Development of a chemically defined medium for Paenibacillus polymyxa by parallel online monitoring of the respiration activity in microtiter plates

Abstract

Background: One critical parameter in microbial cultivations is the composition of the cultivation medium. Nowadays, the application of chemically defined media increases, due to a more defined and reproducible fermentation performance than in complex media. In order, to improve cost-effectiveness of fermentation processes using chemically defined media, the media should not contain nutrients in large excess. Additionally, to obtain high product yields, the nutrient concentrations should not be limiting. Therefore, efficient medium optimization techniques are required which adapt medium compositions to the specific nutrient requirements of microorganisms.

Results: Since most Paenibacillus cultivation protocols so far described in literature are based on complex ingredients, in this study, a chemically defined medium for an industrially relevant Paenibacillus polymyxa strain was developed. A recently reported method, which combines a systematic experimental procedure in combination with online monitoring of the respiration activity, was applied and extended to identify growth limitations for Paenibacillus polymyxa. All cultivations were performed in microtiter plates. By systematically increasing the concentrations of different nutrient groups, nicotinic acid was identified as a growth-limiting component. Additionally, an insufficient buffer capacity was observed. After optimizing the growth in the chemically defined medium, the medium components were systematically reduced to contain only nutrients relevant for growth. Vitamins were reduced to nicotinic acid and biotin, and amino acids to methionine, histidine, proline, arginine, and glutamate. Nucleobases/-sides could be completely left out of the medium. Finally, the cultivation in the reduced medium was reproduced in a laboratory fermenter.

Conclusion: In this study, a reliable and time-efficient high-throughput methodology was extended to investigate limitations in chemically defined media. The interpretation of online measured respiration activities agreed well with the growth performance of samples measured in parallel via offline analyses. Furthermore, the cultivation in microtiter plates was validated in a laboratory fermenter. The results underline the benefits of online monitoring of the respiration activity already in the early stages of process development, to avoid limitations of medium components, oxygen limitation and pH inhibition during the scale-up.

Keywords: Chemically defined media; Growth limitations; Medium optimization; Online monitoring; Paenibacillus polymyxa.

Fig. 1

Cultivation of Paenibacillus polymyxa in complex medium and chemically defined Moppa medium in microtiter plate. Pbp complex medium (specified in Table 1) or chemically defined Moppa medium (specified in Table 2). Initial concentrations were: 56.2–56.8 g/L maltose, 3.5–3.7 g/L glucose, 3.1–3.4 g/L citrate. a Oxygen transfer rate (OTR), b Final optical density (OD) and pH, c Final maltose, glucose and citrate concentration, d Final acetoin, 2,3-butanediol and lactate concentration. a For clarity, only every 10th measuring point over time is marked as a symbol. Mean values for OTR of at least four replicates with standard deviations as shadows are shown. Standard deviations are not well recognizable, because they are small. b-d For offline analysis, samples (wells) of the replicates of the OTR measurement were pooled at the end of the experiments. OD measurement of pooled samples was performed in triplicate and mean values with standard deviations depicted as error bars are shown. pH and concentrations of sugars and metabolites were determined in a single measurement of pooled samples. c, d d.l. means that concentrations of components were lower than the detection limit. Parameters in b-d were determined after 63.3 h. b OD of complex medium prior to inoculation (3.6) is not considered in the final measured optical density. Initial osmolality in Pbp medium is 0.63 osmol/kg and osmolality after 63.3 h is 0.57 osmol/kg. Initial osmolality in Moppa medium is 0.69 osmol/kg and osmolality after 63.3 h is 0.73 osmol/kg. Cultivation conditions: temperature 33 °C, 48-round well plate, filling volume 0.8 mL, shaking frequency 1000 rpm, shaking diameter 3 mm, 0.1 M MES, initial pH 6.5

