Unraveling the potential and constraints associated with corn steep liquor as a nutrient source for industrial fermentations

Costly complex media components such as yeast extract and peptone are still widely used in industrial bioprocesses, despite their ill‐defined composition. Side stream products such as corn steep liquor (CSL) present a compelling economical alternative that contains valuable nutrients required for microbial growth, that is, nitrogen and amino acids, but also vitamins, trace elements, and other minerals. However, as a side stream product, CSL may be subject to batch‐to‐batch variations and compositional heterogeneity. In this study, the Respiration Activity MOnitoring System designed for shake flasks (RAMOS) and 96‐well microtiter plates (μTOM) were applied to investigate the potential and constraints of CSL utilization for two model microorganisms: E. coli and B. subtilis. Considering the dry substance content of complex nutrients involved, CSL‐based media are more efficient in biomass production than the common lysogeny broth (LB) medium, containing 5 g/L yeast extract, 10 g/L peptone, and 5 g/L NaCl. At a glucose to CSL (glucose/CSL, g/g) ratio of 1/1 (g/g) and 2/1 (g/g), a secondary substrate limitation occurred in E. coli and B. subtilis cultivations, respectively. The study sheds light on differences in the metabolic activity of the two applied model organisms between varying CSL batches, which relate to CSL origin and production process, as well as the effect of targeted nutrient supplementation. Through a targeted nutrient supplementation, the most limiting component of the CSL‐glucose medium used for these applied model microorganisms was identified to be ammonium nitrogen. This study proves the suitability of CSL as an alternative nutrient source for E. coli and B. subtilis. The RAMOS and μTOM technique detected differences between CSL batches, allowing easy and early identification of varying batches. A consistent performance of the CSL batches in E. coli and B. subtilis cultivations was demonstrated.


| INTRODUCTION
Choosing the ideal medium for a specific fermentation remains an essential and cost-driving challenge in industrial biotechnological processes. 1,2In addition to macromolecules, such as carbohydrate, phosphorous, and nitrogen, many microorganisms require a plethora of trace elements, vitamins, and amino acids to guarantee optimal growth and production conditions.Despite their ill-defined chemical composition, complex media components are still widely used in industrial fermentation processes, because of their vast nutritional content. 3,4][7][8][9][10] Yeast extract and peptone serve as the basis for both media, while the LB medium additionally contains sodium chloride, and the TB medium is enriched with glycerol and phosphate buffer. 5,6However, the high cost of yeast extract and peptone presents an economic constraint for the industrial large-scale use of complex media. 11Thus, replacing conventional carbon sources with cheaper substrates such as molasses 11 or cheese whey 12 has been in focus in recent years.Less attention has been directed toward replacing other macro-and micronutrients, such as phosphorus, nitrogen, and various metals.
Corn steep liquor (CSL) is an example of a low-cost alternative substrate.During the wet milling process in the corn starch industry, corn kernels undergo a steeping process.By steeping or soaking the corn kernel under a controlled aqueous environment, it is softened and broken down into its constituents, facilitating further milling. 13The remaining steeping water is concentrated by evaporation to a solids concentration of 45%-55%. 14A by-product of the corn steeping process, the concentrated CSL is rich in amino acids, minerals, vitamins, reducing sugars, organic acids, enzymes, and other elemental nutrients. 15Due to its high nutritional content and low cost, CSL has the potential to replace established complex media components. 16The first industrially important application of CSL was reported in 1946 by Moyer and Coghill for penicillin production. 17Nowadays, CSL is a vital organic nitrogen source for various industrial biotechnological processes, such as the production of rhamnolipids, 18 solvents, antibiotics and enzymes. 19However, as known for other complex substrates, a major drawback in using CSL lies in its batch-to-batch variability, due to differences in the steeping process, corn origin, and other seasonal effects.This batch-to-batch variability often leads to inconsistent fermentation performances, when using complex compounds as a substrate for biotechnological processes.For yeast extract, Potvin et al. 20 claimed that different batches from an identical manufacturing process could vary in biomass formation and growth rate by almost 50%.The influence of different batches of yeast extract on final product quality and quantity was also evaluated by Diederichs et al. 21with the help of the respiration activity monitoring system (RAMOS) technology.[24] Consequently, elaborate and repeated screenings of such complex compounds are required to detect the best performing batches for industrial fermentations.A challenge remains in identifying the key compounds, which might dramatically influence the fermentation process.Several screening systems for yeast extract have been previously reported, including a microtiter plate-based automated turbidometry system by Potvin et al. 20 A near-infrared spectroscopy characterization, to correlate fermentation yield with yeast extract composition, was reported by Kasprow et al., 25 while Zhang et al. 4 showed the reliability of fermentation performance predictions based on chemical/biochemical analysis as a screening tool for different yeast extract batches.
To the best of our knowledge, no approach toward performance consistency of side stream products such as CSL has been reported.Moreover, many of the previously mentioned techniques do not allow for high throughput screening, as only single measuring points obtained by manual sampling are generated.The microtiter platebased turbidometry system reported by Potvin et al. 20 enables high throughput, but is met with several major drawbacks associated with common microplate readers.As common microplate readers are not designed for cultivating microorganisms, they lack the appropriate power input for optimal microbial growth and are prone to evaporation effects.Furthermore, the absorbance measurement method in microplate readers requires regular interruption of the shaking movement of the device, leading to hampered growth and metabolism by oxygen limitation. 26,27 this study, to evaluate CSL performance at a higher throughput, the newly developed RAMOS device designed for 96-well microtiter plates (micro(μ)-scale Transfer rate Online Measurement device, μTOM) was applied in addition to the RAMOS shake flask cultivations. 28As the OTR gives valuable insights into the metabolic state of microbial cultivations, 29,30 nutritional requirements and auxotrophies of microorganisms can also be determined, as has been proven for Bacillus pumilus and Sporosarcina pasteurii. 31,32Thus, differences in metabolic activities of varying CSL batches can be detected by means of the RAMOS technology.
This study aims at establishing an efficient CSL screening method by online monitoring of the oxygen transfer rate (OTR).To demonstrate the suitability of CSL as a cost-effective yet efficient nutrient source, the metabolic activity of the two microbial model systems (E. coli and B. subtilis) on carbon-enriched CSL-based media is compared to a common standard complex medium, lysogeny broth (LB), which is equally enriched with carbon.In addition to OTR, important parameters such as pH and OD 600 were evaluated.By introducing secondary substrate-limited conditions (addition of a limited amount of CSL), the influence of batch-to-batch variations of CSL on the microbial systems is investigated, as these batches vary with respect to nutrients other

| Media and solutions
If not otherwise stated, all components were purchased from Carl Roth GmbH & Co. KG.
In this work, lysogeny broth (LB) [5][6][7][8] with 5 g/L yeast extract, 10 g/L peptone, and 5 g/L sodium chloride was used for pre-culture of Escherichia coli and Bacillus subtilis.For some main culture cultivations, both pure LB medium and LB with additional glucose were used.LB medium with glucose consists of 5 g/L yeast extract, 10 g/L peptone, In subsequent investigations, only the dry salt portion of MgSO 4 will be considered, while the crystal water content is deducted from the calculation.A 0.2 μm cutoff filter (VWR International, 0.2 μm PES membrane) was used for all sterile filtrations.

