Macroparticle‐enhanced cultivation of Lentzea aerocolonigenes: Variation of mechanical stress and combination with lecithin supplementation for a significantly increased rebeccamycin production

The actinomycete Lentzea aerocolonigenes produces the antitumor antibiotic rebeccamycin. In previous studies the rebeccamycin production was significantly increased by the addition of glass beads during cultivation in different diameters between 0.5 and 2 mm and the induced mechanical stress by the glass beads was proposed to be responsible for the increased production. Thus, this study was conducted to be a systematic investigation of different parameters for macroparticle addition, such as bead diameter, concentration, and density (glass and ceramic) as well as shaking frequency, for a better understanding of the particle‐induced stress on L. aerocolonigenes. The induced stress for optimal rebeccamycin production can be estimated by a combination of stress energy and stress frequency.


| INTRODUCTION
Filamentous actinomycetes are often a source of bioactive natural products valuable for pharmaceutical industry and medical application (Genilloud, 2017). Therefore, their cultivation is considered quite worthwhile although certain challenges arise in the process. The filamentous actinomycete Lentzea aerocolonigenes for instance, produces the antitumor antibiotic rebeccamycin. Rebeccamycin is a topoisomerase inhibitor and thereby interferes with DNA replication. Despite this activity rebeccamycin itself is not suitable for use in the human body due to the low water-solubility (Bush et al., 1987;Nettleton et al., 1985). To overcome this issue different analogs with increased water-solubility were developed that can easily be derived from rebeccamycin by chemical transformation. The analog becatecarin has already been successfully tested in clinical Phase I and II studies for the treatment of refractory breast cancer, metastatic colorectal cancer, and small-cell lung cancer (Burstein et al., 2007;Goel et al., 2003;Schwandt et al., 2012). In the study of Schwandt et al. (2012) 4200 mg m −2 becatecarin were used for the treatment of small-cell lung cancer. For a person of 1.8 m and 85 kg this would correspond to a total of 8.7 g of substance. Regarding rebeccamycin titers in the literature mostly concentrations of under 100 mg L −1 are achieved in small scales of around 100 ml . Hence, cultivations of L. aerocolonigenes in the bioreactor scale with high productivities are desired. However, the complex cellular morphology of filamentous microorganisms, ranging from freely dispersed mycelium to dense pellets, provides challenges during the cultivation process. Each cell morphological form has certain advantages and disadvantages. The appropriate cell morphology for high productivities strongly depends on the microorganism and the desired product (Walisko et al., 2015;Whitaker, 1992;Wucherpfennig et al., 2010). Walisko et al. (2017) added glass beads with a diameter between 0.25 and 2.1 mm to cultivations of L. aerocolonigenes and thereby increased the rebeccamycin production. The addition of a glass particle concentration of 80 g L −1 with a size range of 0.25-0.5 mm caused a rebeccamycin concentration of 116 mg L −1 which was the highest rebeccamycin concentration achieved in this cultivation approach and a 19-fold increase compared to an unsupplemented control which produced only 6 mg L −1 rebeccamycin. Supplementation of coarser glass particles led to smaller pellets of L. aerocolonigenes and lower rebeccamycin concentrations. The mechanical stress induced by the glass beads was considered to be responsible for this effect, with the proper dose of mechanical stress being an important factor (Walisko et al., 2017). The addition of glass macroparticles to cultivations of L. aerocolonigenes proved to be beneficial in regard to rebeccamycin formation in further studies. Schrader et al. (2019) investigated the influence of different particle diameters ranging from 0.2 to 2.1 mm on the final rebeccamycin concentration, causing differences in mechanical stress. In this case, the addition of glass beads with a mean diameter of 969 µm led to a higher rebeccamycin concentration of approximately 70 mg L −1 than the addition of smaller glass beads with a mean diameter of 540 µm (similar to the beads in Walisko et al. (2017)) with only 57 mg L −1 rebeccamycin. The different effects of glass bead diameters in these two studies arise from different shake flask geometries. In Schrinner et al. (2020) the differences between glass bead supplemented and unsupplemented cultivations over time were considered and additionally cellular morphological changes were investigated.
