Respiration-based investigation of adsorbent-bioprocess compatibility

Abstract

Background: The efficiency of downstream processes plays a crucial role in the transition from conventional petrochemical processes to sustainable biotechnological production routes. One promising candidate for product separation from fermentations with low energy demand and high selectivity is the adsorption of the target product on hydrophobic adsorbents. However, only limited knowledge exists about the interaction of these adsorbents and the bioprocess. The bioprocess could possibly be harmed by the release of inhibitory components from the adsorbent surface. Another possibility is co-adsorption of essential nutrients, especially in an in situ application, making these nutrients unavailable to the applied microorganism.

Results: A test protocol investigating adsorbent-bioprocess compatibility was designed and applied on a variety of adsorbents. Inhibitor release and nutrient adsorption was studied in an isolated manner. Respiratory data recorded by a RAMOS device was used to assess the influence of the adsorbents on the cultivation in three different microbial systems for up to six different adsorbents per system. While no inhibitor release was detected in our investigations, adsorption of different essential nutrients was observed.

Conclusion: The application of adsorption for product recovery from the bioprocess was proven to be generally possible, but nutrient adsorption has to be assessed for each application individually. To account for nutrient adsorption, adsorptive product separation should only be applied after sufficient microbial growth. Moreover, concentrations of co-adsorbed nutrients need to be increased to compensate nutrient loss. The presented protocol enables an investigation of adsorbent-bioprocess compatibility with high-throughput and limited effort.

Keywords: Adsorption; Downstream processing; Integrated bioprocesses; RAMOS.

Fig. 1

Possible interactions between bioprocess and hydrophobic adsorbents. Production of new biomass and product (blue) from nutrients (orange) by microorganisms (green). Product and nutrients may be reversibly adsorbed and released from the adsorbent. Possible complications: Release of inhibitors (red) from adsorbent surface (investigated in protocol step A, Fig. 2), co-adsorption of essential nutrients (investigated in protocol step B, Fig. 2) leading to a limitation of the microorganisms

Fig. 2

Protocol for investigation of adsorbent-bioprocess compatibility. A Investigation of release of inhibitors from adsorbent surface: Heat sterilization of adsorbent in DI water; Evacuation of adsorbent solution for release of residual air from pores; Incubation of adsorbent solution under cultivation conditions; Filtration of adsorbent solution for removal of adsorbents and for sterilization; Nutrient supplementation; Inoculation; Cultivation in a RAMOS device. B Investigation of co-adsorption of nutrients: Mixing of adsorbent in medium without trace elements (for protocol steps B^a, B^b and B^c), without main nutrients (ammonium, magnesium and phosphate salts; for protocol steps B^b and B^c) and without a single nutrient investigated for adsorption (for protocol step B^c); Evacuation of adsorbent solution for release of residual air from pores; Incubation of adsorbent solution under cultivation conditions; Filtration of adsorbent solution for removal of adsorbents and for sterilization; Nutrient supplementation (trace elements, for protocol steps B^a, B^b and B^c; main nutrients, for protocol steps B^b and B^c; single nutrient investigated for adsorption for protocol step B^c); Inoculation; Cultivation in a RAMOS device

Fig. 3

Cultivation of C. glutamicum DM1933 treated with different adsorbents according to Fig. 2 protocol step A, for investigation of release of inhibitors. Depicted is the oxygen transfer rate (OTR) for different sets of adsorbents (A, B) suitable for lysine adsorption. Detailed information about the applied adsorbents is shown in Table 1. Cultivations were performed in a RAMOS device at 30 °C, 350 rpm, V_L = 10 mL in 250 mL RAMOS shake flasks at a shaking diameter of 50 mm, initial pH value 7.25, 20 g/L glucose in CG-XII medium. For clarity, only every fourth measuring point is marked as a symbol. Adsorbents, 6 mg_adsorbent/mL, were added. Final OD₆₀₀ of A Reference: 17.5, Activated carbon 1: 18.1, Activated carbon 2: 18, HCP 1: 17.8; B Reference: 14, Activated carbon 3: 15, Activated carbon 4: 15.1, Zeolite: 14. For all curves, mean values of duplicates are shown