| Microorganisms
This study utilized Escherichia coli BL21 (DE3) pRhotHi-2-EcFbFP and Bacillus subtilis 168 (wt) as model organisms.The Escherichia coli BL21 (DE3) pRhotHi-2-EcFbFP strain contains a lac operon-based T7 promoter encoding a flavin mononucleotide-binding fluorescent protein (EcFbFP). 36,37The production of the encoding recombinant protein required induction by IPTG addition.As IPTG was never added, the production of said recombinant protein was not part of this investigation.

| Cultivation procedure
To establish constant cultivation conditions (temperature and humidity) and ensure required shaking conditions of the cultivation systems, all cultivation experiments were conducted in an incubator shaker (ISF1-X, Kuhner Shaker GmbH Herzogenrath).All E. coli and B. subtilis cultivations were performed at 37 C.All cultivations in the micro(μ)-scale Transfer rate Online Measurement device (μTOM) for 96-deepwell microtiter plates were additionally equipped with constant humidity control at 80% to minimize evaporation of the culture broth.
Cells were harvested during the late exponential growth phase and were stored in a 50% (v/v) glycerol/cell suspension ratio of 3/7 (v/v) in 2 mL vials at À80 C. Pre-cultures were inoculated from a cryo-stock and grown on LB medium.After inoculation, the pre-cultures were monitored in shake flasks via the in-house developed respiration activity monitoring system (RAMOS) device.Upon reaching an oxygen transfer rate (OTR) of >20 mmol/L/h during the late exponential growth phase, cells were harvested, washed, and the pellet was resuspended in fresh main culture medium, to inoculate the main culture at a specific initial cell concentration.As a standard method for approximating the number of cells in a liquid sample, 38,39 the optical density (OD) was measured at a wavelength of 600 nm (OD 600 ).Thus, main cultures were inoculated to an initial OD 600 of 0.1.
All pre-cultures and shake flask cultivations were performed in 250 mL shake flasks at 37 C with a shaking diameter of 50 mm, a shaking frequency of 350 rpm, and a filling volume of 10 mL.In addition, microtiter plate cultivations were carried out in 96-round well microtiter plates (850301, HJ-BIOANALYTIK GmbH) at 37 C with a shaking diameter of 50 mm, a shaking frequency of 350 rpm, and a filling volume of 200 μL-600 μL, depending on the experiment.

| Online measurements of oxygen transfer rate (OTR)
The respiration activity was monitored in all cultivation experiments by applying the respiration activity monitoring system (RAMOS). 22gure S1 shows the RAMOS technology for shake flasks and 96-deepwell microtiter plates.Within the RAMOS device, a specific measuring cycle is automatically repeated, consisting of a flow phase and a stop phase.During the flow phase, the RAMOS flask is flushed with air, while air flow is interrupted by shutting the valves once the stop phase is initiated.The head space of the RAMOS flask is sealed off in the stop phase.By measuring the corresponding drop of the oxygen partial pressure, determined by an oxygen sensor, the OTR is calculated. 22By integrating the measured OTR values over time, the total oxygen (TO) is calculated.
All pre-cultures and shake flask main cultivations were conducted in modified 250 mL shake flasks.The newly developed μTOM device monitors respiration activity in microtiter plates.The μTOM device is able to measure respiration activity in each individual well of a 96-well microtiter plate, following the same principle as described above for the RAMOS device.Detailed information can be obtained from Dinger et al. 28

| pH
The pH value was measured with an InLab Easy pH electrode (Mettler Toledo) with a Cyber-Scan pH 510 meter device (Eutech Instruments, Thermo Scientific).

| Optical density (OD 600 )
The optical density was measured at a wavelength of 600 nm (OD 600 ) 38,39 using a photometer (Genesys 20, Thermo Scientific).To maintain the linear range of OD 600 measurements in the photometer, samples were diluted with 0.9% NaCl to reach values between 0.1 and 0.3.Because fresh CSL medium is already turbid and has an initial optical density, the initial optical density of each CSL-based medium was subtracted from the measured final OD 600 values.

| Glucose, lactate, and glycerol
Measurements of the carbon sources glucose, lactate, and glycerol were performed by high-pressure liquid chromatography (HPLC) (Ultimate, Dionex) using an organic acid resin column (300 Â 7. 8  served as an eluent at a flow rate of 0.5 mL/min and 60 C.

| Primary amino acids
Primary amino acids were derivatized with diethyl ethoxymethylenemalonate (DEEMM) and separated by reversed-phase HPLC using a C18 column (Waters Acquity UHPLC Class H, Waters Chromatography Europe B.V.).The analytes were detected by UV absorbance (Thermo Scientific Ultimate 300 RS Diode Array Detector, Thermo Fisher Scientific Inc.) at 282 nm and quantified by comparing the peak area of each analyte with the corresponding standard.

| Total phosphorus
A sample (0.3-0.4 g) was digested with 6 mL of a 6 M HCl solution and 12 mL of a 8 M HNO 3 solution in 50 mL DigiTubes (SCP Science Ltd.), using a DigiPREP block heater (SCP Science Ltd.).During digestion, the sample was warmed up to 100 C at 25 C/h, followed by a 15 min holding time at 100 C. Upon sample digestion, total phosphorus content was analyzed by inductively coupled plasma optical emission spectrophotometer using Optima 2000-DV ICP-OES (PerkinElmer Inc.).