Macroparticle addition has already been used with other filamentous microorganisms (e.g., Dobson et al., 2008;Lee et al., 2010). Ochi (1986 used 3 mm glass beads for the homogenization of spores and biomass of Streptomyces sp. Sohoni et al. (2012) added glass beads of different diameters between 0.75 and 4 mm to cultivations of Streptomyces coelicolor in microtiter plates. In that study, the cell morphology of the microorganism varied with glass bead size. Until a glass bead diameter of 2 mm pelleted growth was observed, whereas larger glass particles induced mycelial growth. Cultivation of S. coelicolor with glass beads of 3 and 4 mm led to the most reproducible cellular morphology which was accompanied by an enhanced product concentration of actinorhodin and undecylprodigiosin (Sohoni et al., 2012). In the study of Dobson et al. (2008) even larger glass beads with a diameter of 5 mm were added to the main culture of S. hygroscopicus var. geldanus producing geldanamycin. With increasing glass bead number pellet size decreased and the geldanamycin concentration increased (Dobson et al., 2008). Holtmann et al. (2017) used glass beads with a size range of 250-500 µm in cultivations of S. avidinii for the production of streptavidin. The glass bead addition did not increase the final streptavidin concentration, but the production was accelerated. After 72 h of cultivation a 2.2-3.2-fold higher streptavidin concentration in the supplemented approaches compared with an unsupplemented cultivation was achieved, whereas after 120 h of cultivation the streptavidin concentration was similar for all approaches . Hotop et al. (1993) supplemented 4 mm glass beads in pre-cultures of Penicillium chrysogenum producing penicillin V to decrease the pellet size, causing an increased product titer.
In another study, 3 mm glass beads were added to cultivations of Acremonium chrysogenum to enhance cephalosporin C production (Lee et al., 2010). Different numbers of glass beads between two and six beads were added to the shaking flasks and a 30% increase in cephalosporin C production compared to an approach without glass beads was observed.
Since the mechanical stress induced by the glass beads in cultivations of L. aerocolonigenes was proposed to be responsible for the described enhanced rebeccamycin production in different cultivations, Walisko et al. (2017) made a rough estimation of the mechanical stress induced by glass beads based on a stress model for grinding in a ball mill (Kwade, 2003). In this model two characteristic values were determined, the stress energy (SE) (Equation (1)) and the stress frequency (SF) (Equation (2)), describing the comminution in stirred media mills. SE quantifies the maximum amount of energy transferred which is provided for stressing within a single stress event.
where d gm is the diameter of the grinding medium, u t is the stirrer tip speed, and ρ gm is the density of the grinding medium. The stress frequency SF describes the number of stress events (SN) per time.
Varying either SE or SF in a reasonable range, the mechanical stress induced by particles over time can be influenced, especially SCHRINNER ET AL.
| 3985 how many stress events take place to transfer a certain overall energy to a certain product mass. However, both process parameters can be varied to balance each other. An increase in SE by an increased bead density, for example, can be compensated by a decrease in SF caused by a reduced number of beads.
As stated above, this approach can only be used for a rough estimation since the conditions in ball mills and especially stirred media mills essentially differ from those in macroparticle-enhanced cultivation in a shake flask. For example, the degree of filling with beads as grinding media differs. In the case of the stirred media mill, the filling degree is usually 70%-85% of the grinding chamber (Kwade & Schwedes, 1997), corresponding to a volume concentration of 42%-51%, whereas in the shaking flask cultivations of this study volume concentrations of beads between 0.1% and 15% were used.
In this study, the stress energy of beads inside a shaking flask Furthermore, it is assumed in Equation (4) Previous studies of macroparticle addition to L. aerocolonigenes mostly investigated the effects of particle diameter variations (Schrader et al., 2019;Walisko et al., 2017). However, the mechanical stress induced by the macroparticles, that is, beads, cannot only be varied by their diameter. Further parameters, such as macroparticle concentration, macroparticle density, and shaking frequency of a shake flask cultivation, as implemented in Equations (1)-(4), also determine the magnitude of SE b,sh and SF b,sh .
Hence, the aim of the present investigations was the systematic variation of various process parameters such as the bead diameter, concentration and density, as well as the shaking frequency to examine effects of a variation in SE b,sh and SF b,sh . Moreover, the combination of macroparticle-enhanced cultivation with lecithin supplementation was investigated.