Fig. 2

Cultivation of Paenibacillus polymyxa with increased concentrations of nicotinic acid in microtiter plate. Moppa medium (specified in Table 2) without or with increased concentrations of all vitamins of group 1 or of nicotinic acid. Vitamin group (gr.) 1 is specified in Additional file 1: Table S3. nic.: nicotinic. Initial concentrations were: 54.2–56.3 g/L (a-d) and 51.8–53.1 g/L (e–h) maltose, 4.2–4.5 g/L (a-d) and 4.1–4.3 g/L (e–h) glucose, 3.3–3.4 g/L (a-d) and 3.2–3.6 g/L (e–h) citrate. a, e: Oxygen transfer rate (OTR), b, f: Final optical density (OD), total oxygen consumption (TOC) and pH, c, g: Final maltose, glucose and citrate concentration, d, h: Final acetoin, 2,3-butanediol and lactate concentration. a, b: For clarity, only every 10th measuring point over time is marked as a symbol. Mean values for OTR of at least three replicates with standard deviations as shadows are shown. b, f: TOC was determined based on OTR data of the replicates. Mean values for TOC of the replicates with standard deviations depicted as error bars are shown. b-h: For offline analysis, samples (wells) of the replicates of the OTR measurement were pooled at the end of the experiments. OD measurement of pooled samples was performed in triplicate and mean values with standard deviations depicted as error bars are shown. OD of pooled sample of the cultivation w/o nicotinic acid was measured in duplicate and the mean value without standard deviation is shown. pH and concentrations of sugars and metabolites were determined in a single measurement of pooled samples. c – h: d.l. means that concentrations of components were lower than the detection limit. Parameters in c – h were determined after 61 – 66 h. Cultivation conditions: temperature 33 °C, 48-round well plate, filling volume 0.8 mL, shaking frequency 1000 rpm, shaking diameter 3 mm, 0.1 M MES, initial pH 6.5

Fig. 3

Cultivation of Paenibacillus polymyxa with increased pH — buffer capacity in microtiter plate. Moppa medium (specified in Table 2) with 12 × nicotinic acid or Moppa medium with 12 × nicotinic acid and with increased buffer concentration or with increased buffer concentration and initial pH. nic.: nicotinic, pH₀: initial pH. Initial concentrations were: 62.1–63.4 g/L maltose, 1.9–2.1 g/L glucose, 3.7–3.8 g/L citrate. a Oxygen transfer rate (OTR), b Final optical density (OD) and pH, c Final maltose, glucose and citrate concentration, d: Final acetoin and 2,3-butanediol concentration. a For clarity, only every 12th measuring point over time is marked as a symbol. Mean values for OTR of at least four replicates with standard deviations as shadows are shown. b-d For offline analysis, samples (wells) of the replicates of the OTR measurement were pooled at the end of the experiments. OD measurement of pooled samples was performed in triplicate and mean values with standard deviations depicted as error bars are shown. pH and concentrations of sugars and metabolites were determined in a single measurement of pooled samples. c d.l. means that concentrations of components were lower than the detection limit. Final lactate concentrations were lower than the detection limit. Parameters in c-d were determined after 86.3 h. Osmolalities are shown in Additional file 1: Figure S6. Cultivation conditions: temperature 33 °C, 48-round well plate, filling volume 0.8 mL, shaking frequency 1000 rpm, shaking diameter 3 mm

Fig. 4

Cultivation of Paenibacillus polymyxa only with growth relevant vitamins in microtiter plate. Supplemented Moppa medium (specified in Table 2) with or without vitamins (only with nicotinic acid and biotin). nic.: nicotinic. Initial concentrations were: 56.6–57.0 g/L maltose, 3.2–3.3 g/L glucose, 3.0 g/L citrate. a Oxygen transfer rate (OTR), b Final optical density (OD) and pH, c Final maltose and glucose concentration, d Final acetoin and 2,3-butanediol concentration. a For clarity, only every 18th measuring point over time is marked as a symbol. Mean values for OTR of four replicates with standard deviations as shadows are shown. Standard deviations are not well recognizable, because they are small. b-d For offline analysis, samples (wells) of the replicates of the OTR measurement were pooled at the end of the experiments. OD measurement of pooled samples was performed in triplicate and mean values with standard deviations depicted as error bars are shown. pH and concentrations of sugars and metabolites were determined in a single measurement of pooled samples. c d.l. means that concentrations of components were lower than the detection limit. Final citrate and lactate concentrations were lower than the detection limit. Parameters in b-d were determined after 133.3 h. Cultivation conditions: temperature 33 °C, 48-round well plate, filling volume 0.8 mL, shaking frequency 1000 rpm, shaking diameter 3 mm