Fig. 4

Cultivation of C. glutamicum DM1933 treated with different adsorbents according to Fig. 2 protocol step B^a, for investigation of nutrient adsorption. Depicted is the oxygen transfer rate (OTR) for different sets of adsorbents (A, B) suitable for lysine adsorption. Detailed information about the applied adsorbents is shown in Table 1. Cultivations were performed in a RAMOS device at 30 °C, 350 rpm, V_L = 10 mL in 250 mL RAMOS shake flasks at a shaking diameter of 50 mm, initial pH value 7.25, 20 g/L glucose in CG-XII medium. For clarity, only every fourth measuring point is marked as a symbol. Adsorbents, 6 mg_adsorbent/mL, were added. Final OD₆₀₀ of A Reference: 14.3, Activated carbon 1: 18, Zeolite: 18.1 B Reference: 16.1, Activated carbon 2: 16.7, Activated carbon 4: 18.9. For all curves except the zeolite, mean values of duplicates are shown

Fig. 5

Cultivation of U. cynodontis NBRC9727 Δfuz7^r Δcyp3^r P_etefmttA P_ria1ria1 treated with different adsorbents according to Fig. 2 protocol step B^a, for investigation of nutrient adsorption. Depicted are A the oxygen transfer rate (OTR) and B the final itaconic acid concentration and optical density at 600 nm for one set of adsorbents suitable for itaconic acid adsorption. Detailed information about the applied adsorbents is shown in Table 1. Cultivations were performed in a RAMOS device at 30 °C, 350 rpm, V_L = 10 mL in 250 mL RAMOS shake flasks at a shaking diameter of 50 mm, initial pH value 6, 25 g/L glucose in Verduyn medium. For clarity, only every tenth measuring point is marked as a symbol. Adsorbents, 10 mg_adsorbent/mL, were added. B Bars for itaconic acid concentration on the left and for optical density on the right. For all curves, single measurements are shown

Fig. 6

Cultivation of G. oxydans 621H ΔhsdR pBBR1-p264-fdhSCL-ST treated with different adsorbents according to Fig. 2 protocol step B^b, for investigation of nutrient adsorption. Depicted are A the oxygen transfer rate (OTR) and B the final optical density at 600 nm for one set of adsorbents suitable for 5-KF adsorption. Detailed information about the applied adsorbents is shown in Table 1. Cultivations were performed in a RAMOS device at 30 °C, 350 rpm, V_L = 10 mL in 250 mL RAMOS shake flasks at a shaking diameter of 50 mm, initial pH value 6, 60 g/L fructose in Gluconobacter minimal medium. For clarity, only every fifth measuring point is marked as a symbol. Adsorbents, 50 mg_adsorbent/mL, were added. For all curves, mean values of duplicates are shown

Fig. 7

Cultivation of G. oxydans 621H ΔhsdR pBBR1-p264-fdhSCL-ST treated with different adsorbents according to Fig. 2 protocol step B^b/c, for investigation of nutrient adsorption. Depicted are A the oxygen transfer rate (OTR) and B the final optical density at 600 nm and initial fructose concentration for one adsorbent suitable for 5-KF adsorption. Detailed information about the applied adsorbent is shown in Table 1. Cultivations were performed in a RAMOS device at 30 °C, 350 rpm, V_L = 10 mL in 250 mL RAMOS shake flasks at a shaking diameter of 50 mm, initial pH value 6, 60 g/L fructose in Gluconobacter minimal medium. For clarity, only every fifth measuring point is marked as a symbol. Adsorbents, 50 mg_adsorbent/mL, were added. B Bars for optical density on the left and for fructose concentration on the right. For all curves, single measurements are shown