| Total nitrogen
Total nitrogen content was analyzed by the Dumas method using the TruMac C/N analyzer FP628 (LECO Corp.).S1.For all employed media, an initial steep increase of the OTR is observed.When grown in LB medium, E. coli showed increasing respiratory activity over 3 h, reaching an OTR of 30 mmol/L/h (Figure 1a).A total of 81 mmol/L oxygen was consumed during growth on LB medium (Figure 1b).As previously described by Losen et al., 6 growth on complex components found in LB medium result in the release of ammonium ions, thus, increasing the final pH from 7.5 to 8.8.LB medium without any additional carbon source led to a final optical density (OD 600 ) of 3.9 (Figure 1b).
An increase of the maximum OTR to 43 mmol/L/h was observed, when supplementing the LB medium with glucose without any additional buffer.This is contrary to results from Losen et al., 6 where a rapid pH drop was observed in the non-buffered LB-glucose medium, resulting in a low respiratory activity.1][42] As only 5 g/L glucose, thus, half of the glucose concentration employed by Losen et al. 6 was utilized in this study, overflow metabolism was less severe in this case and growth was not hampered.A final pH value of 7.9 was reached (Figure 1b).The additional glucose doubled the total oxygen (TO) consumption to 177 mmol/L, compared to growth on the standard LB medium.The final OD 600 was increased by 25%, compared to standard LB, as a result of the additional carbon source (Figure 1b).
Cultivating E. coli on pure CSL as the sole nutrient source, containing only MOPS buffer as an additive, resulted in a decreased growth rate and a stunted maximum OTR of 17 mmol/L/h (Figure 1a).
The low amount of utilizable carbon source in pure CSL led to a low TO of 32.4 mmol/L (Figure 1b).The resulting low respiratory activity is in line with a low OD 600 of 2.1.Because CSL is a complex nutrient source consisting of many components, such as lactate and reducing sugars, 14,43 it is assumed that lactate and reducing sugars are first consumed, followed by the metabolism of proteins and amino acids.
Contrary to expectations, the final pH value was slightly decreased to 7.2.Higher pH values would have been expected with the consumption of lactate and proteins and amino acids. 44,45 supplementing the CSL medium with glucose, the maximum OTR rose to 19.5 mmol/L/h (Figure 1a).Following the peak, an OTR "plateau" was visible at about 13 mmol/L/h.This OTR behavior indicates a secondary substrate limitation. 22After 12 h, glucose as the primary carbon source is exhausted, leading to an OTR drop.Compared to the pure CSL medium, which resulted in a TO of 32.4 mmol/ L, the additional glucose increased the TO by 3.9-fold, which amounted to 125 mmol/L.Furthermore, a final OD 600 of 2.8 was obtained, in contrast to a final OD 600 of 2.1 obtained on pure CSL medium.This behavior may indicate that growth of E. coli (OD 600 of 2.1), and thus, the resulting TO on the pure CSL medium (32.4 mmol/ L) was limited due to a non-balanced availability of different nutrients.
With the addition of glucose, nutrients contained in the CSL can be further utilized for growth until they become limiting.Furthermore, in contrast to the cultivation on pure CSL medium, the pH was significantly decreased to 6.7 (Figure 1b).The decrease in pH may be explained by the production of overflow metabolites, caused by a high glucose concentration relative to the limiting nutrient. 40Together with the 30% higher biomass, this metabolites production explains the significantly increased total oxygen (TO) observed in those conditions.
As lactate is the most abundant carbohydrate in CSL, 46,47 E. coli was grown on a CSL-lactate medium to further investigate the bacterium's respiratory and metabolic activity.As shown in Figure 1a, growth of E. coli on the CSL-lactate medium resulted in a maximum OTR of 28 mmol/L/h, higher than observed for the CSL-glucose medium (19.5 mmol/L/h).Similar to the respiratory activity shown by the cultivation on CSL-glucose medium, a secondary substrate-limited plateau is indicated by E. coli grown on CSL-lactate medium, followed by an OTR decrease.Despite the different maximum OTR, a similar TO was observed for both enriched CSL media (125 mmol/L for CSL-glucose and 124 mmol/L for CSL-lactate), as an identical amount of carbon source (5 g/L glucose vs. 5 g/L lactate) was employed for both media, respectively.Furthermore, while lactate-enriched CSL medium resulted in a higher maximum OTR (28 mmol/L/h) than glucose-enriched CSL medium (19.5 mmol/L/h), a similar final OD 600 of 2.9 was reached on the CSL-lactate medium.Thus, the maximum OTR is not a measure of the biomass formed.Similar to glucose, additional lactate resulted in a higher nutrient utilization from the CSL.
Due to lactate consumption and, thus, acid intake, counter ions are released, 44 resulting in an increased final pH of 7.9 (Figure 1b).
As the different LB media contain 5 g/L yeast extract and 10 g/L peptone, the amount of complex media components in LB is thrice higher than in the CSL media, the latter employing only 5 g d.s./L CSL.Thus, considering the dry substance content of the complex media components engaged, the CSL-glucose medium offers more efficiency toward biomass production than the LB-glucose medium.

| Influence of CSL batch-to-batch variations on Escherichia coli
As previously mentioned, CSL is a side-stream product, [48][49][50] and as such may suffer from batch-to-batch variations.This phenomenon was further investigated by employing nine different CSL batches.For this, 5 g d.s./L of different CSL batches were each enriched with 5 g/L glucose and 100 mM MOPS.The secondary substrate limitation depicted by E. coli on the CSL-glucose medium (Figure 1a) may also showcase differences in the CSL batches, as varying chemical compositions of the batches are expected to have an impact on E. coli's metabolic activity.These experiments were conducted in 96-deepwell microtiter plates to enable higher experimental throughput.Online monitoring of E. coli on the CSL-glucose medium measured by the well-established RAMOS device was compared to the results obtained by the μTOM device (Figure S2).As already shown by Dinger et al., 28 cultivations in both scales were similar.S2). Figure 2b shows variant 6, which comes from the same production facility as the variants 4 and 5, but was manufactured using an improved process.The CSL variant 1 employed in this experiment (Figure 2a) is identical to the CSL variant in the previous experiment (Figure 1a), as presented in Table S2.As observed in the previous cultivation on the CSL-glucose medium in shake flasks, all nine CSL variants result in a clear peak during the first 3 h, followed by an OTR plateau, which indicates a secondary substrate limitation. 22ntrary to expectations, the differences in the course of the OTR over time of the varying CSL batches are obviously not very large.These findings are not in accordance with results from Diederichs et al., 21 where different yeast extract batches of different and identical suppliers led to considerable differences in respiration behavior of a recombinant protein-producing E. coli strain.In fact, Figure 2a-c show all nine variants leading to an equivalent rate of OTR increase.Moreover, the maximum OTR differs slightly between the variants, which may be explained by the varying concentrations of other nutrients besides glucose in the variants, as reported by Diederichs et al. 21Strikingly, variant 6 shows a higher maximum OTR of 26 mmol/L/h, compared to the other variants.
Furthermore, a higher level of the characteristic OTR plateau is displayed by variants 6-9.Since glucose concentration is identical at 5 g/L for all variants, other nutrients present in the CSL variants must be responsible for the difference in maximum OTR and plateau.As can be seen in Figures S3 and S5, respectively, the CSL variants showed a diversity in lactic acid content, as well as distinct free primary amino acid profile.The difference in the quality of material from the different production facility location, as well as an improvement of the production process, must have greatly influenced the nutritional composition of the CSLs and, thus, result in different OTR profiles.
The similarity in OTR pattern of variants 1-3 (Figure 2a) led to a similar total oxygen (TO), as illustrated in Figure 2d.Moreover, this figure shows that these variants also resulted in similar final optical densities (OD 600 ), giving raise to the assumption that CSL variants 1-3 are highly similar in their compositional make up, as they originate from one production location (Table S2).The same phenomenon can be observed in variants 4 and 5, showing similar values of TO and corresponding OD 600 (Figure 2d).In contrast to variants 4 and 5, variant 6 showed higher TO and OD 600 values, which are attributed to the different OTR pattern shown by variant 6, which is produced at the same location, but underwent an improved production process.
Furthermore, the similarity of OTR patterns depicted by variants 7-9 (Figure 2c) resulted in a similar TO (Figure 2d).However, Figure 2d shows differences in the final OD 600 between these three variants, ranging between 2.8 and 3.3.In line with the higher TO calculated for variant 6 (Figure 2b), variant 6 also resulted in an increased OD 600 at 3.6 (Figure 2d).Compared to the average of OD 600 for all nine variants at 3.18, the OD 600 obtained with variant 6 is increased by 14%.