| Determination of dissolved oxygen tension
In some cultivation approaches the dissolved oxygen tension

| Rebeccamycin and cell dry weight quantification
The 20 ml of cultivation broth were used for rebeccamycin extraction by adding 5 ml ethyl acetate followed by incubation in an overhead shaker (Intelli-Mixer RM-2 M, LTF Labortechnik) for 60 min. The sample was then centrifuged at 4000 min −1 for 10 min (Heraeus Varifuge 3.0R, Thermo Fisher Scientific) and the ethyl acetate phase removed and used for analytic HPLC measurements as described in a previous study (Schrinner et al., 2020). The cell dry weight concentration (CDW) was determined gravimetrically by filtration through filter papers. Since the beads used in this study sedimented quickly, it was possible to take samples of the culture broth without transferring any beads.
The method has been described in more detail previously (Schrinner et al., 2020). For a better overview of the data in the graphs the averaged yield coefficient was calculated by dividing the rebeccamycin concentration by the cell dry weight concentration after ten days of cultivation.

| Macroparticles
Glass (type S and micro glass beads, soda-lime glass) and ceramic (type Z, zirconium silicate) beads as macroparticles were purchased from Sigmund Lindner GmbH. Details on the bead size and density are given in Table 1. For simplicity, the macroparticles will be referred to by the given mean particle size in the following. Beads were either supplemented in certain weight concentrations, which are indicated with the corresponding results, or at a defined bead number calculated from the bead density and size (Table 1).

| Lecithin supplementation
Soy lecithin was purchased from MP Biomedicals. A stock solution of 50 g L −1 lecithin was prepared and diluted with water to set the desired concentration in the shaking flask. 40 ml of a concentrated GYM medium with 20% less water were then added to result in a total filling volume of 50 ml. Inoculation and incubation were performed as described above.

| Variation of stress energy and stress frequency
In this study, shaker frequency, bead size, bead number and bead density were varied to systematically investigate the influence of different SE b,sh and SF b,sh on the production of rebeccamycin. An overview of all experimental parameter combinations together with the calculated values of SE b,sh (Equation (3)) and SF b,sh (Equation (4)) is given in Tables 2a and 2b. For example, SE b,sh and SF b,sh were calculated for different glass bead diameters in a concentration of 100 g L −1 at different shaking frequencies of 100 and 160 min −1 (Table 2a). With increasing glass bead diameter SE b,sh increases while SF b,sh decreases. These parameters were furthermore calculated for a constant glass bead number (Table 2a) as well as different concentrations of glass and ceramic beads (Table 2b). The parameters from this table will be discussed in more detail with the results of the experimental approaches below. The number of particles required for the calculation of SF b,sh was derived from the mass concentration of the beads, the liquid volume, and the mean bead size x ̅ 3.2 .
These characteristic numbers can currently only provide a first qualitative comparison between different cultivations, since the exact dependencies of the characteristic numbers from experimental parameters are not known yet. It should be noted that the modeled SE b,sh are a measure for the maximal value of SE as it was also shown to be very useful in comminution processes (Beinert et al., 2015;Kwade, 2003). In reality, SE b,sh is determined by the distribution of the relative velocities between two colliding beads or a bead and the shaking flask wall. Therefore, the distribution of SE, as well as the T A B L E 1 Particle size range as given by the manufacturer and mean particle size x ̅ 3,2 (calculation see Schrader et al., 2019) of supplemented glass and ceramic beads as well as the calculated mass and volume concentration for 4200 beads Particle size range (µm) Mean particle size x ̅ 3,2 (µm) Shaking frequency (min −1 ) 100 120 160 Bead concentration (g L −1 ) 100 Varying (see Table 1) 100 and 150 g L −1 corresponding to volume concentrations between 10 and 60 ml L −1 , respectively (see Table 2b), since this glass bead diameter generally led to a high rebeccamycin concentration in earlier studies under the same conditions (Schrader et al., 2019;Schrinner et al., 2020).
The resulting rebeccamycin and CDW concentrations are presented in production (Dobson et al., 2008). In this case, the highest number of 55 glass beads with a diameter of 5 mm led to the highest geldanamycin concentration. The course was similar to the results in the present study, with increasing particle concentration or number, the product concentration increases. Only after a certain point, which in this case was a concentration of 125 g L −1 , the product concentration was decreasing. However, larger amounts than 55 glass beads to examine for further production increases were not tested by Dobson et al. (2008 Table 1). An unsupplemented control cultivation (mean glass bead diameter of 0 µm) was added for comparison. The CDW did not show a distinct trend and mostly fluctuated around 3.5 g L −1 . However, the rebeccamycin concentration increased up to a mean glass bead diameter of 969 µm and decreased afterward. This can also be observed for the averaged yield coeffi-

