the reasons for the decreased growth rate, in the later cycles in the batch sequence remain unclear and may be influenced by quorum sensing. Reaching a high cell density in a batch culture is a sign for cells to predict severe competition for nutrients and subsequently lack of nutrients. Entry to the stationary phase is a main solution for bacteria to survive. Therefore, the quorum sensing system (Kaur et al. 2018; Montgomery et al. 2013) regulates the transition into stationary phase by quorum sensing signals which are produced mainly at high cell density (Lazazzera 2000).
Product formation kinetics in the sequential batches
The production of carotenoids and EPSs was evaluated in the sequential batch cultivations of R. marinus DSM 16675 in both LBmalt and MBmalt (Fig. 7).
The production of carotenoids increased as the cell density increased in each batch (Fig. 7). In LBmalt carotenoid production ceased at the end of each cycle, indicating that the stationary phase was reached (Fig. 7a). A corresponding decrease was not observed in MBmalt, in accordance with the production pattern in the single batch. The total carotenoid content in LBmalt increased throughout the complete sequence of batches, as shown by the absorbance at 450 nm from 0.002 at the beginning of the first batch cultivation, to 1.67 at the end of the last batch in the cultivation sequence (Fig. 8a). A similar trend was shown in the batch cultivation sequence of R. marinus DSM 16675 in MBmalt (Fig. 8b). The production of total carotenoids increased with increasing cell density in every batch, which is in accordance with the production trend observed in cultivations using LBmalt. The produced total carotenoids in LBmalt were significantly higher than those in MBmalt (Figs. 8). To increase the production of carotenoids in MBmalt, the cells should be kept in stationary phase for a longer time as seen in the single batch cultivation in MBmalt (Fig. 4b). Moreover, further study is needed to determine the concentration of total produced carotenoids to compare the total carotenoids production by R. marinus DSM 16675 and compare it with other thermophilic carotenoids producers.
In contrast to carotenoid production, there was poor EPSs production in LBmalt in each batch. The highest QE observed in cultivations in LBmalt was in batch 2 and 7, in which the cells remained in the stationary phase for a longer time period than in the other batches (Fig. 7a). The total concentration of produced EPSs was 3.77 (mg/L) with an overall QE of 0.1 (mg/L h) in the complete 7 batch cultivation sequence (of totally 35 h) of R. marinus DSM 16675 in LBmalt.
The EPSs production pattern in the batch sequence grown in MBmalt was different, as expected based on the single batch data (Fig. 3). In batch 1–3, QE increased significantly with growing cells, from 0.02 mg/L h in batch 1–1.42 mg/L h in batch 3. In the last two batches (3 and 4), the productivity was stable (Fig. 9). The total production of EPSs was 19 (mg/L) with an overall QE of 0.67 (mg/L h) during four batch sequences of R. marinus DSM 16675 in MBmalt. Interestingly, the EPSs concentration drastically decreased between batches, meaning that most of the EPSs is in solution and not tightly bound to the cells as tightly bound EPSs. The fact that most detected EPSs, produced by R. marinus DSM 16675, ar excreted into the medium has not previously been reported (Fig. 7B).
The total production of EPSs by R. marinus DSM 16675 was low in comparison to thermophiles with good production of EPSs, shown to reach EPS concentrations in the range of 55–897 mg/L (Kambourova et al. 2016). The reason might be due to harvesting the cells at the beginning of the stationary phase, since all good EPSs producers were reported to synthesize EPSs mainly during a prolonged stationary phase at low cell densities (Kambourova et al. 2016).