Klotz et al. concluded in their study that the productivity of d-lactic
acid production by Sporolactobacillus inulinus was highest when employing yeast extract instead of peptone, due to a higher amount of free amino acids and small peptides in yeast extract. 51As depicted in Figures S5 and S6, a higher concentration of free amino acids for variant 6 has been detected.Variant 6 showed the highest concentration of the free amino acids alanine, glycine, leucine, phenylalanine, methionine, and valine (Figures S5 & S6).These findings are in agreement with those obtained by Klotz et al., 51 as free amino acids and small peptides can easier be transported into the cell than large proteins, 51 resulting in variant 6 showing a higher final OD 600 .
In general, a similar TO is expected, as the same glucose concentration was applied for all nine CSL variants.However, the difference in TO observed in the nine CSL variants corroborates the different WAHJUDI ET AL. concentrations of lactate and acetate (Figure S4), as well as the distinct free primary amino acid profiles exhibited by the CSL variants.For all CSL variants, the final pH lies around 7, as the released overflow metabolites caused acidification of the cultivation broth.Cultivating E. coli under the employed glucose/CSL ratio of 1/1 (g/g) resulted in a secondary substrate limitation for all CSL batches.For example, Potvin et al. detected growth differences in Lactobacillus plantarum, when different yeast extract batches were applied. 20Zhang et al. also observed a similar effect, when employing varying yeast extract lots as medium components for a recombinant yeast fermentation, 4 while Klotz et al.
have reported that productivities may vary with different complex nutrient sources of distinct manufacturers. 51In fact, Diederichs et al.
highlighted that distinct batch-to-batch variations in yeast extract of the same supplier can be detected by the RAMOS technique. 21

| Ammonium sulfate supplementation removes secondary substrate limitation in Escherichia coli
The results presented above suggest that growth of E. coli is limited in the employed glucose-enriched CSL medium containing 5 g d.s./L CSL and 5 g/L glucose.Therefore, to determine the cause of the nutrient limitation, a selective nutrient supplementation was conducted.
As described in the literature, the elemental composition of microorganisms can serve as an indicator of their nutritional requirements. 52In their study, Lawford et al. described that the macronutrients, for example, carbon, nitrogen, and phosphorus, must be sufficiently available in the medium to ensure microbial growth. 52e elemental content of carbon in E. coli is stated to be roughly 50% (dry weight basis), while nitrogen and phosphorus amounts to 15% (dry weight basis) and 3.2% (dry weight basis), respectively. 52sed on our findings from the previous chapter, growth of E. coli on the CSL-glucose medium (Figure 1) results in a final OD 600 of 2.8 (Figure 1b).With a conversion factor of 0.57 g cell dry weight (CDW)/OD 600 for E. coli from the literature, 53 the CDW of E. coli amounts to 1.61 g/L.Considering the elemental composition described above, 52 the calculated content of carbon, nitrogen, and phosphorus is 0.805, 0.242 and 0.0515 g/L, respectively.Since carbon is shown to be abundant in the CSL-glucose medium, further analysis is directed toward the other two macronutrients.Our findings have shown that the total nitrogen content (ammonium, amino acids, peptides, and otherwise bound nitrogen) in CSL amounts ranges from 6.3% to 7.9% (w/w) (Table S3), while the phosphorus concentration in CSL ranges between 3.0 and 3.7% (w/w) (Table S3).By employing 5 g d.s./L CSL in the investigated CSL-glucose medium, the calculated total nitrogen content in the CSL-glucose medium is 0.335 g/L N, whereas the phosphorus concentration in the CSL-glucose medium is 0.175 g/L P. Thus, it can be deduced that the phosphorus content in the CSL medium is sufficient to meet E. coli's requirement (0.175 vs. 0.0515 g/L).However, the nitrogen content calculated for CSL only just exceeds the concentration required for growth (0.335 vs. 0.242 g/L).It is highly likely that not all the nitrogen contained in CSL is bioavailable.Evaporation of ammonium nitrogen during heat sterilization of CSL may also account for the possible nitrogen insufficiency.Thus, as long the cultivation system is sufficiently aerated, it is assumed that nitrogen is the most probable candidate as limiting nutrient in the employed CSL-glucose medium, thereby necessitating the supplementation of nitrogen to the CSL-glucose medium.Furthermore, struvite (MgNH 4 PO 4 Á 6H 2 O) precipitation is known to occur in aqueous culture media, when sufficient amounts of ammonium, magnesium, and phosphate are available. 54This salt has an extremely low solubility and may irreversibly remove ammonium, phosphorus, and magnesium from the medium. 54Thus, with 1 mole struvite formed, 1 mole of nitrogen is removed from medium, further increasing the nitrogen shortage in the CSL-glucose medium.Therefore, phosphorus and magnesium were eventually also investigated as supplements, to ensure their abundance in the CSL-glucose medium.
In this set of experiments, CSL variant 1 (as listed in Table S2) was the sole variant employed for the glucose-enriched CSL medium.(Figure S7), and 0.14-0.57g/L KH 2 PO 4 (Figure S8) separately into glucose-enriched CSL medium did not show a positive impact on the OTR and final cell density.This result proves that a sufficient amount of magnesium, sulfur, potassium, and phosphorus is present in the employed CSL medium.
Figure 3 shows the impact of ammonium sulfate ((NH 4 ) 2 SO 4 ) supplementation to CSL medium on the metabolic activity of E. coli.For better visibility, the OTR data was divided into two figures (Figure 3a,b).As depicted in Figure 3a, adding 0.36 g/L of (NH 4 ) 2 SO 4 led to an increased OTR peak by 67%, compared to the reference.Moreover, once the OTR dropped, instead of a plateau, a "shoulder,"   S7), ammonium nitrogen can clearly be identified as limiting secondary substrate.Thus, the used CSL medium with a glucose/CSL ratio of 1/1 (g/g) cannot meet the ammonium nitrogen demand of E. coli, as all CSL batches show a secondary substrate-limited behavior.In this CSL medium with a glucose/CSL ratio of 1/1 (g/g), it is the nitrogen content of CSL which limits growth.Increasing the CSL concentration to the same nitrogen level as achieved by the ammonium sulfate supplementation might also result in unlimited growth of E. coli.