| Variation of macroparticle density
In a further approach, the particle density was changed by adding ceramic beads (ρ = 3800 kg m −3 ) with an approximately 1.5-fold higher density (Table 1)  with 56 mg L −1 rebeccamycin and was decreasing for higher ceramic bead concentrations. For the addition of similarly sized glass beads, a concentration of 100 g L −1 indicated to be most beneficial in regard to rebeccamycin titer. For ceramic beads the optimal mass concentration of particles with a similar size was only half the value.
Considering volume concentrations, the differences are even larger with 40.0 ml L −1 for glass beads and only 13.2 ml L −1 for ceramic beads (Table 2b). Therefore, if the particle density is changed and the mass concentration is constant, the particle number also differs. This means both SE b,sh and SF b,sh are influenced. For the same particle number of ceramic beads which are included in 100 g L −1 glass beads, a concentration of approximately 130 g L −1 would be required (due to the slightly smaller mean diameter of the ceramic beads it is not the 1.5-fold mass concentration). Hence, increased SE b,sh by an increased particle density can be partly compensated by the reduction of another parameter, for example, the particle number, for maximizing rebeccamycin titers at this SE b,sh . However, the difference between SE b,sh (Table 2b) for glass beads with 0.594 µJ and for F I G U R E 2 Cell dry weight concentration, rebeccamycin concentration, and averaged yield coefficient of a 10-day cultivation with addition of different glass bead sizes with the same glass bead number of 4200 F I G U R E 3 Cell dry weight concentration, rebeccamycin concentration, and averaged yield coefficient of a 10-day cultivation with addition of different concentrations of 918 µm ceramic beads SCHRINNER ET AL.
| 3989 ceramic beads with 0.767 µJ seems not high enough to create this kind of compensation. In this case, the use of a rather simple approach of the comparison of SE b,sh is not sufficient to describe the differences of glass and ceramic beads in cultivation as probably the motion behavior of the beads depends on their density. Here, CFD-DEM simulations could shed light on the underlying differences during cultivation, as these take into account the increased centrifugal forces at higher particle densities.

| Variation of shaking frequency
Another possibility for SE b,sh variation is the cultivation at different shaking frequencies. In cultivations of L. aerocolonigenes in shake flasks presented above, a shaking frequency of 120 min −1 was used.
To investigate the influence of the shaking frequency with glass bead addition on the rebeccamycin production, cultivations with different shaking frequencies of 100 and 160 min −1 were performed. The With a shaking frequency of 160 min −1 (Figure 4b), SE b,sh and SF b,sh is increased compared with conventional cultivations at 120 min −1 (Table 2a). Concluding from the cultivation at 100 min −1 , a shift to a smaller bead diameter than 969 µm would be expected.
However, due to large standard deviations at smaller mean dia- F I G U R E 4 Cell dry weight concentration, rebeccamycin concentration and averaged yield coefficient of a 10-day cultivation with addition of different glass bead diameters with a concentration of 100 g L −1 at different shaking frequencies of (a) 100 min −1 and (b) 160 min −1 . For the frequencies 100, 120, and 160 min −1 (c) the dissolved oxygen tension during cultivation is displayed results indicate that SE b,sh for these glass bead sizes is too high. SE b,sh at 120 min −1 of 1932 µm glass beads is 4.704 µJ while for the same glass beads at 160 min −1 SE b,sh is almost doubled with 8.363 µJ (Table 2a). This could result in cell destruction or even growth inhibition, which in turn reduces production. Since additionally to the application of large glass beads the shaking frequency was increased, both SE b,sh and SF b,sh were increased and, thus, were higher compared with a regular cultivation at 120 min −1 .
The DOT during cultivation differed for the different shaking frequencies (Figure 4c). The DOT for 100, 120, and 160 min −1 is displayed over a cultivation time of 10 days. The minimal DOT was 75% for 160 min −1 , 55% for 120 min −1 , and 43% for 100 min −1 . No oxygen limitations were observed suggesting that the mechanical stress induced by different shaking frequencies is a main reason for the differences in the cultivations described above.
Overall, the CDW for small and medium bead size measurements is rather similar for different SE b,sh and SF b,sh . However, for the three coarser bead sizes and, thus, highest SE b,sh and an increased SF b,sh compared with a lower shaking frequency of 100 min −1 a decreased CDW was observed. Cell destruction or growth inhibition could be a reason for this effect. The mostly similar CDW in the approaches supports the idea that the effects of glass beads are not biomass-related, as already indicated by Schrinner et al. (2020).
Further aspects such as changes in micro-morphology or the inner pellet structure could be of interest in this case (Schrinner et al., 2020). Furthermore, in different Streptomyces spp. a first mycelium is observed, then programmed cell death takes place followed by the differentiation to another secondary metabolite producing mycelium (Manteca & Yagüe, 2018;Manteca et al., 2019). This differentiation could also apply for L. aerocolonigenes and since it is initiated by programmed cell death, the addition of macroparticles would benefit this process.