Fig. 5

Cultivation of Paenibacillus polymyxa with varying amino acid composition in microtiter plate. Supplemented Moppa medium (specified in Table 2) without vitamins (only with nicotinic acid and biotin) and with or without amino acid groups. nic.: nicotinic. Amino acid groups (gr.) are specified in Additional file 1: Table S2. Initial concentrations were: 54.1–56.3 g/L (a-d) and 54.1–56.1 g/L (e–h) maltose, 2.9–3.2 g/L (a-d) and 2.9–3.1 g/L (e–h) glucose, 3.0–3.1 g/L (a-d) and 3.0–3.1 g/L (e–h) citrate. a, e: Oxygen transfer rate (OTR), b, f: Final optical density (OD), total oxygen consumption (TOC) and pH, c, g: Final maltose, glucose and citrate concentration, d, h: Final acetoin and 2,3-butanediol concentration. a, b: For clarity, only every 20th measuring point over time is marked as a symbol. Mean values for OTR of at least three replicates with standard deviations as shadows are shown. b, f: TOC was determined based on OTR data of the replicates. Mean values for TOC of the replicates with standard deviations depicted as error bars are shown. b-h: For offline analysis, samples (wells) of the replicates of the OTR measurement were pooled at the end of the experiments. OD measurement of pooled samples was performed in triplicate and mean values with standard deviations depicted as error bars are shown. OD of pooled sample of the cultivation w/o amino acids was measured in duplicate and the mean value without standard deviation is shown. pH and concentrations of sugars and metabolites were determined in a single measurement of pooled samples. c, d, g: d.l. means that concentrations of components were lower than the detection limit. Final lactate concentrations were lower than the detection limit. Parameters in b-d and f–h were determined after 159.3 h. Cultivation conditions: temperature 33 °C, 48-round well plate, filling volume 0.8 mL, shaking frequency 1000 rpm, shaking diameter 3 mm

Fig. 6

Cultivation of Paenibacillus polymyxa in reduced Moppa medium in microtiter plate and fermenter. Reduced Moppa medium (specified in Table 2). Initial concentrations were: 55.7–58.2 g/L maltose, 2.7–3.3 g/L glucose, 3.1–3.8 g/L citrate. a Oxygen transfer rate (OTR) in microtiter plate or fermenter, b Final optical density (OD) and pH, c Final maltose, glucose and citrate concentration. c d.l. means that concentrations of components were lower than the detection limit. Final lactate concentrations were lower than the detection limit. Parameters in b and c were determined after 133.3 h in microtiter plate (corresponds to 121.0 h cultivation time after shift) and after 119 h in fermenter. Initial osmolality in reduced Moppa medium in microtiter plate is 0.95 osmol/kg and in fermenter is 0.48 osmol/kg. Cultivation conditions: microtiter plate: temperature 33 °C, 48-round well plate, filling volume 0.8 mL, shaking frequency 1000 rpm, shaking diameter 3 mm, 0.2 M MES, initial pH 7.0. For clarity, only every 18th measuring point over time is marked as a symbol. Mean values for OTR of four replicates with standard deviations as shadows are shown for microtiter plate. Standard deviations are not well recognizable, because they are small. b-c For offline analysis, samples (wells) of the replicates of the OTR measurement were pooled at the end of the microtiter plate experiment. OD measurement of pooled sample was performed in triplicate and the mean value with standard deviation depicted as error bar is shown. pH and concentrations of sugars were determined in a single measurement of pooled sample. Fermenter: temperature 33 °C, filling volume 1 L, pH control at pH 6.5 with NH₃ • aq and H₃PO₄ solutions, without MES buffer. For clarity, only every 720th measuring point is marked as a symbol. The cultivation in reduced Moppa medium in microtiter plate is also shown in Additional file 1: Figure S16

Fig. 7

Characterization of the cultivation of Paenibacillus polymyxa in reduced Moppa medium in a fermenter. Reduced Moppa medium (specified in Table 2). a Oxygen transfer rate (OTR), b Optical density (OD) and pH, c Maltose, glucose and citrate concentration, d Lactate concentration. Measuring points in brackets are considered outliers. For clarity, only every 720th measuring point is marked as a symbol for OTR. Cultivation conditions: temperature 33 °C, filling volume 1 L, pH control at pH 6.5 with NH₃ • aq and H₃PO₄ solutions, without MES buffer. The cultivation in the fermenter is also shown in Fig. 6