| Metabolic activity of Bacillus subtilis on LBand CSL-based media
The previous chapters have proven CSL to be a suitable alternative nutrient source for E. coli.Thus, its suitability as a nutrient source for the model organism Bacillus subtilis 168 was also explored.As previously performed for E. coli, the metabolic activity of B. subtilis were compared, when grown on LB medium and different CSL-based media.To maintain the optimal pH range of B. subtilis, the CSL-basedmedia were supplemented with 100 mM MOPS buffer and the initial pH was adjusted with 3 M NaOH to 7 prior to inoculation.The LB medium employed in this experiment was supplemented with 5 g/L glucose.Figure 4 depicts the dependency of OTR, TO, pH, and OD 600 of B. subtilis, when grown on the LB medium and different CSL-based media.
Cultivating B. subtilis in the different media resulted in varying OTR patterns (Figure 4A).Table S4 displays the varying maximum OTRs obtained on the different media.For all CSL-based media, an aligned linear initial increase of OTR was observed.In contrast, the glucose-enriched LB medium led to a 50% higher growth rate than measured in CSL-based media.B. subtilis reached its first OTR peak of 25 mmol/L/h after 3 h, when grown on glucose-enriched LB medium.
Following the first OTR peak, growth on the LB-glucose medium displayed three other peaks after 5, 9, and 16 h.As Bacillus subtilis is known to produce acetate, acetoin, and 2,3-butanediol at excess glucose concentrations, [55][56][57] different respiratory activities may indicate a change in substrate and overflow metabolite consumption.Growth on the complex components of the LB-glucose medium led to ammonium ions being release, 6 causing the pH to be increased to 7.5 (Figure 4b).Moreover, the glucose-enriched LB medium reached a total oxygen (TO) of 238 mmol/L and led to a final optical density of 3.2 (Figure 4b).
Growth on pure CSL medium as the sole nutrient source resulted in a different OTR pattern (Figure 4b).A "shoulder" precedes the maximum OTR of 17 mmol/L/h, after which a drop in OTR was observed, indicating the depletion of the initial carbon source.Since carbon is not abundant in pure CSL, 14,58 the formation of overflow metabolites is not expected.As a result of the consumption of lactate, as well as proteins and amino acids in CSL, 1,44,45 the final pH value was increased (Figure 4b).Furthermore, the low respiratory activity, and thus, the low TO is also reflected in the low total optical density of 1.26, measured at the end of the experiment (Figure 4b).
When glucose was added to the pure CSL medium, B. subtilis showed an increased OTR peak by 24% compared to pure CSL (Figure 4a).This peak is followed by a rounded plateau of 25 mmol/ L/h at 5 h.After obvious carbon source exhaustion, a sharp drop of OTR was observed.The observed OTR plateau cannot be assigned to oxygen limitation, as the maximum oxygen transfer capacity (OTR max ), calculated according to Meier et al., 59 amounts to 81 mmol/L/h for this particular cultivation condition.Instead, the OTR plateau indicates a secondary substrate limitation, as previously shown for E. coli. 22e to the higher amount of available carbon source, a higher TO was observed in the CSL-glucose medium compared to pure CSL medium (Figure 4b).Furthermore, the addition of glucose resulted in a 2.5-fold increase in final optical density to 3.2, compared to pure CSL medium (1.3) (Figure 4b).As observed for E. coli, the low concentration of carbon in the pure CSL medium leads to a growth limitation of B. subtilis.
Thus, the low TO of 57 mmol/L for the pure CSL medium can be explained by the scarcity of carbon sources.Furthermore, with the added 5 g/L glucose, B. subtilis is likely to form its known overflow metabolites, acetate, acetoin, and 2,3-butanediol. 55Due to this overflow metabolites formation, the cultivation broth is acidified to a final pH of 7 (Figure 4b).
Adding lactate to the CSL medium increased the OTR peak by 33%, compared to pure CSL medium (Figure 4a).Furthermore, the OTR peak is followed by a "shoulder" lasting for 5 h.Consistent with our previous results in E. coli (Figure 1), the addition of lactate to the CSL medium also results in more nutrients of CSL becoming available to B. subtilis, leading to a higher TO, compared to the pure CSL medium (Figure 4b).However, unlike in E. coli, in B. subtilis, the addition of lactate to CSL medium did not result in as much biomass formation as with glucose.The OD 600 increase with lactate supplementation was limited to 51% (from 1.26 to 1.9), instead of 151% (from 1.26 to 3.17) with glucose supplementation (Figure 4b).Furthermore, due to the same concentration of carbon source (5 g/L lactate vs. 5 g/L glucose) utilized in the respective enriched CSL medium, a similar TO is observed for the CSL-glucose and CSL-lactate media (Figure 4b).Furthermore, lactate consumption signifies acid uptake, leading to an increased final pH value of 8 (Figure 4b).

Despite the different OTR profiles shown by the cultivation on
the LB-glucose medium and CSL-glucose medium, both media resulted in similar final optical densities (Figure 4b).However, the two media differ in the amount of complex components engaged.As the LB medium contains 5 g d.s./L yeast extract and 10 g d.s./L peptone, the amount of complex components in LB is thrice higher than found in the CSL media (Figure 4b).Thus, CSL was proven to be superior toward biomass production, compared to yeast extract.

| Influence of different glucose/CSL (g/g) ratios on Bacillus subtilis
As growth of B. subtilis on CSL-glucose medium has been successfully demonstrated, the following results will give insight into the influence of varying CSL concentrations on the metabolic activity of B. subtilis.
In this set of experiments, CSL variant 1 was the sole variant employed to prepare the CSL-glucose medium.CSL concentrations ranging between 1.25 g/L and 7.5 g dry substance/L were employed.
For all CSL concentrations, glucose was initially added at 5 g/L, resulting in different glucose/CSL (g/g) ratios with varying CSL concentrations.
Figure 5a displays the different OTR patterns at varying CSL concentrations.The different CSL concentrations show an aligned growth at the beginning of the cultivation.With increasing CSL concentrations, the OTR was increased (Figure 5a).A severe growth limitation indicated by an OTR plateau was observed when 1.25 g/L (glucose/CSL ratio of 4/1, g/g) and 2.5 g/L CSL (ratio of 2/1, g/g) were employed.Decreasing the glucose/CSL ratio further to 1/1 (g/g) with 5 g/L CSL resulted in an OTR pattern familiar from the previous experiment: a "rounded" OTR plateau at around 22 mmol/ L/h, followed by a rapid decrease, indicating carbon source depletion.With a glucose/CSL ratio of 0.7/1 (g/g) with 7.5 g/L CSL, the OTR invoked a "shoulder" preceding the maximum OTR of 37 mmol/L/h.This OTR peak was followed by a second OTR peak of 14.5 mmol/L/h.At this highest CSL concentration, B. subtilis is likely to have produced its known overflow metabolites acetate, acetoin, and 2,3-butanediol, [55][56][57] which are then consumed as indicated by the second peak (Figure 5a).
With increasing CSL concentrations, the final OD 600 was also increased (Figure 5b).By employing 7.5 g/L CSL, the final OD 600 was almost tripled, in comparison to that obtained with 1.25 g/L CSL.
Using a conversion factor of 0.41 g CDW/OD 600 for B. subtilis from the literature, 60 a final CDW of 1.3 g/L was obtained using 7.5 g/L CSL.With increasing CSL concentrations of up to 200 g d.s./L, Zhuangzhuang et al. also reported that the number of viable cells increased, when B. subtilis was cultivated in a high cell density fermentation. 1For Zhuangzhuang et al., higher CSL concentrations had a negative impact on cell viability. 1th increasing CSL concentrations, pH was gradually increased in a narrow range (Figure 5b).The highest CSL concentration with 7.5 g/L reached a final pH of 7.3.This is in accordance with observations from Zhuangzhuang et al., where increasing CSL concentrations up to 400 g/L caused a gradual pH increase. 1us far, the term "glucose to CSL ratio" (glucose/CSL, g/g) has been employed throughout this work.As previously emphasized, CSL is the sole nitrogen and phosphorous source in the employed CSL media.This means that the term "CSL" in the glucose/CSL ratio (g/g) summarizes both the nitrogen, as well as the phosphorous content in the CSL media.As a result, with glucose or (/and) lactate, respectively, being the sole carbon source(s) in the CSL media, the glucose/CSL ratio (g/g) can roughly be translated to C/N (g/g) and C/P (g/g) ratios.In general, previous research suggests that a C/N ratio of around 3/1 (g/g) is typically employed for B. subtilis fermentations in nutrient media. 61,62However, a higher C/N (g/g) ratio is applied for the targeted production of overflow metabolites, such as acetoin or 2,3-butanediol, and for biosurfactant production. 62As our present study revolves around the use of CSL, it is important to note that the available nitrogen inside CSL is "limited in its utilization for the synthesis of metabolites," as reported in the literature. 1 Hence, the glucose/CSL (g/g) ratio only gives an estimation of the biologically available nitrogen content.In general, a glucose/CSL ratio greater than 1/1 (g/g) invokes a significant secondary substrate-limited OTR pattern in B. subtilis (Figure 5a).Out of the investigated concentrations, 1.25 g/L CSL shows the most severe secondary substrate limitation, while 5 g/L CSL leads to the least severe limited appearance (Figure 5a).A quantity of 7.5 g/L CSL proves to be largely sufficient to meet the requirements of B. subtilis, as growth does not seem severely limited (Figure 5a).For the following investigation of the different CSL batches, a significant secondary substrate-limited system is required, as this specific condition would result in a higher sensitivity toward supplementations of the limiting components.Consequently, 2.5 g/L CSL was chosen for subsequent experiments for identifying the nutrients required for a non-limited growth of B. subtilis.