| Soy lecithin supplementation with L. aerocolonigenes
The addition of lecithin to cultivations of different actinomycetes isolated from soil was beneficial in previous studies regarding product formation (Adelson et al., 1957;Brock, 1956;Choi & Cho, 2004;Schatz et al., 1956). Due to the positive effects for similar microorganisms as L. aerocolonigenes lecithin supplemented cultivations were performed.
The addition of soy lecithin to the cultivation of L. aerocolonigenes was first conducted in different concentrations between 0 and 10 g L −1 (Figure 5). This provides an overview of whether lecithin is beneficial and how much lecithin needs to be reasonably added for further cultivations that are described below.
The CDW increased with increasing lecithin concentration. The addition of 2.5 g L −1 lecithin nearly doubled the biomass compared to an unsupplemented control, 10 g L −1 lecithin even resulted in an almost three-fold CDW. The rebeccamycin titer, however, increased until 7.5 g L −1 lecithin (with about 103 mg L −1 rebeccamycin) and is significantly lower for 10 g L −1 lecithin. The rebeccamycin concentration of 18 mg L −1 for the approach with the addition of 10 g L −1 lecithin was similar to the rebeccamycin concentration in the unsupplemented control with 15 mg L −1 rebeccamycin. This course can also be observed for the averaged yield coefficient. The increasing CDW indicates the metabolization of lecithin as an additional carbon source leading to enhanced growth. Higher CDWs can produce higher amounts of rebeccamycin, explaining the increasing rebeccamycin titer with increasing lecithin concentration. The decreased rebeccamycin titer for 10 g L −1 lecithin might be explained with a prolonged exponential growth due to larger amounts of substrate. Since rebeccamycin is a secondary metabolite and is only produced after substrate deprivation, this could lead to a delayed start of rebeccamycin production. To further investigate this hypothesis, the growth kinetics of cultivation without and with 5 g L −1 soy lecithin were compared ( Figure 6). Additionally, 100 g L −1 of 969 µm glass beads were supplemented in both approaches.
Both approaches showed a similar course of CDW, but the lecithin supplementation resulted in higher overall concentrations ( Figure 6a). The glucose consumption was slower for the lecithin supplemented approach. The glucose was fully depleted after 3 days, whereas without lecithin the glucose was nearly completely  These results support the hypothesis of prolonged exponential growth of L. aerocolonigenes with lecithin addition. Since more oxygen is consumed by the microorganism in the exponential growth phase the DOT significantly decreases in this time period.
The DOT increases after this phase since the growth is slowed down. Hence, a faster increase of the DOT can indicate a rather abrupt growth slowdown. The specific rebeccamycin productivity q P (Figure 6c) was additionally calculated. Without lecithin addition the maximum specific productivity was reached on Day 5 with around 6.1 mg g −1 d −1 . With lecithin addition the specific productivity was highest on Day 6 with around 10.5 mg g −1 d −1 . Here again a delay in the rebeccamycin production with lecithin addition compared with an unsupplemented approach was observed, but also a higher maximum specific productivity. Brock (1956) investigated the influence of different oils and fatty acids on the filipin production in S. filipinensis. Among others, the effect of the addition of a vegetable and animal-based lecithin was investigated. Both supplementations led to an increased product concentration during cultivation compared to the cultivation approach with glucose added as a carbon source. The vegetable lecithin even led to a two-fold higher filipin concentration than the animal lecithin. The lecithin, which was chosen in the current study to increase rebeccamycin production was derived from soybean and might therefore potentially lead to higher product titers than animal lecithin. Lam et al. (1989) investigated different carbon sources for L. aerocolonigenes and found starch, which is also a plant-based compound, to be most beneficial in regard to rebeccamycin production. Choi and Cho (2004) added lecithin to cultivations of S. lincolnensis and thereby increased lincomycin production. Adelson et al. (1957) used lecithin in cultivations of a soil actinomycete as it provided fast and abundant growth accompanied by increased oxygen uptake, as also observed for L. aerocolonigenes in this study.

| Combination of soy lecithin supplementation with macroparticle addition
In Figure 5 the addition of 5 and 7.5 g L −1 of lecithin led to around 100 mg L −1 of rebeccamycin, which is a rather high product titer compared with literature data with mostly 10-50 mg/L (see Pommerehne et al. (2019)). Since lecithin acts as a suitable additional carbon source, the combination of lecithin supplementation and mechanical stress caused by glass beads could lead to even higher rebeccamycin titers as both methods employ different mode of actions for an increased rebeccamycin production. Glass beads with a diameter of 969 µm were already added in the cultivation approach for which the results are shown in Figure 6. In that case, the final rebeccamycin concentration with 5 g L −1 lecithin and glass bead supplementation was even higher with 259 mg L −1 . However, since both experiments are different biological approaches started from different pre-cultures, a direct comparison is not entirely reasonable.
Therefore, a direct comparison with and without glass beads and different concentrations of lecithin starting from the same preculture was conducted for reliable results (Figure 7). The CDW increased with increasing lecithin concentration. For the addition of 5 g L −1 of lecithin, the CDW of the cultivation with glass beads is lower than of the cultivation without glass beads, whereas for 10 g L −1 lecithin a similar level was observed. The rebeccamycin concentration for the cultivation with 5 g L −1 lecithin and no glass beads was around 220 mg L −1 and was therefore significantly higher F I G U R E 6 (a) Cell dry weight concentration, (b) glucose and rebeccamycin concentrations as well as (c) dissolved oxygen tension and specific rebeccamycin productivity of a 10-day cultivation without and with 5 g L −1 soy lecithin. 100 g L −1 glass beads with a mean diameter of 969 µm were added in both approaches lower for both approaches. Furthermore, the averaged yield coefficient is higher for both approaches with the combination of lecithin and glass beads compared to approaches in which only lecithin was supplemented. This was likely the effect of a delayed start of the rebeccamycin production due to larger amounts of substrate, as it was suggested by the results shown in Figure 6. However, further effects can not be completely excluded by the presented results at this point.
In this approach, the favorable substrate soy lecithin was combined with glass macroparticle addition and resulted in an extremely high rebeccamycin concentration. The highest rebeccamycin titer in shaking flasks achieved in literature was 183 mg L −1 (Nettleton, Jr. et al., 1985;Pommerehne et al., 2019). The given 388 mg L −1 rebeccamycin by addition of lecithin and glass particles is more than a two-fold increase compared to this literature value.
Looking at the microscopic images of L. aerocolonigenes pellets in Figure 8 some differences between the above-described approaches become apparent. Without glass bead addition, pellets from the unsupplemented cultivation and the cultivation with 5 g L −1 lecithin ( Figure 8a,b) looked similar. However, the combination of 100 g L −1 glass beads and 5 g L −1 soy lecithin resulted in pellets that, in some cases, appeared less dense. This can be observed by a lighter color in the microscopic images (Figure 8c). At higher magnification ( Figure 8d) a dense core and a less dense peripheral area of these pellets were visible. These pellets suggest changes in the micromorphology or the inner pellet structure that might be connected to the significantly increased rebeccamycin formation.

| CONCLUSIONS AND FUTURE PERSPECTIVES
The addition of macroparticles (e.g., glass beads) to cultivations of the filamentous growing actinomycete L. aerocolonigenes can significantly enhance the rebeccamycin concentration. Walisko et al. (2017) proposed the mechanical stress induced on the microorganism by the beads to be responsible for the increased product concentration. Hence, this study was performed to be a systematic investigation of different parameters for macroparticle addition, such as bead diameter, concentration, and density as well as shaking frequency, for a better understanding of the particle-induced stress on L. aerocolonigenes. The proper level of particle-induced stress for optimal rebeccamycin production can be estimated by a combination of stress energy and stress frequency. Different combinations of these parameters are possible to achieve high a rebeccamycin concentration during cultivation. Regarding different concentrations of