| Influence of CSL batch-to-batch variations on Bacillus subtilis
As conducted for E. coli, the underlying batch-to-batch variations in CSL was further investigated by growing B. subtilis on nine different CSL batches.In this experiment, the glucose-enriched medium composed of 5 g/L glucose, 2.5 g/L of the respective CSL batch, and 100 mM MOPS buffer was applied.To enable a higher throughput, these experiments were conducted in 96-deepwell microtiter plates and measured by a μTOM device.As conducted for E. coli, cultivations of B. subtilis in the RAMOS and μTOM devices were compared and proven to be similar in both scales (Figure S9).
Figures 6a-c showcase the different metabolic activities of B. subtilis, when grown on distinct CSL batches.A depiction of all nine CSL variants in one graph can be found in Figure S10. Figure 6b shows variant 6, which was produced using an improved process, but originates from the same production facility as variants 4 and 5. Similar to the result obtained with 2.5 g/L CSL in the previous experiment (Figure 5), all CSL variants show an aligned exponential increase, leading to a plateau after 3.5 h. Figure 6a-c shows overlapping OTR patterns for all nine CSL variants.In general, the differences in OTR behavior of B. subtilis, when grown on distinct CSL variants, are of minor nature.
Despite the use of the same carbon source concentration on all CSL variants, a wider range of measured total oxygen (TO) was detected, ranging from 106.7 to 138.6 mmol/L (±4-12) (Figure 6d).
Similarly, the different variants resulted in a resembling final optical density (Figure 6d), ranging from 2.2 to 2.9, with an average of 2.5.
Moreover, the final pH value lies at 7 for all variants.
The results of B. subtilis are not in line with findings from Zhang et al. 4 They encountered significant differences in biomass and antigen productivities between varying yeast extract batches.When compared with our findings in E. coli, where significant differences were observed between the CSL variants (Figure 2a-d), it can be deduced that B. subtilis is less sensitive toward changes in the CSL variants than E. coli.

| Ammonium sulfate supplementation removes secondary substrate limitation in Bacillus subtilis
As previously shown, B. subtilis is limited by a secondary substrate, when grown in glucose-enriched CSL medium with a glucose/CSL (g/g) ratio of 2/1 (g/g).To identify the limiting components, a selective nutrient supplementation was performed in microtiter plates.
As described in the previous chapter, the elemental composition of microorganisms may indicate their nutritional requirements. 52Since our method focusing on the macronutrients nitrogen, phosphorus, and magnesium has been proven successful in E. coli, the same macro-  S11).Furthermore, some negative impact on the OTR patterns and final cell densities was observed by supplementing 0.14-0.57g/L potassium dihydrogen phosphate (KH 2 PO 4 , Figure S12) to the glucose-enriched CSL medium.Consequently, it is concluded that magnesium, sulfur, potassium, and phosphorus are not limited in the employed CSL medium.A (NH 4 ) 2 SO 4 concentration of 0.36 g/L increased the final optical density by 41%, compared to the reference (Figure 7c).Higher (NH 4 ) 2 SO 4 concentrations could not improve the final optical density, confirming that 0.36 g/L is sufficient to meet the nitrogen demand of B. subtilis.As expected by the identical amount of carbon source engaged (5 g/L glucose), the calculated total oxygen (TO) was similar, while the final pH was not influenced by the supplementation and was similar for all cultivations (Figure 7c).Despite being able to improve the overall OTR pattern and final cell density, (NH 4 ) 2 SO 4 supplementation alone could not bring the cultivation on 2.5 g/L CSL medium to the OTR levels observed for the glucose-enriched LB medium, with 5 g/L CSL and 7.5 g/L CSL (Figure S13).This result suggests that at least one additional nutrient is limited in the selected 2.5 g/L CSL medium.As B. subtilis 168 and its derivatives have been successfully grown in Spizisen's minimal salts (SM) medium, 63 SM medium can serve as a basis for further studies on possible limiting nutrients or auxotrophies.Nevertheless, it is notable that the addition of only 0.36 g/L (NH 4 ) 2 SO 4 to the 2.5 g/L CSL medium increased the final OD 600 from 2.1 to 3.This OD 600 value is similar to those obtained on the LB medium (Figure 4b) and 7.5 g/L CSL medium (Figure 5b), both also enriched with 5 g/L glucose.

| CONCLUSION
In this study, the respiration activity monitoring system designed for shake flasks (RAMOS) and 96-well plates (μTOM) was employed to develop a screening method for different CSL batches, as an alternative complex nutrient source in fermentation processes.For the two model organisms, E. coli and B. subtilis, the employed glucose-enriched CSL medium proved to be more efficient than the glucose-enriched LB medium, in terms of biomass production.Thus, CSL proves to be a serious alternative to yeast extract and peptone.At a glucose/CSL ratio of 1/1 (g/g), E. coli showed a clear secondary substrate-limited behavior, whereas this type of limitation was displayed by B. subtilis at a glucose/ CSL ratio of 2/1 (g/g).E. coli and B. subtilis were grown on different CSL batches from varying locations and processes, investigating the extent of the secondary substrate limitation and performance inconsistencies of the CSL variants.The Respiration Activity MOnitoring System, designed for shake flasks (RAMOS) and 96-well microtiter plates (μTOM), was able to detect differences in OTR between CSL batches, allowing "fingerprinting" of the CSL batches.In E. coli, significant differences were found between CSL variants in their OTR behavior and biomass production.However, a rather similar fermentation performance than carbon.Using the online monitoring device for 96-well microtiter plates, μTOM, the varying CSL batches sourced from different locations are ranked based on their performance.The different microbial systems may have varying sensitivities to changes in the composition of the CSL batches used.Based on the elemental composition of E. coli and other bacteria, a systematic nutrient supplementation (MgSO 4 Á 7H 2 O, KH 2 PO 4 , and (NH 4 ) 2 SO 4 ) is performed to identify the limiting secondary substrate in the CSL media used.Batch-to-batch variations of CSL may be overcome with targeted nutrient supplementation.
5 g/L sodium chloride, and 5 g/L glucose.The pH of these LB media was set to 7.0 with a 5 M NaOH solution at the beginning of E. coli cultivations, while B. subtilis cultivations required an adjustment of pH for LB medium to 7.5 (with 5 M NaOH solution) at the start of the experiment.Aside from buffering the culture medium by 0.1 M MOPS buffer, pH was not otherwise maintained throughout the experiment.Next to LB media, CSL media were also used in main cultures.Cargill provided the corn steep liquor (CSL) batches from two different manufacturing plants and third-party market reference batches, with a dry substance content of 43.8%-49.7%(w/w).The crude CSL stock solutions were heat sterilized via a steam autoclave.1 bar overpressure was used to raise the steam temperature to 121 C inside the autoclave chamber.Each heat sterilization cycle was performed for 20 min.Heat-sterilized CSL stock solutions were stored at 4 C. Pure CSL medium consists of 5 g/L of one type of CSL variant (1-9, respectively) and 0.1 M MOPS buffer.CSL-glucose medium consists of 5 g/L of one type of CSL variant (1-9, respectively), 1.25-7.5 g/L glucose, and 0.1 M MOPS buffer.CSL-lactate medium consists of 5 g/L of one type of CSL variant (1-9, respectively) and 5 g/L lactate and 0.1 M MOPS buffer.For CSL media supplementations, MgSO 4 Á 7H 2 O (calculated as 0.25-1.0g/L MgSO 4 ), 0.14-0.57g/L KH 2 PO 4 , and 0.36-3.66g/L (NH 4 ) 2 SO 4 were added to the CSL-glucose medium.All CSLbased media were freshly prepared before the experiments by diluting the heat-sterilized CSL stock solution with demineralized water and adding MOPS buffer, glucose, or other nutrients, for example, lactate or KH 2 PO 4 , to reach the desired concentrations in the media.The pH of the 1 M MOPS buffer solution was adjusted to 7.0 with a 5 M NaOH solution prior to sterile filtration.Stock solutions of glucose, lactate, KH 2 PO 4 , (NH 4 ) 2 SO 4 , and MgSO 4 Á 7H 2 O were sterile filtered.

3 | RESULTS AND DISCUSSION 3 . 1 |
Metabolic activity of Escherichia coli on LB-and CSL-based media CSL-based media and commonly used LB media were compared to assess the suitability of CSL as sole nutrient or nitrogen source for E. coli BL21 DE3 pRotHi-2-EcFbFP.In all CSL media, a CSL amount of 5 g dry substance (d.s.)/L was employed.The metabolic activities of E. coli on CSL-based and LB-based media are shown in Figure 1.Cultivating E. coli in varying media led to clear differences in the course of the oxygen transfer rate (OTR) (Figure 1a).The resulting maximum OTRs obtained on the different media are displayed on Table

F
I G U R E 1 Characterization of Escherichia coli metabolic activity on lysogeny broth (LB)-based media and corn steep liquor (CSL)-based media in RAMOS flasks.(a) Oxygen transfer rate of E. coli BL21 DE3 pRotHi-2-EcFbFP.Data points shown are mean values, ±half the amplitude between duplicate cultivations is shown in shadows.(b) Optical density at 600 nm wavelength (OD 600 ), total oxygen (TO), and final pH values, obtained after 18.5 h.OD 600 and final pH values were measured in triplicates, whereas one value for TO was calculated from each OTR value per cultivation.Error bars for OD 600 and pH in (b) indicate the respective standard deviation.Error bars for pH values are barely visible, due to low standard deviations.Experimental parameters: 250 mL RAMOS flask; LB medium = 5 g/L yeast extract, 10 g/L peptone, and 5 g/L NaCl; CSL medium = 5 g dry substance (d.s.)/L CSL 1 (Table S2), 0.1 M MOPS buffer; glucose concentration c glucose (if applied) = 5 g/L, c lactate (if applied) = 5 g/L, initial pH = 7.5, culture volume V L = 10 mL, shaking frequency n = 350 rpm, shaking diameter d 0 = 50 mm, temperature T = 37 C, and initial OD 600 = 0.1.

Figures
Figures 2a-c depict the metabolic activities of E. coli on different CSL variants.For easy comparison, the CSL batches are shown in three separate graphs (Figure 2a-c) corresponding to three different production locations (TableS2).Figure2bshows variant 6, which comes from Metabolic activity of Escherichia coli grown on various CSL variants of different origins in the μTOM device.(a-c) Oxygen transfer rate of E. coli BL21 DE3 pRotHi-2-EcFbFP.Different CSL variants (as listed in Table S2) are grouped into separate graphs based on their respective origin.All nine CSL batches in one graph are depicted in Figure S3.All data points are mean values measured from 9 to 11 individual wells.Shadows in (a-c) indicate respective standard deviation for mentioned replicates.For some CSL variants, the respective shadows are barely visible, due to low standard deviations <0.5.(d) Optical density at 600 nm wavelength (OD 600 ), total oxygen (TO), and final pH values, obtained after 22 h.Error bars in (d) indicate the respective standard deviation.OD 600 and final pH values were each measured from three individual wells.TO values were calculated from OTRs in 9-11 replicates.Experimental parameters: 96-well microtiter plate (MTP), CSL concentration c CSL, 1-9 = 5 g dry substance (d.s.)/L, glucose concentration c glucose = 5 g/L, MOPS buffer concentration c MOPS = 0.1 M, initial pH = 7.5, culture volume V L = 500 μL/well, shaking frequency n = 350 rpm, shaking diameter d 0 = 50 mm, humidity = 80%, temperature T = 37 C, and initial OD 600 = 0.1.

F
I G U R E 3 Metabolic activity of Escherichia coli in the μTOM device on CSL medium supplemented with ammonium sulfate.(a,b) Oxygen transfer rate of E. coli BL21 DE3 pRotHi-2-EcFbFP.OTR curves of different ammonium sulfate supplementations are split into graphs (a) and (b) for a better overview.For comparison, the reference OTR curve with 0 g/L ammonium sulfate is depicted in both graphs (a) and (b).Data points shown are mean values measured from 5 to 6 individual wells.Shadows, indicating the respective standard deviation, are barely visible, due to low standard deviations <0.5.(c) Optical density at 600 nm wavelength (OD 600 ), total oxygen (TO), and final pH values, obtained after 20 h.OD 600 and final pH values were each measured from three individual wells.TO values were calculated from OTRs in 5-6 replicates.Error bars in (c) indicate the respective standard deviation.Experimental parameters: 96-well microtiter plate (MTP), CSL 1 concentration c CSL1 = 5 g dry substance/L, glucose concentration c glucose = 5 g/L, MOPS buffer concentration c MOPS = 0.1 M, initial pH = 7.5, culture volume V L = 200 μL/well, shaking frequency n = 350 rpm, shaking diameter d 0 = 50 mm, humidity = 80%, temperature T = 37 C, and initial OD 600 = 0.1.still indicating a secondary substrate limitation, is visible.With increasing (NH 4 ) 2 SO 4 concentrations, the OTR was further increased and the "shoulder" became less pronounced, the latter being invisible at (NH 4 ) 2 SO 4 concentrations higher than 1.46 g/L.Increasing the (NH 4 ) 2 SO 4 concentration to more than 1.46 g/L does not further improve the OTR course, as shown in Figure 3b.

Figure
Figure 3c shows the dependence of the OD, calculated total oxygen (TO) and pH on the added (NH 4 ) 2 SO 4 concentrations.The reference cultivation resulted in a secondary substrate limitation, leading to a TO of 112 mmol/L.By supplementing up to 0.73 g/L (NH 4 ) 2 SO 4 , a 13% increase of TO was evaluated.(NH 4 ) 2 SO 4 supplementations above 0.73 g/L decrease the TO to around 100 mmol/L.As observed in Figure 3c, an increase in final optical density was observed with increasing supplementation.In fact, an 86% increase of optical density was enabled by supplementing 1.46 g/L (NH 4 ) 2 SO 4 .Higher (NH 4 ) 2 SO 4 supplementation than 1.46 g/L did not significantly increase cell density.The final pH was not significantly influenced by (NH 4 ) 2 SO 4 supplementation and showed a rather constant value of 7.2.Supplementing the specific glucose-enriched CSL medium with ammonium sulfate led to unlimited growth of E. coli.Based on these supplementation results, it is concluded that 1.46 g/L (NH 4 ) 2 SO 4 suffices to prevent the secondary substrate limitation in this CSL medium.As sulfate has already been ruled out by adding MgSO 4 Á 7H 2 O, (calculated as MgSO 4 , Figure S7), ammonium nitro-

F I G U R E 4
Characterization of Bacillus subtilis metabolic activity on LB medium and CSL-based media in RAMOS flasks.(a) Oxygen transfer rate of B. subtilis 168 (wt).Data points shown are mean values, ±half the amplitude between duplicate cultivations is shown as shadows.(b) Optical density at 600 nm wavelength (OD 600 ), total oxygen (TO), and final pH values, obtained after 24 h.Error bars for OD 600 and final pH values in (b) indicate the respective standard deviation.Error bars in (B) for pH are barely visible, due to low standard deviations <0.5.Experimental parameters: 250 mL RAMOS flask, LB medium = 5 g/L yeast extract, 10 g/L peptone, 5 g/L NaCl; CSL medium = 5 g dry substance/L CSL 1, 0.1 M MOPS buffer; glucose concentration c glucose (if applied) = 5 g/L, c lactate (if applied) = 5 g/L, MOPS buffer concentration c MOPS = 0.1 M, initial pH = 7.0, culture volume V L = 10 mL, shaking frequency n = 350 rpm, shaking diameter d 0 = 50 mm, temperature T = 37 C, and initial OD 600 = 0.1.

F I G U R E 5
Influence of different CSL concentrations on Bacillus subtilis in RAMOS flasks.(a) Oxygen transfer rate of B. subtilis 168 (wt).Data points shown are mean values, ±half the amplitude between duplicate cultivations is shown as shadows.(b) Optical density at 600 nm wavelength (OD 600 ), total oxygen (TO), and pH, obtained after 40 h.OD 600 and final pH values were measured in triplicates, whereas one value for TO was calculated from each OTR measured per cultivation.Error bars for OD 600 and final pH values in (b) indicate the respective standard deviation.Error bars for final pH values are barely visible, due to low standard deviations <0.5.Experimental parameters: 96-well microtiter plate (MTP), CSL 1 concentration c CSL, 1 = 1.25-7.5 g dry substance/L, glucose concentration c glucose = 5 g/L, MOPS buffer concentration c MOPS = 0.1 M, initial pH = 6.0, culture volume V L = 200 μL/well, shaking frequency n = 350 rpm, shaking diameter d 0 = 50 mm, humidity = 80%, temperature T = 37 C, and initial OD 600 = 0.1.
nutrients were analyzed as supplements to identify the limiting nutrients in the CSL-glucose medium employed in B. subtilis.As performed with E. coli, this set of experiments used only CSL variant 1 to prepare the glucose-enriched CSL medium.These targeted supplementation experiments were conducted in 96-deepwell microtiter plates and measured by a μTOM device.For the nutrient supplementation experiments, B. subtilis was grown in CSL medium containing 2.5 g dry substance/L CSL, 5 g/L glucose, and 100 mM MOPS buffer.In addition, the medium was supplemented with varying concentrations of only one type of nutrient.Simultaneously, a reference cultivation without additional supplements was performed.No significant improvement in OTR and final cell density was observed when adding magnesium sulfate heptahydrate (MgSO 4 Á 7H 2 O, calculated as 0.25-1.0g/L MgSO 4 , Figure

Figures
Figures 7a,b depict the influence of ammonium sulfate ((NH 4 ) 2 SO 4 ) supplementation to glucose-enriched CSL medium (with was observed for B. subtilis for all CSL variants.These results show the varying sensitivity of different microorganisms toward changes in the CSL composition.B. subtilis was proven more tolerant to changes than E. coli.Based on the elemental composition of the biomass of E. coli and other bacteria, selected nutrients (MgSO 4 Á 7H 2 O, KH 2 PO 4 , and (NH 4 ) 2 SO 4 ) were supplemented to the CSL-glucose medium.This study shows that ammonium nitrogen becomes limiting in the employed CSLglucose medium, depicted by the positive impact on the respiration F I G U R E 7 Growth of Bacillus subtilis in the μTOM device on supplemented CSL medium with ammonium sulfate.(a,b) Oxygen transfer rate of B. subtilis 168 (wt).OTR curves of different ammonium sulfate supplementations are split into graphs (a) and (b) for a better overview.For comparison, the reference OTR curve with 0 g/L ammonium sulfate is depicted in both graphs (a) and (b).Data points shown are mean values measured from 5 to 6 individual wells.Shadows indicating the respective standard deviation are barely visible due to the low standard deviations <0.5.(c) Optical density at 600 nm wavelength (OD 600 ), total oxygen (TO), and final pH values, obtained after 20 h.OD 600 and final pH values were each measured from three individual wells.TO values were calculated from OTRs in 5-6 replicates.Error bars in (C) indicate the respective standard deviation.Experimental parameters: 96-well microtiter plate (MTP), CSL 1 concentration c CSL 1 = 2.5 g dry substance/L, glucose concentration c glucose = 5 g/L, MOPS buffer concentration c MOPS = 0.1 M, initial pH = 7.0, culture volume V L = 200 μL/well, shaking frequency n = 350 rpm, shaking diameter d 0 = 50 mm, humidity = 80%, temperature T = 37 C, and initial OD 600 = 0.1.